Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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TRANSGENIC MICE CONTAINING TARGETED GENE DISRUPTIONS
Field of the Invention
The present invention relates to transgenic animals, compositions and methods
relating to the
characterization of gene function.
Background of the Invention
Experimental animal models are important tools for understanding the role of
genes. More
particularly, the ability to develop animals with specific genes altered or
inactivated has been
invaluable to the study of gene function, and has lead to unexpected
discoveries of a gene and/or
mechanisms responsible for disease with similar manifestations in humans.
These genetically
engineered animals are also useful for identifying and testing therapeutic
treatments for a variety of
diseases and disorders.
The identification of the function of numerous genes has been useful in
ascertaining the roles
of these genes in disease. Because of the high level of homology between
humans and mice, for
example, it is possible to define the function of individual human genes by
making targeted germline
mutations in selected genes in the animal. The phenotype of the resulting
mutant animal can be used
to help define the phenotype in humans.
Several interesting genes have recently been discovered belonging to various
families
encoding G-protein coupled receptors (GPCRs), LDL receptors, cerberus and
cerberus-like proteins,
and proteins involved in carbohydrate metabolism, particularly those involved
in epithelial develop-
ment. Identifying the roles of these genes and their expression products may
permit the definition of
disease pathways and the identification of diagnostically and therapeutically
useful targets.
GPCRs have been characterized as having seven putative transmembrane domains
(desig-
nated TM1, TM2, TM3, TM4, TMS, TM6, and TM7) which are believed to represent
transmembrane
a-helices connected by extracellular or cytoplasmic loops. Most G-protein
coupled receptors have
single conserved cysteine residues in each of the first two extracellular
loops which form disulfide
bonds that are believed to stabilize functional protein structure. G-protein
coupled receptors can be
intracellularly coupled by heterotrimeric G-proteins to various intracellular
enzymes, ion channels and
transporters. Different G-protein a-subunits preferentially stimulate
particular effectors to modulate
various biological functions in a cell.
Platelet-activating factor (PAF) has been implicated as a mediator in diverse
pathologic
processes, such as allergy, asthma, septic shock, arterial thrombosis, and
inflammatory processes
(Prescott et al., J. Biol. Chem. 265:17381-17384 (1990)). PAF is a
phospholipid (1-0-alkyl-2-acetyl-
sn-glycero-3-phosphorylcholine) and exerts its various effects via specific
cell surface receptors that
bind PAF with high affinity. Using a guinea pig probe, Seyfried et al.
(Gezzomics 13: 832-834 (1992))
isolated the gene for human PAF receptor (PTAFR). The coding sequence contains
no introns. The
encoded protein is highly homologous to the guinea pig PAF receptor (82%
identity) and contains 7
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putative transmembrane domains. The PAF receptor (PAFR) therefore appears to
be a member of the
G protein-coupled family of receptors and exhibits significant similarity to
many members of this
family. By analysis of rodent/human somatic cell hybrids, Seyfried et al.
concluded that the PTAFR
gene is located on human chromosome 1.
Recently, marine ESTs were identified (accession AA170490; GI:1749042;
accession
AA162789; GI:1738456) bearing sequence similarity to human platelet activating
receptor homolog
(accession NM 013308; GI:7019400). Additionally, a marine EST was identified
(accession
AA274112; GI:1912557) bearing sequence similarity to a human GPCR (KIAA0001;
Nomura et al.,
DNA Res 2(4):210 (1995)) and to human platelet-activating factor receptor. The
start of the EST
corresponds to approximately position 85 of PAF receptor which is in the 1st
extracellular loop. The
strongest homology is to KIAA0001, which is a randomly identified GPCR
sequence with similarity
to anaphylatoxin CSa receptor and PAF receptor. Over the past 15 years, nearly
350 therapeutic
agents targeting 7 transmembrane receptors have been successfully introduced
onto the market. As
these receptors have an established, proven history as therapeutic targets, a
clear need exists for
identification and characterization of GPCRs which can play a role in
preventing, ameliorating or
correcting dysfunctions or diseases.
Epidermal growth factor (EGF) has a characteristic structure, which comprises
three disulfide
bridges that are essential for the protein's growth-stimulating activity. Many
other proteins, however,
have similar EGF domains (EGF-like domains). These proteins include both
growth factors and other
proteins having functions unrelated to EGF. (See, e.g., Van Zoelen et al.,
Vitamins and Horr~zorees
59:99-131 (2000)).
Calcium binding EGF-like domains are present in many extracellular proteins
that perform a
diverse range of functions. These proteins include, for example, fibrillin-1,
Notch-3, protein S, factor
IX, and the low-density lipoprotein (LDL) receptor. (See, e.g., Smallridge et
al., Journal of
Molecular Biology 286(3):661-668 (1999)).
The LDL receptor family is known to bind and internalize apoE-rich
lipoproteins and thought
to play a role in lipoprotein metabolism. (See, e.g., Bujo, Nippofa rinslZO
57(12):2690-2695 (1999);
Gliemann, Biological Chemistry 379:951-964 (1998)). Mutations cause familial
hypercholesterolemia and premature coronary artery disease. LDL receptor-
related proteins play an
important role in the clearance of plasma-activated alpha 2-acroglobulin and
apolipoprotein E-
enriched lipoproteins. It is essential for fetal development and has been
associated with Alzheimer's
disease (Hussain, et al., Arcra. Rev. Nutr.19:141-72 (1999)). Recent studies
have shown that this
receptor family is important in neural development, suggesting that the LDL
receptor family
comprises multifunctional receptors involved in intracellular signal
transduction, neuron migration,
vascular smooth muscle cell proliferation, and lipoprotein incorporation.
(See, e.g., Bujo (1999),
supra).
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Recently, an expressed sequence tag (EST) (GenBank Accession No. AA482431)
having
EGF-like domains and showing similarities to LDL receptor-related proteins was
identified. The gene,
termed low-density lipoprotein receptor related protein 5 (LRPS), has been
implicated as a candidate
for conferring susceptibility to diabetes based on its chromosomal
localization within the insulin-
dependent diabetes mellitus (IDDM) locus (see Hey, et al., Gene 216:103-111
(1998)).
Cerberus and cerberus-like proteins comprise a protein family expressed in the
embryo, that
are able to induce, enhance, and/or inhibit the formation, growth, and
maintenance of neurons and
related neural cells. The cerberus gene encodes a secreted protein that is
expressed in the anterior
endomesoderm, and when microinjected into Xenopus embryos, induces ectopic
heads, and dupli-
Gated hearts and livers. (See, e.g., Bouwmeester et al., Nature 382(6592):595-
601 (1996)). The
cerberus-like (cerrl) gene was subsequently isolated in mice, and encodes a
novel secreted protein
specifically expressed in the anterior visceral endoderm during early
gastrulation. (See, e.g., Belo et
al., Mechanisms of Developmefat 68:45-57 (1997)). In Xenopus assays, cer-1
acts as a potent
neutralizing factor that induces forebrain markers and endoderm, but is unable
to induce ectopic head-
like structures as cerberus. Cer-1 encodes a putative secreted protein that is
48% identical to cerberus
over a 110-amino acid region. (See, e.g., Shawlot et al., ProceediJZgs of the
National Academy of
Scieyaces, USA 95(11):6198-6203 (1998)). These proteins appear to have a 9
cysteine residue pattern.
(See, e.g., U.S. Patent No. 5,935,852).
The cerberus protein has also shown to be a multifunctional antagonist of
Nodal, BMP, and
Wnt signals. Specifically, the cerberus protein functions as a multivalent
growth factor antagonist in
the extracellular space, binding to Nodal, BMP, and Wnt proteins through
independent sites. (See,
e.g., Piccolo et al., Nature 382 (6592):595-601 (1996)). Reported studies
indicated that cerberus does
not have a receptor or a dedicated transduction pathway, but acts as an
extracellular inhibitor. (See,
e.g., Agius et al., Journal de la Societe de biologie 193:347-354 (1999)).
The human cerberus protein has been isolated and reported. (See, e.g., PCT
Patent
Publication No.: WO 98492967. As described in the art, these proteins are
capable of inducing
endodermal, cardiac, and neural tissue development in vertebrates when
expressed, and may be useful
in applications that require regeneration, differentiation, or repair of
tissues, such as wound repair,
neuronal regeneration or transplantation, supplementation of heart muscle
differentiation, and
differentiation of the pancreas. (See, e.g., PCT Patent Publication No.: WO
9748275). These
proteins may also be useful in the treatment of BMP-related disorders. (See,
e.g., WO 984929.
Recently, an expressed sequence tag (EST) (GenBank Accession No. AA120122)
bearing high
similarities to cerberus and cerberus-like genes was identified.
Successful development of fertile eggs involves the direct interaction between
germline cells
and follicle cells. (See, e.g., McLaren and Wylie, CurrefZt Problems ira Germ
Cell Differentiatiofa,
Cambridge University Press (1992)). Epithelial morphogenesis and the
development of the follicular
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epithelium and germline differentiation that occurs in oogenesis have been
studied well in Drosophila
melanogaster. (See, e.g., King, Ovarian Development In Drosophila
Melanogaster, New York,
Academic Press, (1970)). More particularly, the monolayer follicular
epithelium has developmental,
morphological, and molecular properties of vertebrate epithelia, and develops
in concert with the
differentiating germline during oogenesis in Drosophila Melazzogaster. (See,
e.g., Goode et al.,
Development 122:3863-3879 (1996)).
Several classes of cell adhesion molecules are essential for epithelial
development in both
invertebrates and vertebrates. (See, e.g., Gumbiner et al., Cell 69:385-387
(1992)). For example, e-
cadhedrin cell adhesion molecules are required for the formation and
maintenance of epithelial
structure in vertebrates and Drosophila. (See, e.g., Takeichie, Development
102:639-655 (1995);
Tepass et al., Genes Dev. 10:672-685 (1996); and Uemural et al., Genes Dev.
10:659-671 (1996)).
Receptor tyrosine kinases and their ligands are cell surface factors that are
also necessary for
epithelial development. (See, e.g., Naldini et al., EMBO J. 10:2867-2878
(1991); Miettinen et al.,
Nature 376:337-341 (1995); Luetteke et al., Cell 73:249-261 (1993); Schiibach,
Cell 49:699-707
(1987); Goode et al., Dev. Biol. 178:35-50 (1996)).
Neurogenic genes encode another class of molecules important in the
development of the
epithelium. Drosoph.ila neurogenic genes, including, Notch (n), Delta,
neuralized, and Enhancer split
are necessary for development of epithelial characteristics in embryonic
tissue. (See, e.g., Artavanis-
Tsakonas et al., Science 268:225-232 (1995); Hartenstein et al.,
Developzzzezzt 116:1203-1220 (1992);
Coffman et al., Cell 73:659-671 (1993); Ruohola et al., Cell 66:1-20 (1991);
and Xu et al.,
Development 115:913-922 (1992)). Although these Drosophila neurogenic genes
contribute to
epithelial characteristics in embryonic tissue, little is known about their
role in follicular epithelium
morphogenesis.
One neurogenic gene, in particular, known as brainiac has shown to play a
specific role in
epithelial development. The brainiac gene has been reported to encode a novel,
putative secreted
protein required in the germline for establishing the follicular epithelium
and for determining its
dorsal-ventral polarity. (See, e.g., Goode et al., (1992) and Goode et al.,
Dev. Biol. (1996) supra).
More specifically, the brainiac gene is present on the X chromosome and
encodes a 325 amino acid
protein with a putative signal sequence. The brainiac gene is expressed
constitutivelyin the germline
during the first 12 hours of embryogenesis. (See, e.g., Goode, S. et al.
(1992) supra; Goode et al.,
Dev. Biol. (1996) supra; and Goode et al., Development (1996) supra) Mutations
in the brainiac gene
demonstrate synergistic genetic interactions with mutations in signaling
molecules, including,
transforming growth factor alpha (TGF-a) and epidermal growth factor receptor
(Egfr). TGF-a and
Egfr mutant mice suffer from multi-organ failure due to widespread impairment
of epithelial
development (See, e.g., Miettinen et al., (1995) supra).
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Brainiac genes' are important for correct development of the follicular
epithelium. Brainiac
mutant females and their offspring have multiple defects including
ventralization of the eggshell, gaps
in the follicular epithelium, and multiple layers of follicle cells around
oocytes. In the absence of
brainiac gene function, less efficiency in follicular epithelium formation
occurs, resulting in a failure
to individuate germline cysts. Once the follicular epithelium is formed,
brainiac genes are essential
for maintenance of its epithelial characteristics. In addition, disruption in
brainiac germline function
results in loss of apicalbasal polarity and an accumulation of multiple layers
of follicle cells,
particularly around the oocyte. Brainiac is essential for efficient migration
and maintenance of border
cells and main body epithelial cells moving towards the oocyte during the late
phases of oogenesis.
(See, e.g., Goode et al., Development (1996) supra). An additional gene known
as egghead has
shown to function similarly in epithelial development. Moreover, both genes
appear to be compo-
nents of novel signaling pathways essential for epithelial development and
maintenance. Most
recently, an expressed sequence tag (EST) was isolated bearing sequence
similarity to genes encoding
neurogenic secreted signaling proteins, and more particularly to the brainiac
gene (GenBank
Accession No.: AA133340; EST name: zn92h01.s1).
Summary of the Invention
The present invention generally relates to transgenic animals, as well as to
compositions and
methods relating to the characterization of gene function.
The present invention provides transgenic cells comprising a disruption in a
targeted gene.
The transgenic cells of the present invention are comprised of any cells
capable of undergoing
homologous recombination. Preferably, the cells of the present invention are
stem cells and more
preferably, embryonic stem (ES) cells, and most preferably, marine ES cells.
According to one
embodiment, the transgenic cells are produced by introducing a targeting
construct into a stem cell to
produce a homologous recombinant, resulting in a mutation of the targeted
gene. In another
embodiment, the transgenic cells are derived from the transgenic animals
described below. The cells
derived from the transgenic animals includes cells that are isolated or
present in a tissue or organ, and
any cell lines or any progeny thereof.
The present invention also provides a targeting construct and methods of
producing the
targeting construct that when introduced into stem cells produces a homologous
recombinant. In one
embodiment, the targeting construct of the present invention comprises first
and second polynucleo-
tide sequences that are homologous to the targeted gene. The targeting
construct also comprises a
polynucleotide sequence that encodes a selectable marker that is preferably
positioned between the
two different homologous polynucleotide sequences in the construct. The
targeting construct may
also comprise other regulatory elements that may enhance homologous
recombination.
The present invention further provides non-human transgenic animals and
methods of
producing such non-human transgenic animals comprising a disruption in a
target gene. The
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transgenic animals of the present invention include transgenic animals that
are heterozygous and
homozygous for a mutation in the target gene. In one aspect, the transgenic
animals of the present
invention are defective in the function of the target gene. In another aspect,
the transgenic animals of
the present invention comprise a phenotype associated with having a mutation
in a target gene.
The present invention also provides methods of identifying agents capable of
affecting a
phenotype associated with a disruption of a target gene in a transgenic
animal. For example, a
putative agent is administered to the transgenic animal and a response of the
transgenic animal to the
putative agent is measured and compared to the response of a "normal" or wild
type mouse, or
alternatively compared to a transgenic animal control (without agent
administration). The invention
further provides agents identified according to such methods. The present
invention also provides
methods of identifying agents useful as therapeutic agents for treating
conditions associated with a
disruption of the target gene.
The present invention further provides a method of identifying agents having
an effect on
target gene expression or function. The method includes administering an
effective amount of the
agent to a transgenic animal, preferably a mouse. The method includes
measuring a response of the
transgenic animal, for example, to the agent, and comparing the response of
the transgenic animal to a
control animal, which may be, for example, a wild-type animal or
alternatively, a transgenic animal
control. Compounds that may have an effect on target gene expression or
function may also be
screened against cells in cell-based assays, for example, to identify such
compounds.
The invention also provides cell lines comprising nucleic acid sequences of a
target gene.
Such cell lines may be capable of expressing such sequences by virtue of
operable linkage to a
promoter functional in the cell line. Preferably, expression of the target
gene sequence is under the
control of an inducible promoter. Also provided are methods of identifying
agents that interact with
the target gene, comprising the steps of contacting the target gene or target
protein with an agent and
detecting an agent! target gene or agent/target protein complex. Such
complexes can be detected by,
for example, measuring expression of an operably linked detectable marker.
The invention further provides methods of treating diseases or conditions
associated with a
disruption in a target gene, and more particularly, to a disruption in the
expression or function of the
target gene or the target protein encoded by the target gene. In a preferred
embodiment, methods of
the present invention involve treating diseases or conditions associated with
a disruption in the target
gene's expression or function, including administering to a subject in need, a
therapeutic agent which
effects target gene or target protein expression or function. In accordance
with this embodiment, the
method comprises administration of a therapeutically effective amount of a
natural, synthetic, semi-
synthetic, or recombinant target gene, target gene products or fragments
thereof as well as natural,
synthetic, semi-synthetic or recombinant analogs.
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The present invention further provides methods of treating diseases or
conditions associated
with disrupted targeted gene expression or function, wherein the methods
comprise detecting and
replacing through gene therapy mutated target genes.
Definitions
The term "gene" refers to (a) a gene containing at least one of the DNA
sequences disclosed
herein; (b) any DNA sequence that encodes the amino acid sequence encoded by
the DNA sequences
disclosed herein andlor; (c) any DNA sequence that hybridizes to the
complement of the coding
sequences disclosed herein. Preferably, the term includes coding as well as
noncoding regions, and
preferably includes all sequences necessary for normal gene expression
including promoters,
enhancers and other regulatory sequences.
The terms "polynucleotide" and "nucleic acid molecule" are used
interchangeably to refer to
polymeric forms of nucleotides of any length. The polynucleotides may contain
deoxyribonucleo-
tides, ribonucleotides and/or their analogs. Nucleotides may have any three-
dimensional structure,
and may perform any function, known or unknown. The term "polynucleotide"
includes single-,
double-stranded and triple helical molecules.
"Oligonucleotide" refers to polynucleotides of between 5 and about 100
nucleotides of single- or
double-stranded DNA. Oligonucleotides are also known as oligomers or oligos
and may be isolated
from genes, or chemically synthesized by methods known in the art. A "primer"
refers to an
oligonucleotide, usually single-stranded, that provides a 3'-hydroxyl end for
the initiation of enzyme-
mediated nucleic acid synthesis. The following are non-limiting embodiments of
polynucleotides: a
gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA,
recombinant
polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of
any sequence, isolated
RNA of any sequence, nucleic acid probes and primers. A nucleic acid molecule
may also comprise
modified nucleic acid molecules, such as methylated nucleic acid molecules and
nucleic acid
molecule analogs. Analogs of purines and pyrimidines are known in the art, and
include, but are not
limited to, aziridinycytosine, 4-acetylcytosine, 5-fluorouracil, 5-
bromouracil, 5-carboxymethylamino-
methyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, inosine, N6-
isopentenyladenine, 1-methyl-
adenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-
dimethylguanine, 2-methyl-
adenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, pseudouracil, 5-
pentylnyluracil and
2,6-diaminopurine. The use of uracil as a substitute for thymine in a
deoxyribonucleic acid is also
considered an analogous form of pyrimidine.
A "fragment" of a polynucleotide is a polynucleotide comprised of at least 9
contiguous
nucleotides, preferably at least 15 contiguous nucleotides and more preferably
at least 45 nucleotides,
of coding or non-coding sequences.
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The term "gene targeting" refers to a type of homologous recombination that
occurs when a
fragment of genomic DNA is introduced into a mammalian cell and that fragment
locates and
recombines with endogenous homologous sequences.
The term "homologous recombination" refers to the exchange of DNA fragments
between
two DNA molecules or chromatids at the site of homologous nucleotide
sequences.
The term "homologous" as used herein denotes a characteristic of a DNA
sequence having at least
about 70 percent sequence identity as compared to a reference sequence,
typically at least about 85
percent sequence identity, preferably at least about 95 percent sequence
identity, and more preferably
about 98 percent sequence identity, and most preferably about 100 percent
sequence identity as
compared to a reference sequence. Homology can be determined using a "BLASTN"
algorithm. It is
understood that homologous sequences can accommodate insertions, deletions and
substitutions in the
nucleotide sequence. Thus, linear sequences of nucleotides can be essentially
identical even if some
of the nucleotide residues do not precisely correspond or align. The reference
sequence may be a
subset of a larger sequence, such as a portion of a gene or flanking sequence,
or a repetitive portion of
a chromosome.
The term "target gene" (alternatively referred to as "target gene sequence" or
"target DNA
sequence" or "target sequence") refers to any nucleic acid molecule or
polynucleotide of any gene to
be modified by homologous recombination. The target sequence includes an
intact gene, an exon or
intron, a regulatory sequence or any region between genes. The target gene
comprises a portion of a
particular gene or genetic locus in the individual's genomic DNA. As provided
herein, the target gene
of the present invention is a platelet activating receptor gene, a PAF
receptor gene, an LRPS gene, a
cerberus gene, and a brainiac gene.
A "platelet activating receptor gene" refers to a sequence comprising SEQ ID
NO:1 or
comprising the sequence identified in Genebank as Accession No. AA162789;
GI:1738456. In one
aspect, the coding sequence of the platelet activating receptor gene comprises
SEQ ID NO:1 or
comprises the gene identified in Genebank as Accession No.: AA162789;
GI:1738456.
An "PAF receptor gene" refers to a sequence comprising SEQ ID N0:4 or
comprising the
sequence identified in Genebank as Accession No. AA274112; GI:1912557. In one
aspect, the coding
sequence of the PAF receptor gene comprises SEQ ID N0:4 or comprises the gene
identified in
Genebank as Accession No. AA274112; GI:1912557.
An "LPRS gene" refers to a sequence comprising refers to a sequence comprising
SEQ ID
N0:7 or comprising the sequence identified in Genebank as Accession No.
NM_008513; GI:6678715.
In one aspect, the coding sequence of the LPRS gene comprises SEQ ID N0:7 or
comprises the gene
identified in Genebank as Accession No. NM_008513; GI:6678715 .
A "cerberus gene" refers to a sequence comprising SEQ ID NO:10 or comprising
the
sequence identified in Genebank as Accession No. NM_009887; GI:6753409 . In
one aspect, the
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coding sequence of the cerberus gene comprises SEQ ID N0:7 or comprises the
gene identified in
Genebank as Accession No. NM_009887; GI:6753409.
A "brainiac gene" refers to a sequence comprising SEQ ID N0:13 or comprising
the sequence
identified in GenBank as Accession No.: AA133340; EST name: zn92hOl.sl. In one
aspect, the
coding sequence of the platelet activating receptor gene comprises SEQ ID
N0:13 or comprises the
gene identified in Genebank as Accession No.: AA133340; EST name: zn92hOl.sl.
"Disruption" of a target gene occurs when a fragment of genomic DNA locates
and
recombines with an endogenous homologous sequence. These sequence disruptions
or modifications
may include insertions, missense, frameshift, deletion, or substitutions, or
replacements of DNA
sequence, or any combination thereof. Insertions include the insertion of
entire genes which may be
of animal, plant, prokaryotic, or viral origin. Disruption, for example, can
alter or replace a promoter,
enhancer, or splice site of a target gene, and can alter the normal gene
product by inhibiting its
production partially or completely or by enhancing the normal gene product's
activity.
The term, "transgenic cell", refers to a cell containing within its genome a
target gene that has
been disrupted, modified, altered, or replaced completely or partially by the
method of gene targeting.
The term "transgenic animal" refers to an animal that contains within its
genome a specific
gene that has been disrupted by the method of gene targeting. The transgenic
animal includes both the
heterozygote animal (i.e., one defective allele and one wild-type allele) and
the homozygous animal
(i.e., two defective alleles).
As used herein, the terms "selectable marker" or "positive selection marker"
refers to a gene
encoding a product that enables only the cells that carry the gene to survive
and/or grow under certain
conditions. For example, plant and animal cells that express the introduced
neomycin resistance
(Neon) gene are resistant to the compound 6418. Cells that do not carry the
Neor gene marker are
killed by 6418. Other positive selection markers will be known to those of
skill in the art.
A "host cell" includes an individual cell or cell culture which can be or has
been a recipient
for vectors) or for incorporation of nucleic acid molecules and/or proteins.
Host cells include
progeny of a single host cell, and the progeny may not necessarily be
completely identical (in
morphology or in total DNA complement) to the original parent due to natural,
accidental, or
deliberate mutation. A host cell includes cells transfected with the
constructs of the present invention.
The term "modulates" as used herein refers to the inhibition, reduction,
increase or
enhancement of a target gene or target protein function, expression, activity,
or alternatively a
phenotype associated with a disruption in a target gene.
The term "ameliorates" refers to a decreasing, reducing or eliminating of a
condition, disease,
disorder, or phenotype, including an abnormality or symptom associated with a
disruption in a target
gene.
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The term "abnormality" refers to any disease, disorder, condition, or
phenotype in which a
disruption of a target gene is implicated, including pathological conditions.
Brief Description of the Drawings
Figure 1 shows the polynucleotide sequence for a platelet activating receptor
gene (SEQ ID
N0:1).
Figure 2A-2B shows design of the targeting construct used to disrupt platelet
activating
receptor genes. Figure 2B shows the sequences identified as SEQ m N0:2 and SEQ
m N0:3, which
were used as the targeting arms (homologous sequences) in the targeting
construct.
Figure 3 shows a graph relating to the hot plate testing on mice having a
disruption in a
platelet activating receptor genes.
Figure 4 shows a graph relating to the open field testing on mice having a
disruption in a
platelet activating receptor genes.
Figure 5 shows the polynucleotide sequence for a PAF receptor gene (SEQ ID
N0:4).
Figure 6A-6B shows design of the targeting construct used to disrupt PAF
genes. Figure 6B
shows the sequences identified as SEQ ID N0:5 and SEQ m N0:6, which were used
as the targeting
arms (homologous sequences) in the targeting construct.
Figure 7A-7C shows the polynucleotide sequence for a LPRS gene (SEQ ID N0:7).
Figure 8 shows the amino acid sequence for a LPRS polypeptide (SEQ m N0:8).
Figure 9A-9C shows design of the targeting construct used to disrupt LPRS
genes. Figure 9C
shows the sequences identified as SEQ ID N0:9 and SEQ ID NO:10, which were
used as the
targeting arms (homologous sequences) in the targeting construct.
Figure 10 shows a graph relating to the open field testing (time spent in the
central region) of
mice having a disruption in a LPRS gene.
Figure 11 shows a graph relating to the open field testing (total distance
traveled) of mice
having a disruption in a LPRS gene.
Figure 12 shows the polynucleotide sequence for the cerberus gene (SEQ ID
N0:11) and the
amino acid sequence of a cerberus polypeptide (SEQ ID N0:12).
Figure 13A-13B shows design of the targeting construct used to disrupt
cerberus genes.
Figure 13B shows the sequences identified as SEQ m N0:13 and SEQ ID N0:14,
which were used
as the targeting arms (homologous sequences) in the targeting construct.
Figure 14 shows a graph relating to the open field testing of mice having a
disruption in a
cerberus gene.
Figure 15 shows a graph relating to the tail suspension testing of mice having
a disruption in a
cerberus gene.
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Figure 16 shows the polynucleotide sequence of a brainiac gene (SEQ ID N0:17)
and SEQ
ID N0:16 and SEQ ID N0:17 which were used as the targeting arms (homologous
sequences) in the
brainiac gene targeting construct.
Detailed Description of the Invention
The invention is based, in part, on the evaluation of the expression and role
of genes and gene
expression products, primarily those associated with a target gene. Among
others, the invention
permits the definition of disease pathways and the identification of
diagnostically and therapeutically
useful targets. For example, genes which are mutated or down-regulated under
disease conditions
may be involved in causing or exacerbating the disease condition. Treatments
directed at up-
regulating the activity of such genes or treatments which involve alternate
pathways, may ameliorate
the disease condition.
Generation of Targeting Construct
The targeting construct of the present invention may be produced using
standard methods
known in the art. (See, e.g., Sambrook, et al., 1989, Molecular Clo>zi>zg: A
Laboratory Manual,
Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New
York; E.N. Glover
(eds.), 1985, DNA Clorzihg: A Practical Approach, Volumes I and II; M.J. Gait
(ed.), 1984,
Oligonucleotide Synthesis; B.D. Hames & S.J. Higgins (eds.), 1985, Nucleic
Acid Hybridization; B.D.
Hames & S.J. Higgins (eds.), 1984, Transcription and Translation; R.I.
Freshney (ed.), 1986, Animal
Cell Culture; Immobilized Cells and Enzymes,1RL Press, 1986; B. Perbal, 1984,
A Practical Guide
To Molecular Cloning; F.M. Ausubel et al., 1994, Current Protocols in
Molecular Biology, John
Wiley & Sons, Inc.). For example, the targeting construct may be prepared in
accordance with
conventional ways, where sequences may be synthesized, isolated from natural
sources, manipulated,
cloned, ligated, subjected to in vitro mutagenesis, primer repair, or the
like. At various stages, the
joined sequences may be cloned, and analyzed by restriction analysis,
sequencing, or the like.
The targeting DNA can be constructed using techniques well known in the art.
For example,
the targeting DNA may be produced by chemical synthesis of oligonucleotides,
nick-translation of a
double-stranded DNA template, polymerase chain-reaction amplification of a
sequence (or ligase
chain reaction amplification), purification of prokaryotic or target cloning
vectors harboring a
sequence of interest (e.g., a cloned cDNA or genomic DNA, synthetic DNA or
from any of the
aforementioned combination) such as plasmids, phagemids, YACs, cosmids,
bacteriophage DNA,
other viral DNA or replication intermediates, or purified restriction
fragments thereof, as well as other
sources of single and double-stranded polynucleotides having a desired
nucleotide sequence.
Moreover, the length of homology may be selected using known methods in the
art. For example,
selection may be based on the sequence composition and complexity of the
predetermined
endogenous target DNA sequence(s).
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The targeting construct of the present invention typically comprises a first
sequence
homologous to a portion or region of the target gene and a second sequence
homologous to a second
portion or region of the target gene. The targeting construct further
comprises a positive selection
marker, which is preferably positioned in between the first and the second DNA
sequence that are
homologous to a portion or region of the target DNA sequence. The positive
selection marker may be
operatively linked to a promoter and a polyadenylation signal.
Other regulatory sequences known in the art may be incorporated into the
targeting construct
to disrupt or control expression of a particular gene in a specific cell type.
In addition, the targeting
construct may also include a sequence coding for a screening marker, for
example, green fluorescent
protein (GFP), or another modified fluorescent protein.
Although the size of the homologous sequence is not critical and can range
from as few as 50
base pairs to as many as 100 kb, preferably each fragment is greater than
about 1 kb in length, more
preferably between about 1 and about 10 kb, and even more preferably between
about 1 and about 5
kb. One of skill in the art will recognize that although larger fragments may
increase the number of
homologous recombination events in ES cells, larger fragments will also be
more difficult to clone.
In a preferred embodiment of the present invention, the targeting construct is
prepared
directly from a plasmid genomic library using the methods described in pending
U.S. Patent Applica-
tion No.: 08/971,310, filed November 17, 1997, the disclosure of which is
incorporated herein in its
entirety. Generally, a sequence of interest is identified and isolated from a
plasmid library in a single
step using, for example, long-range PCR. Following isolation of this sequence,
a second polynucleo-
tide that will disrupt the target sequence can be readily inserted between two
regions encoding the
sequence of interest. In accordance with this aspect, the construct is
generated in two steps by
(1) amplifying (for example, using long-range PCR) sequences homologous to the
target sequence,
and (2) inserting another polynucleotide (for example a selectable marker)
into the PCR product so
that it is flanked by the homologous sequences. Typically, the vector is a
plasmid from a plasmid
genomic library. The completed construct is also typically a circular plasmid.
In another embodiment, the targeting construct is designed in accordance with
the regulated
positive selection method described in U.S. Application No. 60/232,957, filed
September 15, 2000,
the disclosure of which is incorporated herein in its entirety. The targeting
construct is designed to
include a PGK-~eeo fusion gene having two lac0 sites, positioned in the PGK
promoter and an NLS-
lacl gene comprising a lac repressor fused to sequences encoding the NLS from
the SV40 T antigen.
In another embodiment, the targeting construct may contain more than one
selectable maker
gene, including a negative selectable marker, such as the herpes simplex virus
tk (HSV-tk) gene. The
negative selectable marker may be operatively linked to a promoter and a
polyadenylation signal.
(See, e.g., U.S. Patent No. 5,464,764; U.S. Patent No. 5,487,992; U.S. Patent
No. 5,627,059; and U.S.
Patent No. 5,631,153).
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Generation of Cells and Confirmation of Homologous Recombination Events
Once an appropriate targeting construct has been prepared, the targeting
construct may be
introduced into an appropriate host cell using any method known in the art.
Various techniques may
be employed in the present invention, including, for example, pronuclear
microinjection; retrovirus
mediated gene transfer into germ lines; gene targeting in embryonic stem
cells; electroporation of
embryos; sperm-mediated gene transfer; and calcium phosphate/DNA co-
precipitates, microinjection
of DNA into the nucleus, bacterial protoplast fusion with intact cells,
transfection, polycations, e.g.,
polybrene, polyornithine, etc., or the like (See, e.g., U.S. Pat. No.
4,873,191; Van der Putten, et al.,
1985, Proc. Natl. Acad. Sci., USA 82:6148-6152; Thompson, et al., 1989, Cell
56:313-321; Lo, 1983,
Mol Cell. Biol. 3:1803-1814; Lavitrano, et a1.,1989, Cell, 57:717-723).
Various techniques for
transforming mammalian cells are known in the art. (See, e.g., Gordon, 1989,
Intl. Rev. Cytol.,
115:171-229; Keown et al., 1989, Methods in Enzymology; Keovn et al., 1990,
Methods and
Enzymology, Vol. 185, pp. 527-537; Mansour et al., 1988, Nature, 336:348-352).
In a preferred aspect of the present invention, the targeting construct is
introduced into host
cells by electroporation. In this process, electrical impulses of high field
strength reversibly
permeabilize biomembranes allowing the introduction of the construct. The
pores created during
electroporation permit the uptake of macromolecules such as DNA. (See, e.g.,
Potter, H., et al., 1984,
Proc. Nat'l. Acad. Sci. U.S.A. 81:7161-7165).
Any cell type capable of homologous recombination may be used in the practice
of the
present invention. Examples of such target cells include cells derived from
vertebrates including
mammals such as humans, bovine species, ovine species, marine species, simian
species, and ether
eucaryotic organisms such as filamentous fungi, and higher multicellular
organisms such as plants.
Preferred cell types include embryonic stem (ES) cells, which are typically
obtained from pre-
implantation embryos cultured in vitro. (See, e.g., Evans, M. J., et al.,
1981, Nature 292:154-156;
Bradley, M. O., et al., 1984, Nature 309:255-258; Gossler et al., 1986, Proc.
Natl. Acad. Sci. USA
83:9065-9069; and Robertson, et al., 1986, Nature 322:445-448). The ES cells
are cultured and
prepared for introduction of the targeting construct using methods well known
to the skilled artisan.
(See, e.g., Robertson, E. J. ed. "Teratocarcinomas and Embryonic Stem Cells, a
Practical Approach",
IRL Press, Washington D.C., 1987; Bradley et al., 1986, Current Topics ira
Devel. Biol. 20:357-371;
by Hogan et al. in "Manipulating the Mouse Embryo": A Laboratory Manual, Cold
Spring Harbor
Laboratory Press, Cold Spring Harbor N.Y., 1986; Thomas et al., 1987, Cell
51:503; Koller et al.,
1991, Proc. Natl. Acad. Sci. USA, 88:10730; Dorin et al., 1992, Transgenic
Res. 1:101; and Veis et
al., 1993, Cell 75:229). The ES cells that will be inserted with the targeting
construct are derived
from an embryo or blastocyst of the same species as the developing embryo into
which they are to be
introduced. ES cells are typically selected for their ability to integrate
into the inner cell mass and
contribute to the germ line of an individual when introduced into the mammal
in an embryo at the
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blastocyst stage of development. Thus, any ES cell line having this capability
is suitable for use in the
practice of the present invention.
The present invention may also be used to knockout genes in other cell types,
such as stem
cells. By way of example, stem cells may be myeloid, lymphoid, or neural
progenitor and precursor
cells. These cells comprising a disruption or knockout of a gene may be
particularly useful in the
study of target gene function in individual developmental pathways. Stem cells
may be derived from
any vertebrate species, such as mouse, rat, dog, cat, pig, rabbit, human, non-
human primates and the
like.
After the targeting construct has been introduced into cells, the cells where
successful gene
targeting has occurred are identified. Insertion of the targeting construct
into the targeted gene is
typically detected by identifying cells for expression of the marker gene. In
a preferred embodiment,
the cells transformed with the targeting construct of the present invention
are subjected to treatment
with an appropriate agent that selects against cells not expressing the
selectable marker. Only those
cells expressing the selectable marker gene survive and/or grow under certain
conditions. For
example, cells that express the introduced neomycin resistance gene are
resistant to the compound
6418, while cells that do not express the neo gene marker are killed by 6418.
If the targeting
construct also comprises a screening marker such as GFP, homologous
recombination can be
identified through screening cell colonies under a fluorescent light. Cells
that have undergone
n homologous recombination will have deleted the GFP gene and will not
fluoresce.
If a regulated positive selection method is used in identifying homologous
recombination
events, the targeting construct is designed so that the expression of the
selectable marker gene is
regulated in a manner such that expression is inhibited following random
integration but is permitted
(derepressed) following homologous recombination. More particularly, the
transfected cells are
screened for expression of the neo gene, which requires that (1) the cell was
successfully
electroporated, and (2) lac repressor inhibition of rteo transcription was
relieved by homologous
recombination. This method allows for the identification of transfected cells
and homologous
recombinants to occur in one step with the addition of a single drug.
Alternatively, a positive-negative selection technique may be used to select
homologous
recombinants. This technique involves a process in which a first drug is added
to the cell population,
for example, a neomycin-like drug to select for growth of transfected cells,
i.e. positive selection. A
second drug, such as FIAU is subsequently added to kill cells that express the
negative selection
marker, i. e. negative selection. Cells that contain and express the negative
selection marker are killed
by a selecting agent, whereas cells that do not contain and express the
negative selection marker
survive. For example, cells with non-homologous insertion of the construct
express HSV thymidine
kinase and therefore are sensitive to the herpes drugs such as gancyclovir
(GANG) or FIAU (1-(2-
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deoxy 2-fluoro-B-D-arabinofluranosyl)-5-iodouracil). (See, e.g., Mansour et
al., Nature 336:348-352:
(1988); Capecchi, Science 244:1288-1292, (1989); Capecchi, Trends izz Gezzet.
5:70-76 (1989)).
Successful recombination may be identified by analyzing the DNA of the
selected cells to
confirm homologous recombination. Various techniques known in the art, such as
PCR and/or
Southern analysis may be used to confirm homologous recombination events.
Homologous recombination may also be used to disrupt genes in stem cells, and
other cell
types, which are not totipotent embryonic stem cells. By way of example, stem
cells may be myeloid,
lymphoid, or neural progenitor and precursor cells. Such transgenic cells may
be particularly useful
in the study of gene function in individual developmental pathways. Stem cells
may be derived from
any vertebrate species, such as mouse, rat, dog, cat, pig, rabbit, human, non-
human primates and the
like.
In cells which are not totipotent it may be desirable to knock out both copies
of the target
using methods which are known in the art. For example, cells comprising
homologous recombination
at a target locus which have been selected for expression of a positive
selection marker (e.g., Neor)
and screened for non-random integration, can be further selected for multiple
copies of the selectable
marker gene by exposure to elevated levels of the selective agent (e.g.,
G418). The cells are then
analyzed for homozygosity at the target locus. Alternatively, a second
construct can be generated
with a different positive selection marker inserted between the two homologous
sequences. The two
constructs can be introduced into the cell either sequentially or
simultaneously, followed by
appropriate selection for each of the positive marker genes. The final cell is
screened for homologous
recombination of both alleles of the target.
Production of Trans~enic Animals
Selected cells are then injected into a blastocyst (or other stage of
development suitable for
the purposes of creating a viable animal, such as, for example, a morula) of
an animal (e.g., a mouse)
to form chimeras (see e.g., Bradley, A. ih Teratocarcifzonzas and Ernbryonic
Stem Cells: A Practical
Approach, E. J. Robertson, ed., IRL, Oxford, pp. 113-152 (1987)).
Alternatively, selected ES cells
can be allowed to aggregate with dissociated mouse embryo cells to form the
aggregation chimera. A
chimeric embryo can then be implanted into a suitable pseudopregnant female
foster animal and the
embryo brought to term. Chimeric progeny harboring the homologously recombined
DNA in their
germ cells can be used to breed animals in which all cells of the animal
contain the homologously
recombined DNA. In one embodiment, chimeric progeny mice are used to generate
a mouse with a
heterozygous disruption in the gene. Heterozygous transgenic mice can then be
mated. It is well
know in the art that typically 1/a of the offspring of such matings will have
a homozygous disruption in
the target gene.
The heterozygous and homozygous transgenic mice can then be compared to
normal, wild
type mice to determine whether disruption of the target gene causes phenotypic
changes, especially
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pathological changes. For example, heterozygous and homozygous mice may be
evaluated for
phenotypic changes by physical examination, necropsy, histology, clinical
chemistry, complete blood
count, body weight, organ weights, and cytological evaluation of bone marrow.
In one embodiment, the phenotype (or phenotypic change) associated with a
disruption in the
target gene is placed into or stored in a database. Preferably, the database
includes: (i) genotypic data
(e.g., identification of the disrupted gene) and (ii) phenotypic data (e.g.,
phenotype(s) resulting from
the gene disruption) associated with the genotypic data. The database is
preferably electronic. In
addition, the database is preferably combined with a search tool so that the
database is searchable.
Conditional Trans~enic Animals
The present invention further contemplates conditional transgenic or knockout
animals, such
as those produced using recombination methods. Bacteriophage P1 Cre
recombinase and flp
recombinase from yeast plasmids are two non-limiting examples of site-specific
DNA recombinase
enzymes which cleave DNA at specific target sites (lox P sites for cre
recombinase and frt sites for flp
recombinase) and catalyze a ligation of this DNA to a second cleaved site. A
large number of suitable
alternative site-specific recombinases have been described, and their genes
can be used in accordance
with the method of the present invention. Such recombinases include the Int
recombinase of bacterio-
phage 7~ (with or without Xis) (Weisberg, R. et. al., in Lambda 11, (Hendrix,
R., et al., Eds.), Cold
Spring Harbor Press, Cold Spring Harbor, NY, pp. 211-50 (1983), herein
incorporated by reference);
TpnI and the (3-lactamase transposons (Mercier, et al., J. Bacteriol.,
172:3745-57 (1990)); the Tn3
resolvase (Flanagan & Fennewald J. Moles. Biol., 206:295-304 (1989); Stark, et
al., Cell, 58:779-90
(1989)); the yeast recombinases (Matsuzaki, et al., J. Bacteriol., 172:610-18
(1990)); the B. subtilis
SpoIVC recombinase (Sato, et al., J. Bacteriol. 172:1092-98 (1990)); the Flp
recombinase (Schwartz
& Sadowski, J. Molec.Biol., 205:647-658 (1989); Parsons, et al., J. Biol.
Clzezzz., 265:4527-33 (1990);
Golic & Lindquist, Cell, 59:499-509 (1989); Amin, et al., J. Moles. Biol.,
214:55-72 (1990)); the Hin
recombinase (Glasgow, et al., J. Biol. Chezrz., 264:10072-82 (1989));
immunoglobulin recombinases
(Malynn, et al., Cell, 54:453-460 (1988)); and the Cin recombinase (Haffter &
Bickle, EMBO J.,
7:3991-3996 (1988); Hubner, et al., J. Moles. Biol., 205:493-500 (1989)), all
herein incorporated by
reference. Such systems are discussed by Echols (J. Biol. Chem. 265:14697-
14700 (1990)); de
Villartay (Nature, 335:170-74 (1988)); Craig, (Ann. Rev. Gehet., 22:77-105
(1988)); Poyart-Salmeron,
et al., (EMBO J. 8:2425-33 (1989)); Hunger-Bertling, et al. (Mol Cell.
Biocherzz., 92:107-16 (1990));
and Cregg & Madden (Mol. Gezz. Gezzet., 219:320-23 (1989)), all herein
incorporated by reference.
Cre has been purified to homogeneity, and its reaction with the loxP site has
been extensively
characterized (Abremski & Hess J. Mol. Biol. 259:1509-14 (1984), herein
incorporated by reference).
Cre protein has a molecular weight of 35,000 and can be obtained commercially
from New England
Nuclear/DuPont. The cre gene (which encodes the Cre protein) has been cloned
and expressed
(Abremski, et al. Cell 32:1301-11 (1983), herein incorporated by reference).
The Cre protein
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mediates recombination between two loxP sequences (Sternberg, et al. Cold
Spring Harbor Symp.
Quaut. Biol. 45:297-309 (1981)), which may be present on the same or different
DNA molecule.
Because the internal spacer sequence of the loxP site is asymmetrical, two
loxP sites can exhibit
directionality relative to one another (Hoess & Abremski Proc. Natl. Acad.
Sci. U.S.A. 81:1026-29
(1984)). Thus, when two sites on the same DNA molecule are in a directly
repeated orientation, Cre
will excise the DNA between the sites (Abremski, et al. Cell 32:1301-11
(1983)). However, if the
sites are inverted with respect to each other, the DNA between them is not
excised after recombina-
tion but is simply inverted. Thus, a circular DNA molecule having two loxP
sites in direct orientation
will recombine to produce two smaller circles, whereas circular molecules
having two loxP sites in an
inverted orientation simply invert the DNA sequences flanked by the loxP
sites. In addition,
recombinase action can result in reciprocal exchange of regions distal to the
target site when targets
are present on separate DNA molecules.
Recombinases have important application for characterizing gene function in
knockout
models. When the constructs described herein are used to disrupt target genes,
a fusion transcript can
be produced when insertion of the positive selection marker occurs downstream
(3') of the translation
initiation site of the target gene. The fusion transcript could result in some
level of protein expression
with unknown consequence. It has been suggested that insertion of a positive
selection marker gene
can affect the expression of nearby genes. These effects may make it difficult
to determine gene
function after a knockout event since one could not discern whether a given
phenotype is associated
with the inactivation of a gene, or the transcription of nearby genes. Both
potential problems are
solved by exploiting recombinase activity. When the positive selection marker
is flanked by
recombinase sites in the same orientation, the addition of the corresponding
recombinase will result in
the removal of the positive selection marker. In this way, effects caused by
the positive selection
marker or expression of fusion transcripts are avoided.
In one embodiment, purified recombinase enzyme is provided to the cell by
direct
microinjection. In another embodiment, recombinase is expressed from a co-
transfected construct or
vector in which the recombinase gene is operably linked to a functional
promoter. An additional
aspect of this embodiment is the use of tissue-specific or inducible
recombinase constructs which
allow the choice of when and where recombination occurs. One method for
practicing the inducible
forms of recombinase-mediated recombination involves the use of vectors that
use inducible or tissue-
specific promoters or other gene regulatory elements to express the desired
recombinase activity. The
inducible expression elements are preferably operatively positioned to allow
the inducible control or
activation of expression of the desired recombinase activity. Examples of such
inducible promoters or
other gene regulatory elements include, but are not limited to, tetracycline,
metallothionine, ecdysone,
and other steroid-responsive promoters, rapamycin responsive promoters, and
the like (No, et al.
Proc. Natl. Acad. Sci. USA, 93:3346-51 (1996); Furth, et al. Proc. Natl. Acad.
Sci. USA, 91:9302-6
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(1994)). Additional control elements that can be used include promoters
requiring specific
transcription factors such as viral, promoters. Vectors incorporating such
promoters would only
express recombinase activity in cells that express the necessary transcription
factors.
Models for Disease
The cell- and animal-based systems described herein can be utilized as models
for diseases.
Animals of any species, including, but not limited to, mice, rats, rabbits,
guinea pigs, pigs, micro-pigs,
goats, and non-human primates, e.g., baboons, monkeys, and chimpanzees may be
used to generate
disease animal models. In addition, cells from humans may be used. These
systems may be used in a
variety of applications. Such assays may be utilized as part of screening
strategies designed to
identify agents, such as compounds which are capable of ameliorating disease
symptoms. Thus, the
animal-,and cell-based models may be used to identify drugs, pharmaceuticals,
therapies and
interventions which may be effective in treating disease.
Cell-based systems may be used to identify compounds which may act to
ameliorate disease
symptoms. For example, such cell systems may be exposed to a compound
suspected of exhibiting an
ability to ameliorate disease symptoms, at a sufficient concentration and for
a time sufficient to elicit
such an amelioration of disease symptoms in the exposed cells. After exposure,
the cells are
examined to determine whether one or more of the disease cellular phenotypes
has been altered to
resemble a more normal or more wild type, non-disease phenotype.
In addition, animal-based disease systems, such as those described herein, may
be used to
identify compounds capable of ameliorating disease symptoms. Such animal
models may be used as
test substrates for the identification of drugs, pharmaceuticals, therapies,
and interventions which may
be effective in treating a disease or other phenotypic characteristic of the
animal. For example, animal
models may be exposed to a compound or agent suspected of exhibiting an
ability to ameliorate
disease symptoms, at a sufficient concentration and for a time sufficient to
elicit such an amelioration
of disease symptoms in the exposed animals. The response of the animals to the
exposure rnay be
monitored by assessing the reversal of disorders associated with the disease.
Exposure may involve
treating mother animals during gestation of the model animals described
herein, thereby exposing
embryos or fetuses to the compound or agent which may prevent or ameliorate
the disease or
phenotype. Neonatal, juvenile, and adult animals can also be exposed.
More particularly, using the animal models of the invention, specifically,
transgenic mice,
methods of identifying agents, including compounds are provided, preferably,
on the basis of the
ability to affect at least one phenotype associated with a disruption in a
target gene. In one embodi-
ment, the present invention provides a method of identifying agents having an
effect on target gene or
alternatively, target protein expression or function. The method includes
measuring a physiological
response of the animal, for example, to the agent, and comparing the
physiological response of such
animal to a control animal, wherein the physiological response of the animal
comprising a disruption
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in a target gene as compared to the control animal indicates the specificity
of the agent. A "physiolo-
gical response" is any biological or physical parameter of an animal which can
be measured.
Molecular assays (e.g., gene transcription, protein production and degradation
rates), physical
parameters (e.g., exercise physiology tests, measurement of various parameters
of respiration,
measurement of heart rate or blood pressure, measurement of bleeding time,
aPTT.T, or TT), and
cellular assays (e.g.,. immunohistochemical assays of cell surface markers, or
the ability of cells to
aggregate or proliferate) can be used to assess a physiological response. The
transgenic animals and
cells of the present invention may by utilized as models for diseases,
disorders, or conditions
associated with phenotypes relating to a disruption in a target gene.
The present invention provides a unique animal model for testing and
developing new
treatments relating to the behavioral phenotypes. Analysis of the behavioral
phenotype allows for the
development of an animal model useful for testing, for instance, the efficacy
of proposed genetic and
pharmacological therapies for human genetic diseases, such as neurological,
neuropsychological, or
psychotic illnesses.
A statistical analysis of the various behaviors measured can be carried out
using any
conventional statistical program routinely used by those skilled in the art
(such as, for example,
"Analysis of Variance" or ANOVA). A "p" value of about 0.05 or less is
generally considered to be
statistically significant, although slightly higher p values may still be
indicative of statistically
significant differences. To statistically analyze abnormal behavior, a
comparison is made between the
behavior of a transgenic animal (or a group thereof) to the behavior of a wild-
type mouse (or a group
thereof), typically under certain prescribed conditions. "Abnormal behavior"
as used herein refers to
behavior exhibited by an animal having a disruption in the target gene, e.g.
transgenic animal, which
differs from an animal without a disruption in the target gene, e.g. wild-type
mouse. Abnormal
behavior consists of any number of standard behaviors that can be objectively
measured (or observed)
and compared. In the case of comparison, it is preferred that the change be
statistically significant to
confirm that there is indeed a meaningful behavioral difference between the
knockout animal and the
wild-type control animal. Examples of behaviors which may be measured or
observed include, but
are not limited to, ataxia, rapid limb movement, eye movement, breathing,
motor activity, cognition,
emotional behaviors, social behaviors, hyperactivity, hypersensitivity,
anxiety, impaired learning,
abnormal reward behavior, and abnormal social interaction, such as aggression.
A series of tests may be used to measure the behavioral phenotype of the
animal models of
the present invention, including neurological and neuropsychological tests to
identify abnormal
behavior. These tests may be used to measure abnormal behavior relating to,
for example, learning
and memory, eating, pain, aggression, sexual reproduction, anxiety,
depression, schizophrenia, and
drug abuse. (See, e.g., Crawley and Paylor, Hormones ahd Behavior 31:197-211
(1997)).
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The social interaction test involves exposing a mouse to other animals in a
variety of settings.
The social behaviors of the animals (e.g., touching, climbing, sniffing, and
mating) are subsequently
evaluated. Differences in behaviors can then be statistically analyzed and
compared (See, e.g., S. E.
File, et al., Pharfnacol. Bioch. Behav. 22:941-944 (1985); R. R. Holson, Phys.
Behav. 37:239-247
(1986)). Examplary behavioral tests include the following.
The mouse startle response test typically involves exposing the animal to a
sensory (typically
auditory) stimulus and measuring the startle response of the animal (see,
e.g., M. A. Geyer, et al.,
Brain Res. Bull. 25:485-498 (1990); Paylor and Crawley, PsyclZOpharrnacology
132:169-180 (1997)).
A pre-pulse inhibition test can also be used, in which the percent inhibition
(from a normal startle
response) is measured by "cueing" the animal first with a brief low-intensity
pre-pulse prior to the
startle pulse.
The electric shock test generally involves exposure to an electrified surface
and measurement
of subsequent behaviors such as, for example, motor activity, learning, social
behaviors. The behavi-
ors are measured and statistically analyzed using standard statistical tests.
(See, e.g., G. J. Kant, et al.,
Pharnz. Bioch. Behav. 20:793-797 (1984); N. J. Leidenheimer, et al.,
Pharmacol. Bioch. Behav.
30:351-355 (1988)).
The tail-pinch or immobilization test involves applying pressure to the tail
of the animal
and/or restraining the animal's movements. Motor activity, social behavior,
and cognitive behavior
are examples of the areas that are measured. (See, e.g., M. Bertolucci
D'Angic, et al., Neurochena.
55:1208-1214 (1990)).
The novelty test generally comprises exposure to a novel environment andlor
novel objects.
The animal's motor behavior in the novel environment and/or around the novel
object are measured
and statistically analyzed. (See, e.g., D. I~. Reinstein, et al., Pharnz.
Bioch. Behav. 17:193-202 (1982);
B. Poucet, Behav. Neurosci. 103:1009-10016 (1989); R. R. Holson, et al.,
Plzys. Behav. 37:231-238
(1986)). This test may be used to detect visual processing deficiencies or
defects.
The learned helplessness test involves exposure to stresses, for example,
noxious stimuli,
which cannot be affected by the animal's behavior. The animal's behavior can
be statistically
analyzed using various standard statistical tests. (See, e.g., A. Leshner, et
al., Belzav. Neural Biol.
26:497-501 (1979)).
Alternatively, a tail suspension test may be used, in which the "immobile"
time of the mouse
is measured when suspended "upside-down" by its tail. This is a measure of
whether the animal
struggles, an indicator of depression. In humans, depression is believed to
result from feelings of a
lack of control over one's life or situation. It is believed that a depressive
state can be elicited in
animals by repeatedly subjecting them to aversive situations over which they
have no control. A
condition of "learned helplessness" is eventually reached, in which the animal
will stop trying to
change its circumstances and simply accept its fate. Animals that stop
struggling sooner are believed
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to be more prone to depression. Studies have shown that the administration of
certain antidepressant
drugs prior to testing increases the amount of time that animals struggle
before giving up.
The Morns water-maze test comprises learning spatial orientations in water and
subsequently
measuring the animal's behaviors, such as, for example, by counting the number
of incorrect choices.
The behaviors measured are statistically analyzed using standard statistical
tests. (See, e.g., E. M.
Spruijt, et al., Braizz Res. 527:192-197 (1990)).
Alternatively, a Y-shaped maze may be used (see, e.g., McFarland, D.J.,
Pharzzzacology,
Biochemistry and Behavior 32:723-726 (1989); Dellu, F., et al., Neurobiology
of Learn.i>zg azzd
Memory 73:31-48 (2000)). The Y-maze is generally believed to be a test of
cognitive ability. The
dimensions of each arm of the Y-maze can be, for example, approximately 40 cm
x 8 cm x 20 cm,
although other dimensions may be used. Each arm can also have, for example,
sixteen equally spaced
photobeams to automatically detect movement within the arms. At least two
different tests can be
performed using such a Y-maze. In a continuous Y-maze paradigm, mice are
allowed to explore all
three arms of a Y-maze for, e.g., approximately 10 minutes. The animals are
continuously tracked
using photobeam detection grids, and the data can be used to measure
spontaneous alteration and
positive bias behavior. Spontaneous alteration refers to the natural tendency
of a "normal" animal to
visit the least familiar arm of a maze. An alternation is scored when the
animal makes two
consecutive turns in the same direction, thus representing a sequence of
visits to the least recently
entered arm of the maze. Position bias determines egocentrically defined
responses by measuring the
animal's tendency to favor turning in one direction over another. Therefore,
the test can detect
differences in an animal's ability to navigate on the basis of allocentric
or~egocentric mechanisms.
The two-trial Y-maze memory test measures response to novelty and spatial
memory based on a free-
choice exploration paradigm. During the first trial (acquisition), the animals
are allowed to freely
visit two arms of the Y-maze for, e.g., approximately 15 minutes. The third
arm is blocked off during
this trial. The second trial (retrieval) is performed after an intertrial
interval of, e.g., approximately 2
hours. During the retrieval trial, the blocked arm is opened and the animal is
allowed access to all
three arms for, e.g., approximately 5 minutes. Data are collected during the
retrieval trial and
analyzed for the number and duration of visits to each arm. Because the three
arms of the maze are
virtually identical, discrimination between novelty and familiarity is
dependent on "environmental"
spatial cues around the room relative to the position of each arm. Changes in
arm entry and duration
of time spent in the novel arm in a transgenic animal model may be indicative
of a role of that gene in
mediating novelty and recognition processes.
The passive avoidance or shuttle box test generally involves exposure to two
or more
environments, one of which is noxious, providing a choice to be learned by the
animal. Behavioral
measures include, for example, response latency, number of correct responses,
and consistency of
response. (See, e.g., R. Ader, et al., Psychozz. Sci. 26:125-128 (1972); R. R.
Holson, Phys. Behav.
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37:221-230 (1986)). Alternatively, a zero-maze can be used. In a zero-maze,
the animals can, for
example, be placed in a closed quadrant of an elevated annular platform
having, e.g., 2 open and 2
closed quadrants, and are allowed to explore for approximately 5 minutes. This
paradigm exploits an
approach-avoidance conflict between normal exploratory activity and an
aversion to open spaces in
rodents. This test measures anxiety levels and can be used to evaluate the
effectiveness of anti-
anxiolytic drugs. The time spent in open quadrants versus closed quadrants may
be recorded
automatically, with, for example, the placement of photobeams at each
transition site.
The food avoidance test involves exposure to novel food and objectively
measuring, for
example, food intake and intake latency. The behaviors measured are
statistically analyzed using
standard statistical tests. (See, e.g., B. A. Campbell, et al., J. Comp.
Physiol. Psychol. 67:15-22
( 1969)).
The elevated plus-maze test comprises exposure to a maze, without sides, on a
platform, the
animal's behavior is objectively measured by counting the number of maze
entries and maze learning.
The behavior is statistically analyzed using standard statistical tests. (See,
e.g., H. A. Baldwin, et al.,
Brairt Res. Bull, 20:603-606 (1988)).
The stimulant-induced hyperactivity test involves injection of stimulant drugs
(e.g.,
amphetamines, cocaine, PCP, and the like), and objectively measuring, for
example, motor activity,
social interactions, cognitive behavior. The animal's behaviors are
statistically analyzed using
standard statistical tests. (See, e.g., P. B. S. Clarke, et al.,
Psychopharmacology 96:511-520 (1988); P.
Kuczenski, et al., J. Neuroscience 11:2703-2712 (1991)).
The self stimulation test generally comprises providing the mouse with the
opportunity to
regulate electrical and/or chemical stimuli to its own brain. Behavior is
measured by frequency and
pattern of self-stimulation. Such behaviors are statistically analyzed using
standard statistical tests.
(See, e.g., S. Nassif, et al., Braira Res., 332:247-257 (1985); W. L. Isaac,
et al., Beh.av. Neurosci.
103:345-355 (1989)).
The reward test involves shaping a variety of behaviors, e.g., motor,
cognitive, and social,
measuring, for example, rapidity and reliability of behavioral change, and
statistically analyzing the
behaviors measured. (See, e.g., L. E. Jarrard, et al., Exp. Brain Res. 61:519-
530 (1986)).
The DRL (differential reinforcement to low rates of responding) performance
test involves
exposure to intermittent reward paradigms and measuring the number of proper
responses, e.g., lever
pressing. Such behavior is statistically analyzed using standard statistical
tests. (See, e.g., J. D. Sinden,
et al., Behav. Neurosci. 100:320-329 (1986); V. Nalwa, et al., Behav Brain
Res. 17:73-76 (1985); and
A. J. Nonneman, et al., J. Comp. Physiol. Psych. 95:588-602 (1981)).
The spatial learning test involves exposure to a complex novel environment,
measuring the
rapidity and extent of spatial learning, and statistically analyzing the
behaviors measured. (See, e.g.,
N. Pitsikas, et al., Pharm. Baoch. Behav. 38:931-934 (1991); B. Poucet, et
al., Braih Res. 37:269-280
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(1990); D. Christie, et al., Braifa Res. 37:263-268 (1990); and F. Van Haaren,
et al., Behav. Neurosci.
102:481-488 (1988)). Alternatively, an open-field (of) test may be used, in
which the greater distance
traveled for a given amount of time is a measure of the activity level and
anxiety of the animal. When
the open field is a novel environment, it is believed that an approach-
avoidance situation is created, in
which the animal is "torn" between the drive to explore and the drive to
protect itself. Because the
chamber is lighted and has no places to hide other than the corners, it is
expected that a "normal"
mouse will spend more time in the corners and around the periphery than it
will in the center where
there is no place to hide. "Normal" mice will, however, venture into the
central regions as they
explore more and more of the chamber. It can then be extrapolated that
especially anxious mice will
spend most of their time in the corners, with relatively little or no
exploration of the central region,
whereas bold (i.e., less anxious) mice will travel a greater distance, showing
little preference for the
periphery versus the central region.
The visual, somatosensory and auditory neglect tests generally comprise
exposure to a
sensory stimulus, objectively measuring, for example, orientating responses,
and statistically
analyzing the behaviors measured. (See, e.g., J. M. Vargo, et al., Exp.
Neurol. 102:199-209 (1988)).
The consummatory behavior test generally comprises feeding and drinking, and
objectively
measuring quantity of consumption. The behavior measured is statistically
analyzed using standard
statistical tests. (See, e.g., P. J. Fletcher, et al., Psychopharmacol.
102:301-308 (1990); M. G. Corda,
et al." Proc. Nat'1 Acad. Sci. USA 80:2072-2076 (1983)).
A visual discrimination test can also be used to evaluate the visual
processing of an animal.
One or two similar objects are placed in an open field and the animal is
allowed to explore for about
5-10 minutes. The time spent exploring each object (proximity to, i.e.,
movement within, e.g., about
3-5 cm of the object is considered exploration of an object) is recorded. The
animal is then removed
from the open field, and the objects are replaced by a similar object and a
novel object. The animal is
returned to the open field and the percent time spent exploring the novel
object over the old object is
measured (again, over about a 5-10 minute span). "Normal" animals will
typically spend a higher
percentage of time exploring the novel object rather than the old object. If a
delay is imposed
between sampling and testing, the memory task becomes more hippocampal-
dependent. If no delay is
imposed, the task is more based on simple visual discrimination. This test can
also be used for
olfactory discrimination, in which the objects (preferably, simple blocks) can
be sprayed or otherwise
treated to hold an odor. This test can also be used to determine if the animal
can make gustatory
discriminations; animals that return to the previously eaten food instead of
novel food exhibit
gustatory neophobia.
A hot plate analgesia test can be used to evaluate an animal's sensitivity to
heat or painful
stimuli. For example, a mouse can be placed on an approximately 55°C
hot plate and the mouse's
response latency (e.g., time to pick up and lick a hind paw) can be recorded.
These responses are not
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reflexes, but rather "higher" responses requiring cortical involvement. This
test may be used to
evaluate a nociceptive disorder.
An accelerating rotarod test may be used to measure coordination and balance
in mice.
Animals can be, for example, placed on a rod that acts like a rotating
treadmill (or rolling log). The
rotarod can be made to rotate slowly at first and then progressively faster
until it reaches a speed of,
e.g., approximately 60 rpm. The mice must continually reposition themselves in
order to avoid falling
off. The animals are preferably tested in at least three trials, a minimum of
20 minutes apart. Those
mice that are able to stay on the rod the longest are believed to have better
coordination and balance.
A metrazol administration test can be used to screen animals for varying
susceptibilities to
seizures or similar events. For example, a 5mg/ml solution of metrazol can be
infused through the tail
vein of a mouse at a rate of, e.g., approximately 0.375 ml/min. The infusion
will cause all mice to
experience seizures, followed by death. Those mice that enter the seizure
stage the soonest are
believed to be more prone to seizures. Four distinct physiological stages can
be recorded: soon after
the start of infusion, the mice will exhibit a noticeable "twitch", followed
by a series of seizures,
ending in a final tensing of the body known as "tonic extension", which is
followed by death.
Target Gene Products
The present invention further contemplates use of the target gene sequence to
produce target
gene products. Target gene products may include proteins that represent
functionally equivalent gene
products. Such an equivalent gene product may contain deletions, additions or
substitutions of amino
acid residues within the amino acid sequence encoded by the gene sequences
described herein, but
which result in a silent change, thus producing a functionally equivalent
target gene product. Amino
acid substitutions may be made on the basis of similarity in polarity, charge,
solubility, hydrophobi-
city, hydrophilicity, and/or the amphipathic nature of the residues involved.
For example, nonpolar (hydrophobic) amino acids include alanine, leucine,
isoleucine, valine,
proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids
include glycine, serine,
threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged
(basic) amino acids
include arginine, lysine, and histidine; and negatively charged (acidic) amino
acids include aspartic
acid and glutamic acid. "Functionally equivalent", as utilized herein, refers
to a protein capable of
exhibiting a substantially similar in vivo activity as the endogenous gene
products encoded by the
target gene sequences. Alternatively, when utilized as part of an assay,
"functionally equivalent" may
refer to peptides capable of interacting with other cellular or extracellular
molecules in a manner
substantially similar to the way in which the corresponding portion of the
endogenous gene product
would.
Other protein products useful according to the methods of the invention are
peptides derived
from or based on the target gene produced by recombinant or synthetic means
(derived peptides).
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Target gene products may be produced by recombinant DNA technology using
techniques
well known in the art. Thus, methods for preparing the gene polypeptides and
peptides of the
invention by expressing nucleic acid encoding gene sequences are described
herein. Methods which
are well known to those skilled in the art can be used to construct expression
vectors containing gene
protein coding sequences and appropriate transcriptional/translational control
signals. These methods
include, for example, in vitro recombinant DNA techniques, synthetic
techniques and in vivo
recombination/genetic recombination (see, e.g., Sambrook, et al., 1989, supra,
and Ausubel, et al.,
1989, supra). Alternatively, RNA capable of encoding gene protein sequences
may be chemically
synthesized using, for example, automated synthesizers (see, e.g.
Oligonucleotide Synthesis: A
Practical Approach, Gait, M. J. ed., IRL Press, Oxford (1984)).
A variety of host-expression vector systems may be utilized to express the
gene coding
sequences of the invention. Such host-expression systems represent vehicles by
which the coding
sequences of interest may be produced and subsequently purified, but also
represent cells which may,
when transformed or transfected with the appropriate nucleotide coding
sequences, exhibit the gene
protein of the invention in situ. These include but are not limited to
microorganisms such as bacteria
(e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA,
plasmid DNA or cosmid
DNA expression vectors containing gene protein coding sequences; yeast (e.g.
Saccharomyces,
Pichia) transformed with recombinant yeast expression vectors containing the
gene protein coding
sequences; insect cell systems infected with recombinant virus expression
vectors (e.g., baculovirus)
containing the gene protein coding sequences; plant cell systems infected with
recombinant virus
expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic
virus, TMV) or trans-
formed with recombinant plasmid expression vectors (e. g., Ti plasmid)
containing gene protein
coding sequences; or mammalian cell systems (e.g. COS, CHO, BHK, 293, 3T3)
harboring
recombinant expression constructs containing promoters derived from the genome
of mammalian
cells (e.g., metallothionine promoter) or from mammalian viruses (e.g., the
adenovirus late promoter;
the vaccinia virus 7.5 K promoter).
In bacterial systems, a number of expression vectors may be advantageously
selected
depending upon the use intended for the gene protein being expressed. For
example, when a large
quantity of such a protein is to be produced, for the generation of antibodies
or to screen peptide
libraries, for example, vectors which direct the expression of high levels of
fusion protein products
that are readily purified may be desirable. Such vectors include, but are not
limited, to the E, coli
expression vector pUR278 (Ruther et al., EMBO J.; 2:1791-94 (1983)), in which
the gene protein
coding sequence may be ligated individually into the vector in frame with the
lac Z coding region so
that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids
Res., 13:3101-09
(1985); Van Heeke et al., J. Biol. Chern., 264:5503-9 (1989)); and the like.
pGEX vectors may also be
used to express foreign polypeptides as fusion proteins with glutathione S-
transferase (GST). In
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general, such fusion proteins are soluble and can easily be purified from
lysed cells by adsorption to
glutathione-agarose beads followed by elution in the presence of free
glutathione. The pGEX vectors
are designed to include thrombin or factor Xa protease cleavage sites so that
the cloned target gene
protein can be released from the GST moiety.
In a preferred embodiment, full length cDNA sequences are appended with in-
frame Bam HI
sites at the amino terminus and Eco RI sites at the carboxyl terminus using
standard PCR methodo-
logies (Innis, et al. (eds) PCR Protocols: A Guide to Methods and
Applications, Academic Press, San
Diego (1990)) and ligated into the pGEX-2TK vector (Pharmacia, Uppsala,
Sweden). The resulting
cDNA construct contains a kinase recognition site at the amino terminus for
radioactive labeling and
glutathione S-transferase sequences at the carboxyl terminus for affinity
purification (Nilsson, et al.,
EMBO J., 4: 1075-80 (1985); Zabeau et al., EMBO J., 1: 1217-24 (1982)).
In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV)
is used as a
vector to express foreign genes. The virus grows in Spodoptera frugiperda
cells. The gene coding
sequence may be cloned individually into non-essential regions (for example
the polyhedrin gene) of
the virus and placed under control of an AcNPV promoter (for example the
polyhedrin promoter).
Successful insertion of gene coding sequence will result in inactivation of
the polyhedrin gene and
production of non-occluded recombinant virus (i.e., virus lacking the
proteinaceous coat coded for by
the polyhedrin gene). These recombinant viruses are then used to infect
Spodoptera frugiperda cells
in which the inserted gene is expressed (see, e.g., Smith, et al.., J. Virol.
46: 584-93 (1983); U.S. Pat.
No.4,745,051).
In mammalian host cells, a number of viral-based expression systems may be
utilized. In
cases where an adenovirus is used as an expression vector, the gene coding
sequence of interest may
be ligated to an adenovirus transcription/translation control complex, e.g.,
the late promoter and
tripartite leader sequence. This chimeric gene may then be inserted in the
adenovirus genome by in
vitro or in vivo recombination. Insertion in a non-essential region of the
viral genome (e.g., region E1
or E3) will result in a recombinant virus that is viable and capable of
expressing gene protein in
infected hosts. (e.g., see Logan et al., Proc. Natl. Acad. Sci. USA, 81:3655-
59 (1984)). Specific
initiation signals may also be required for efficient translation of inserted
gene coding sequences.
These signals include the ATG initiation codon and adjacent sequences. In
cases where an entire
gene, including its own initiation codon and adjacent sequences, is inserted
into the appropriate
expression vector, no additional translational control signals may be needed.
However, in cases
where only a portion of the gene coding sequence is inserted, exogenous
translational control signals,
including, perhaps, the ATG initiation codon, must be provided. Furthermore,
the initiation codon
must be in phase with the reading frame of the desired coding sequence to
ensure translation of the
entire insert. These exogenous translational control signals and initiation
codons can be of a variety of
origins, both natural and synthetic. The efficiency of expression may be
enhanced by the inclusion of
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appropriate transcription enhancer elements, transcription terminators, etc.
(see Bitter, et al., Methods
ifa Ehzyjnol., 153:516-44 (1987)).
In addition, a host cell strain may be chosen which modulates the expression
of the inserted
sequences, or modifies and processes the gene product in the specific fashion
desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein
products may be
important for the function of the protein. Different host cells have
characteristic and specific
mechanisms for the post-translational processing and modification of proteins.
Appropriate cell lines
or host systems can be chosen to ensure the correct modification and
processing of the foreign protein
expressed. To this end, eukaryotic host cells which possess the cellular
machinery for proper
processing of the primary transcript, glycosylation, and phosphorylation of
the gene product may be
used. Such mammalian host cells include but are not limited to CHO, VERO, BHK,
HeLa, COS,
MDCK, 293, 3T3, WI38, etc.
For long-term, high-yield production of recombinant proteins, stable
expression is preferred.
For example, cell lines which stably express the gene protein may be
engineered. Rather than using
expression vectors which contain viral origins of replication, host cells can
be transformed with DNA
controlled by appropriate expression control elements (e.g., promoter,
enhancer, sequences,
transcription terminators, polyadenylation sites, etc.), and a selectable
marker. Following the
introduction of the foreign DNA, engineered cells may be allowed to grow for 1-
2 days in an enriched
media, and then are switched to a selective media. The selectable marker in
the recombinant plasmid
confers resistance to the selection and allows cells which stably integrate
the plasmid into their
chromosomes and grow, to form foci which in turn can be cloned and expanded
into cell lines. This
method may advantageously be used to engineer cell lines which express the
gene protein. Such
engineered cell lines may be particularly useful in screening and evaluation
of compounds that affect
the endogenous activity of the gene protein.
In a preferred embodiment, control of timing and/or quantity of expression of
the recombinant
protein can be controlled using an inducible expression construct. Inducible
constructs and systems
for inducible expression of recombinant proteins will be well known to those
skilled in the art.
Examples of such inducible promoters or other gene regulatory elements
include, but are not limited
to, tetracycline, metallothionine, ecdysone, and other steroid-responsive
promoters, rapamycin
responsive promoters, and the like (No, et al., Proc. Natl. Acad. Sci. USA,
93:3346-51 (1996); Furth,
et al., Proc. Natl. Acad. Sci. USA, 91:9302-6 (1994)). Additional control
elements that can be used
include promoters requiring specific transcription factors such as viral,
particularly HIV, promoters.
In one in embodiment, a Tet inducible gene expression system is utilized.
(Gossen et al., Proc. Natl.
Acad. Sci. USA, 89:5547-51 (1992); Gossen, et al., Science, 268:1766-69
(1995)). Tet Expression
Systems are based on two regulatory elements derived from the tetracycline-
resistance operon of the
E. coli TnlO transposon-the tetracycline repressor protein (TetR) and the
tetracycline operator
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sequence (tet0) to which TetR binds. Using such a system, expression of the
recombinant protein is
placed under the control of the tet0 operator sequence and transfected or
transformed into a host cell.
In the presence of TetR, which is co-transfected into the host cell,
expression of the recombinant
protein is repressed due to binding of the TetR protein to the tet0 regulatory
element. High-level,
regulated gene expression can then be induced in response to varying
concentrations of tetracycline
(Tc) or Tc derivatives such as doxycycline (Dox), which compete with tet0
elements for binding to
TetR. Constructs and materials for tet inducible gene expression are available
commercially from
CLONTECH Laboratories, Inc., Palo Alto, CA.
When used as a component in an assay system, the gene protein may be labeled,
either
directly or indirectly, to facilitate detection of a complex formed between
the gene protein and a test
substance. Any of a variety of suitable labeling systems may be used including
but not limited to
radioisotopes such as 125I; enzyme labeling systems that generate a detectable
calorimetric signal or
light when exposed to substrate; and fluorescent labels. Where recombinant DNA
technology is used
to produce the gene protein for such assay systems, it may be advantageous to
engineer fusion
proteins that can facilitate labeling, immobilization and/or detection.
Indirect labeling involves the use of a protein, such as a labeled antibody,
which specifically
binds to the gene product. Such antibodies include but are not limited to
polyclonal, monoclonal,
chimeric, single chain, Fab fragments and fragments produced by a Fab
expression library.
Production of Antibodies
2.0 Described herein are methods for the production of antibodies capable of
specifically
recognizing one or more epitopes. Such antibodies may include, but are not
limited to polyclonal
antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies,
single chain antibodies,
Fab fragments, F(ab')2 fragments, fragments produced by a Fab expression
library, anti-idiotypic
(anti-Id) antibodies, and epitope-binding fragments of any of the above. Such
antibodies may be used,
for example, in the detection of a target gene in a biological sample, or,
alternatively, as a method for
the inhibition of abnormal target gene activity. Thus, such antibodies may be
utilized as part of
disease treatment methods, and/or may be used as part of diagnostic techniques
whereby patients may
be tested for abnormal levels of target gene proteins, or for the presence of
abnormal forms of such
proteins.
For the production of antibodies, various host animals may be immunized by
injection with
the target gene, its expression product or a portion thereof. Such host
animals may include but are not
limited to rabbits, mice, and rats, to name but a few. Various adjuvants may
be used to increase the
immunological response, depending on the host species, including but not
limited to Freund's
(complete and incomplete), mineral gels such as aluminum hydroxide, surface
active substances such
as lysolecithin, platonic polyols, polyanions, peptides, oil emulsions,
keyhole limpet hemocyanin,
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dinitrophenol, and potentially useful human adjuvants such as BCG (bacille
Calmette-Gueriu) and
Coryzxebacterium parvum.
Polyclonal antibodies are heterogeneous populations of antibody molecules
derived from the
sera of animals immunized with an antigen, such as target gene product, or an
antigenic functional
derivative thereof. For the production of polyclonal antibodies, host animals
such as those described
above, may be immunized by injection with gene product supplemented with
adjuvants as also
described above.
Monoclonal antibodies, which are homogeneous populations of antibodies to a
particular
antigen, may be obtained by any technique which provides for the production of
antibody molecules
by continuous cell lines in culture. These include, but are not limited to the
hybridoma technique of
Kohler and Milstein, Nature, 256:495-7 (1975); and U.S. Pat. No. 4,376,110),
the human B-cell
hybridoma technique (Kosbor, et al., Immunology Today, 4:72 (1983); Cote, et
al., Proc. Natl. Acad.
Sci. USA, 80:2026-30 (1983)), and the EBV-hybridoma technique (Cole, et al.,
in Monoclonal
Antibodies And Cancer Therapy, Alan R. Liss, Inc., New York, pp. 77-96
(1985)). Such antibodies
may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any
subclass thereof. The
hybridoma producing the mAb of this invention may be cultivated in vitro or in
vivo. Production of
high titers of mAbs in vivo makes this the presently preferred method of
production.
In addition, techniques developed for the production of "chimeric antibodies"
(Morrison, et
al., Proc. Natl. Acad. Sci., 81:6851-6855 (1984); Takeda, et al., Nature,
314:452-54 (1985)) by
splicing the genes from a mouse antibody molecule of appropriate antigen
specificity together with
genes from a human antibody molecule of appropriate biological activity can be
used. A chimeric
antibody is a molecule in which different portions are derived from different
animal species, such as
those having a variable region derived from,a murine mAb and a human
immunoglobulin constant
region.
Alternatively, techniques described for the production of single chain
antibodies (U.S. Pat.
No. 4,946,778; Bird, Science 242:423-26 (1988); Huston, et al., Proc. Natl.
Acad. Sci. USA, 85:5879-
83 (1988); and Ward, et al., Nature, 334:544-46 (1989)) can be adapted to
produce gene-single chain
antibodies. Single chain antibodies are formed by linking the heavy and light
chain fragments of the
Fv region via an amino acid bridge, resulting in a single chain polypeptide.
Antibody fragments which recognize specific epitopes may be generated by known
tech-
niques. For example, such fragments include but are not limited to: the
F(ab')2 fragments which can
be produced by pepsin digestion of the antibody molecule and the Fab fragments
which can be
generated by reducing the disulfide bridges of the F(ab')2 fragments.
Alternatively, Fab expression
libraries may be constructed (Huse, et al., Science, 246:1275-81 (1989)) to
allow rapid and easy
identification of monoclonal Fab fragments with the desired specificity.
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Screening Methods
The present invention may be employed in a process for screening for agents
such as agonists,
i. e. agents that bind to and activate target gene polypeptides, or
antagonists, i. e. inhibit the activity or
interaction of target gene polypeptides with its ligand. Thus, polypeptides of
the invention may also
be used to assess the binding of small molecule substrates and ligands in, for
example, cells, cell-free
preparations, chemical libraries, and natural product mixtures as known in the
art. Any methods
routinely used to identify and screen for agents that can modulate receptors
may be used in
accordance with the present invention.
The present invention provides methods for identifying and screening for
agents that
modulate target gene expression or function. More particularly, cells that
contain and express target
gene sequences may be used to screen for therapeutic agents. Such cells may
include non-
recombinant monocyte cell lines, such as U937 (ATCC# CRL-1593), THP-1 (ATCC#
TIB-202), and
P388D1 (ATCC# TIB-63); endothelial cells such as HUVEC's and bovine aortic
endothelial cells
(BAEC's); as well as generic mammalian cell lines such as HeLa cells and COS
cells, e.g., COS-7
(ATCC# CRL-1651). Further, such cells may include recombinant, transgenic cell
lines. For
example, the transgenic mice of the invention may be used to generate cell
lines, containing one or
more cell types involved in a disease, that can be used as cell culture models
for that disorder. While
cells, tissues, and primary cultures derived from the disease transgenic
animals of the invention may
be utilized, the generation of continuous cell lines is preferred. For
examples of techniques which
may be used to derive a continuous cell line from the transgenic animals, see
Small, et al., Mol. Cell
Biol., 5:642-48 (1985).
Target gene sequences may be introduced into, and overexpressed in, the genome
of the cell
of interest. In order to overexpress a target gene sequence, the coding
portion of the target gene
sequence may be ligated to a regulatory sequence which is capable of driving
gene expression in the
cell type of interest. Such regulatory regions will be well known to those of
skill in the art, and may
be utilized in the absence of undue experimentation. Target gene sequences may
also be disrupted or
underexpressed. Cells having target gene disruptions or underexpressed target
gene sequences may
be used, for example, to screen for agents capable of affecting alternative
pathways which compensate
for any loss of function attributable to the disruption or underexpression.
In vitYO systems may be designed to identify compounds capable of binding the
target gene
products. Such compounds may include, but are not limited to, peptides made of
D-and/or L-configu-
ration amino acids (in, for example, the form of random peptide libraries; see
e.g., Lam, et al., Nature,
354:82-4 (1991)), phosphopeptides (in, for example, the form of random or
partially degenerate,
directed phosphopeptide libraries; see, e.g., Songyang, et al., Cell, 72:767-
78 (1993)), antibodies, and
small organic or inorganic molecules. Compounds identified may be useful, for
example, in modu-
lating the activity of target gene proteins, preferably mutant target gene
proteins; elaborating the
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biological function of the target gene protein; or screening for compounds
that disrupt normal target
gene interactions or themselves disrupt such interactions.
The principle of the assays used to identify compounds that bind to the target
gene protein
involves preparing a reaction mixture of the target gene protein and the test
compound under
conditions and for a time sufficient to allow the two components to interact
and bind, thus forming a
complex which can be removed and/or detected in the reaction mixture. These
assays can be
conducted in a variety of ways. For example, one method to conduct such an
assay would involve
anchoring the target gene protein or the test substance onto a solid phase and
detecting target protein/
test substance complexes anchored on the solid phase at the end of the
reaction. In one embodiment
of such a method, the target gene protein may be anchored onto a solid
surface, and the test
compound, which is not anchored, may be labeled, either directly or
indirectly.
In practice, microtitre plates are conveniently utilized. The anchored
component may be
immobilized by non-covalent or covalent attachments. Non-covalent attachment
may be accom-
plished simply by coating the solid surface with a solution of the protein and
drying. Alternatively, an
immobilized antibody, preferably a monoclonal antibody, specific for the
protein may be used to
anchor the protein to the solid surface. The surfaces may be prepared in
advance and stored.
In order to conduct the assay, the nonimmobilized component is added to the
coated surface
containing the anchored component. After the reaction is complete, unreacted
components are
removed (e.g., by washing) under conditions such that any complexes formed
will remain
immobilized on the solid surface. The detection of complexes anchored on the
solid surface can be -
accomplished in a number of ways. Where the previously nonimmobilized
component is pre-labeled,
the detection of label immobilized on the surface indicates that complexes
were formed. Where the
previously nonimmobilized component is not pre-labeled, an indirect label can
be used to detect
complexes anchored on the surface; e.g., using a labeled antibody specific for
the previously nonim-
mobilized component (the antibody, in turn, may be directly labeled or
indirectly labeled with a
labeled anti-Ig antibody).
Alternatively, a reaction can be conducted in a liquid phase, the reaction
products separated
from unreacted components, and complexes detected; e.g., using an immobilized
antibody specific for
target gene product or the test compound to anchor any complexes formed in
solution, and a labeled
antibody specific for the other component of the possible complex to detect
anchored complexes.
Compounds that are shown to bind to a particular target gene product through
one of the
methods described above can be further tested for their ability to elicit a
biochemical response from
the target gene protein. Agonists, antagonists and/or inhibitors of the
expression product can be
identified utilizing assays well la~own in the art.
Antisense, Ribozymes, and Antibodies
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Other agents which may be used as therapeutics include the target gene, its
expression
products) and functional fragments thereof. Additionally, agents which reduce
or inhibit mutant
target gene activity may be used to ameliorate disease symptoms. Such agents
include antisense,
ribozyme, and triple helix molecules. Techniques for the production and use of
such molecules are
well known to those of skill in the art.
Anti-sense RNA and DNA molecules act to directly block the translation of mRNA
by
hybridizing to targeted mRNA and preventing protein translation. With respect
to antisense DNA,
oligodeoxyribonucleotides derived from the translation initiation site, e.g.,
between the -10 and +10
regions of the target gene nucleotide sequence of interest, are preferred.
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence-specific hybridization
of the ribozyme
molecule to complementary target RNA, followed by an endonucleolytic cleavage.
The composition
of ribozyme molecules must include one or more sequences complementary to the
target gene mRNA,
and must include the well known catalytic sequence responsible for mRNA
cleavage. For this
sequence, see U.S. Pat. No. 5,093,246, which is incorporated by reference
herein in its entirety. As
such within the scope of the invention are engineered hammerhead motif
ribozyme molecules that
specifically and efficiently catalyze endonucleolytic cleavage of RNA
sequences encoding target gene
proteins.
Specific ribozyme cleavage sites within any potential RNA target are initially
identified by
scanning the molecule of interest for ribozyme cleavage sites which include
the following sequences,
GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20
ribonucleotides
corresponding to the region of the target gene containing the cleavage site
may be evaluated for
predicted structural features, such as secondary structure, that may render
the oligonucleotide
sequence unsuitable. The suitability of candidate sequences may also be
evaluated by testing their
accessibility to hybridization with complementary oligonucleotides, using
ribonuclease protection
assays.
Nucleic acid molecules to be used in triple helix formation for the inhibition
of transcription
should be single stranded and composed of deoxyribonucleotides. The base
composition of these
oligonucleotides must be designed to promote triple helix formation via
Hoogsteen base pairing rules,
which generally require sizeable stretches of either purines or pyrimidines to
be present on one strand
of a duplex. Nucleotide sequences may be pyrimidine-based, which will result
in TAT and CGC
triplets across the three associated strands of the resulting triple helix.
The pyrimidine-rich molecules
provide base complementarity to a purine-rich region of a single strand of the
duplex in a parallel
orientation to that strand. In addition, nucleic acid molecules may be chosen
that are purine-rich, for
example, containing a stretch of G residues. These molecules will form a
triple helix with a DNA
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duplex that is rich in GC pairs, in which the majority of the purine residues
are located on a single
strand of the targeted duplex, resulting in GGC triplets across the three
strands in the triplex.
Alternatively, the potential sequences that can be targeted for triple helix
formation may be
increased by creating a so called "switchback" nucleic acid molecule.
Switchback molecules are
synthesized in an alternating 5'-3', 3'-5' manner, such that they base pair
with first one strand of a
duplex and then the other, eliminating the necessity for a sizeable stretch of
either purines or
pyrimidines to be present on one strand of a duplex.
It is possible that the antisense, ribozyme, and/or triple helix molecules
described herein may
reduce or inhibit the transcription (triple helix) and/or translation
(antisense, ribozyme) of mRNA
produced by both normal and mutant target gene alleles. In order to ensure
that substantially normal
levels of target gene activity are maintained, nucleic acid molecules that
encode and express target
gene polypeptides exhibiting normal activity may be introduced into cells that
do not contain
sequences susceptible to whatever antisense, ribozyme, or triple helix
treatments are being utilized.
Alternatively, it may be preferable to coadminister normal target gene protein
into the cell or tissue in
order to maintain the requisite level of cellular or tissue target gene
activity.
Anti-sense RNA and DNA, ribozyme, and triple helix molecules of the invention
may be
prepared by any method known in the art for the synthesis of DNA and RNA
molecules. These
include techniques for chemically synthesizing oligodeoxyribonucleotides and
oligoribonucleotides
well known in the art such as for example solid phase phosphoramidite chemical
synthesis.
Alternatively, RNA molecules may be generated by in vitro and in vivo
transcription of DNA
sequences encoding the antisense RNA molecule. Such DNA sequences may be
incorporated into a
wide variety of vectors which incorporate suitable RNA polymerise promoters
such as the T7 or SP6
polymerise promoters. Alternatively, antisense cDNA constructs that synthesize
antisense RNA
constitutively or inducibly, depending on the promoter used, can be introduced
stably into cell lines.
Various well-known modifications to the DNA molecules may be introduced as a
means of
increasing intracellular stability and half-life. Possible modifications
include but are not limited to the
addition of flanking sequences of ribonucleotides or deoxyribonucleotides to
the 5' and/or 3' ends of
the molecule or the use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages
within the oligodeoxyribonucleotide backbone.
Antibodies that are both specific for target gene protein, and in particular,
mutant gene
protein, and interfere with its activity may be used to inhibit mutant target
gene function. Such
antibodies may be generated against the proteins themselves or against
peptides corresponding to
portions of the proteins using standard techniques known in the art and as
also described herein. Such
antibodies include but are not limited to polyclonal, monoclonal, Fab
fragments, single chain
antibodies, chimeric antibodies, etc.
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In instances where the target gene protein is intracellular and whole
antibodies are used,
internalizing antibodies may be preferred. However, lipofectin liposomes may
be used to deliver the
antibody or a fragment of the Fab region which binds to the target gene
epitope into cells. Where
fragments of the antibody are used, the smallest inhibitory fragment which
binds to the target or
expanded target protein's binding domain is preferred. For example, peptides
having an amino acid
sequence corresponding to the domain of the variable region of the antibody
that binds to the target
gene protein may be used. Such peptides may be synthesized chemically or
produced via
recombinant DNA technology using methods well known in the art (see, e.g.,
Creighton, Proteins:
Structures and Molecular Principles (1984) W.H. Freeman, New York 1983, supYa;
and Sambrook, et
a
al., 1989, supra). Alternatively, single chain neutralizing antibodies which
bind to intracellular target
gene epitopes may also be administered. Such single chain antibodies may be
administered, for
example, by expressing nucleotide sequences encoding single-chain antibodies
within the target cell
population by utilizing, for example, techniques such as those described in
Marasco, et al., Proc. Natl.
Acad. Sci. USA, 90:7889-93 (1993).
RNA sequences encoding TARGET gene protein may be directly administered to a
patient
exhibiting disease symptoms, at a concentration sufficient to produce a level
of target gene protein
such that disease symptoms are ameliorated. Patients may be treated by gene
replacement therapy.
One or more copies of a normal target gene, or a portion of the gene that
directs the production of a
normal target gene protein with target gene function, may be inserted into
cells using vectors which
include, but are not limited to adenovirus, adeno-associated virus, and
retrovirus vectors, in addition
to other particles that introduce DNA into cells, such as liposomes.
Additionally, techniques such as
those described above may be utilized for the introduction of normal target
gene sequences into
human cells.
Cells, preferably, autologous cells, containing normal target gene expressing
gene sequences
may then be introduced or reintroduced into the patient at positions which
allow for the amelioration
of disease symptoms.
Pharmaceutical Compositions, Effective Dosages, and Routes of Administration
The identified compounds that inhibit target mutant gene expression, synthesis
and/or activity
can be administered to a patient at therapeutically effective doses to treat
or ameliorate the disease. A
therapeutically effective dose refers to that amount of the compound
sufficient to result in
amelioration of symptoms of the disease.
Toxicity and therapeutic efficacy of such compounds can be determined by
standard pharma-
ceutical procedures in cell cultures or experimental animals, e.g., for
determining the LDSO (the dose
lethal to 50% of the population) and the EDSo (the dose therapeutically
effective in 50% of the
population). The dose ratio between toxic and therapeutic effects is the
therapeutic index and it can
be expressed as the ratio LDSO/ED50. Compounds which exhibit large therapeutic
indices are preferred.
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While compounds that exhibit toxic side effects may be used, care should be
taken to design a
delivery system that targets such compounds to the site of affected tissue in
order to minimize
potential damage to uninfected cells and, thereby, reduce side effects.
The data obtained from the cell culture assays and animal studies can be used
in formulating a
range of dosage for use in humans. The dosage of such compounds lies
preferably within a range of
circulating concentrations that include the EDso with little or no toxicity.
The dosage may vary within
this range depending upon the dosage form employed and the route of
administration utilized. For
any compound used in the method of the invention, the therapeutically
effective dose can be estimated
initially from cell culture assays. A dose may be formulated in animal models
to achieve a circulating
plasma concentration range that includes the ICSO (i.e., the concentration of
the test compound which
achieves a half-maximal inhibition of symptoms) as determined in cell culture.
Such information can
be used to more accurately determine useful doses in humans. Levels in plasma
may be measured, for
example, by high performance liquid chromatography.
Pharmaceutical compositions for use in accordance with the present invention
may be
formulated in conventional manner using one or more physiologically acceptable
carriers or
excipients. Thus, the compounds and their physiologically acceptable salts and
solvates may be
formulated for administration by inhalation or insufflation (either through
the mouth or the nose) or
oral, buccal, parenteral, topical, subcutaneous, intraperitoneal, intravenous,
intrapleural, intraoccular,
intraarterial, or rectal administration. It is also contemplated that
pharmaceutical compositions may
be administered with other products that potentiate the activity of the
compound and optionally, may
include other therapeutic ingredients.
For oral administration, the pharmaceutical compositions may take the form of,
for example,
tablets or capsules prepared by conventional means with pharmaceutically
acceptable excipients such
as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or
hydroxypropyl
methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or
calcium hydrogen phosphate);
lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g.,
potato starch or sodium starch
glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may
be coated by methods
well known in the art. Liquid preparations for oral administration may take
the form of, for example,
solutions, syrups or suspensions, or they may be presented as a dry product
for constitution with water
or other suitable vehicle before use. Such liquid preparations may be prepared
by conventional means
with pharmaceutically acceptable additives such as suspending agents (e.g.,
sorbitol syrup, cellulose
derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin
or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated
vegetable oils); and preservatives
(e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations
may also contain buffer
salts, flavoring, coloring and sweetening agents as appropriate.
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Preparations for oral administration may be suitably formulated to give
controlled release of
the active compound.
For buccal administration the compositions may take the form of tablets or
lozenges
formulated in conventional manner.
For administration by inhalation, the compounds for use according to the
present invention
are conveniently delivered in the form of an aerosol spray presentation from
pressurized packs or a
nebulizer, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case
of a pressurized aerosol
the dosage unit may be determined by providing a valve to deliver a metered
amount. Capsules and
cartridges of e.g. gelatin for use in an inhaler or insufflator may be
formulated containing a powder
mix of the compound and a suitable powder base such as lactose or starch.
The compounds may be formulated for parenteral administration by injection,
e.g., by bolus
injection or continuous infusion. Formulations for injection may be presented
in unit dosage form,
e.g., in ampoules or in mufti-dose containers, with an added preservative. The
compositions may take
such forms as suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain
formulatory agents such as suspending, stabilizing and/or dispersing agents.
Alternatively, the active
ingredient may be in powder form for constitution with a suitable vehicle,
e.g., sterile pyrogen-free
water, before use.
The compounds may also be formulated in rectal compositions such as
suppositories or
retention enemas, e.g., containing conventional suppository bases such as
cocoa butter or other
glycerides. Oral ingestion is possibly the easiest method of taking any
medication. Such a route of
administration, is generally simple and straightforward and is frequently the
least inconvenient or
unpleasant route of administration from the patient's point of view. However,
this involves passing
the material through the stomach, which is a hostile environment for many
materials, including
proteins and other biologically active compositions. As the acidic, hydrolytic
and proteolytic
environment of the stomach has evolved efficiently to digest proteinaceous
materials into amino acids
and oligopeptides for subsequent anabolism, it is hardly surprising that very
little or any of a wide
variety of biologically active proteinaceous material, if simply taken orally,
would survive its passage
through the stomach to be taken up by the body in the small intestine. The
result, is that many
proteinaceous medicaments must be taken in through another method, such as
parenterally, often by
subcutaneous, intramuscular or intravenous injection.
Pharmaceutical compositions may also include various buffers (e.g., Tris,
acetate, phosphate),
solubilizers (e.g., Tween, Polysorbate), carriers such as human serum albumin,
preservatives
(thimerosol, benzyl alcohol) and anti-oxidants such as ascorbic acid in order
to stabilize
pharmaceutical activity. The stabilizing agent may be a detergent, such as
tween-20, tween-80, NP
or Triton X-100., EBP may also be incorporated into particulate preparations
of polymeric
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compounds for controlled delivery to a patient over an extended period of
time. A more extensive
survey of components in pharmaceutical compositions is found in Remington's
Pharmaceutical
Sciences, 18th ed., A. R. Gennaro, ed., Mack Publishing, Easton, Pa. (1990).
In addition to the formulations described previously, the compounds may also
be formulated
as a depot preparation. Such long acting formulations may be administered by
implantation (for
example subcutaneously or intramuscularly) or by intramuscular injection.
Thus, for example, the
compounds may be formulated with suitable polymeric or hydrophobic materials
(for example as an
emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble
derivatives, for example,
as a sparingly soluble salt.
The compositions may, if desired, be presented in a pack or dispenser device
which may
contain one or more unit dosage forms containing the active ingredient. The
pack may for example
comprise metal or plastic foil, such as a blister pack. The pack or dispenser
device may be
accompanied by instructions for administration.
Dia ostics
A variety of methods may be employed to diagnose disease conditions associated
with the
target gene. Specifically, reagents may be used, for example, for the
detection of the presence of
target gene mutations, or the detection of either over or under expression of
target gene mRNA.
According to the diagnostic and prognostic method of the present invention,
alteration of the
wild-type target gene locus is detected. In addition, the method can be
performed by detecting the
wild-type target gene locus and confirming the lack of a predisposition or
neoplasia. "Alteration of a
wild-type gene" encompasses all forms of mutations including deletions,
insertions and point
mutations in the coding and noncoding regions. Deletions may be of the entire
gene or only a portion
of the gene. Point mutations may result in stop codons, frameshift mutations
or amino acid
substitutions. Somatic mutations are those which occur only in certain
tissues, e.g., in the tumor
tissue, and are not inherited in the gertnline. Germline mutations can be
found in any of a body's
tissues and are inherited. If only a single allele is somatically mutated, an
early neoplastic state is
indicated. However, if both alleles are mutated, then a late neoplastic state
may be indicated. The
finding of gene mutations thus provides both diagnostic and prognostic
information. A target gene
allele which is not deleted (e.g., that found on the sister chromosome to a
chromosome carrying a
target gene deletion) can be screened for other mutations, such as insertions,
small deletions, and
point mutations. Mutations found in tumor tissues may be linked to decreased
expression of the target
gene product. However, mutations leading to non-functional gene products may
also be linked to a
cancerous state. Point mutational events may occur in regulatory regions, such
as in the promoter of
the gene, leading to loss or diminution of expression of the mRNA. Point
mutations may also abolish
proper RNA processing, leading to loss of expression of the target gene
product, or a decrease in
mRNA stability or translation efficiency.
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One test available for detecting mutations in a candidate locus is to directly
compare genomic
target sequences from cancer patients with those from a control population.
Alternatively, one could
sequence messenger RNA after amplification, e.g., by PCR, thereby eliminating
the necessity of
determining the exon structure of the candidate gene. Mutations from cancer
patients falling outside
the coding region of the target gene can be detected by examining the non-
coding regions, such as
introns and regulatory sequences near or within the target gene. An early
indication that mutations in
noncoding regions are important may come from Northern blot experiments that
reveal messenger
RNA molecules of abnormal size or abundance in cancer patients as compared to
control individuals.
The methods described herein may be performed, for example, by utilizing pre-
packaged
diagnostic kits comprising at least one specific gene nucleic acid or anti-
gene antibody reagent
described herein, which may be conveniently used, e.g., in clinical settings,
to diagnose patients
exhibiting disease symptoms or at risk for developing disease.
Any cell type or tissue, preferably monocytes, endothelial cells, or smooth
muscle cells, in
which the gene is expressed may be utilized in the diagnostics described
below.
DNA or RNA from the cell type or tissue to be analyzed may easily be isolated
using
procedures which are well known to those in the art. Diagnostic procedures may
also be performed in
situ directly upon tissue sections (fixed and/or frozen) of patient tissue
obtained from biopsies or
resections, such that no nucleic acid purification is necessary. Nucleic acid
reagents may be used as
probes and/or primers for such in situ procedures (see, for example, Nuovo,
PCR In Situ
Hybridization: Protocols and Applications, Raven Press, N.Y. (1992)).
Gene nucleotide sequences, either RNA or DNA, may, for example, be used in
hybridization
or amplification assays of biological samples to detect disease-related gene
structures and expression.
Such assays may include, but are not limited to, Southern or Northern
analyses, restriction fragment
length polymorphism assays, single stranded conformational polymorphism
analyses, ih situ
hybridization assays, and polymerase chain reaction analyses. Such analyses
may reveal both
quantitative aspects of the expression pattern of the gene, and qualitative
aspects of the gene
expression and/or gene composition. That is, such aspects may include, for
example, point mutations,
insertions, deletions, chromosomal rearrangements, and/or activation or
inactivation of gene
expression.
Preferred diagnostic methods for the detection of gene-specific nucleic acid
molecules may
involve for example, contacting and incubating nucleic acids, derived from the
cell type or tissue
being analyzed, with one or more labeled nucleic acid reagents under
conditions favorable for the
specific annealing of these reagents to their complementary sequences within
the nucleic acid
molecule of interest. Preferably, the lengths of these nucleic acid reagents
are at least 9 to 30
nucleotides. After incubation, all non-annealed nucleic acids are removed from
the nucleic
acid:fingerprint molecule hybrid. The presence of nucleic acids from the
fingerprint tissue which
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have hybridized, if any such molecules exist, is then detected. Using such a
detection scheme, the
nucleic acid from the tissue or cell type of interest may be immobilized, for
example, to a solid
support such as a membrane, or a plastic surface such as that on a microtitre
plate or polystyrene
beads. In this case, after incubation, non-annealed, labeled nucleic acid
reagents are easily removed.
Detection of the remaining, annealed, labeled nucleic acid reagents is
accomplished using standard
techniques well-known to those in the art.
Alternative diagnostic methods for the detection of gene-specific nucleic acid
molecules may
involve their amplification, e.g., by PCR (the experimental embodiment set
forth in Mullis U.S. Pat.
No. 4,683,202 (1987)), ligase chain reaction (Barany, Proc. Natl. Acad. Sci.
USA, 88:189-93 (1991)),
self sustained sequence replication (Guatelli, et al., Proc. Natl. Acad. Sci.
USA, 87:1874-78 (1990)),
transcriptional amplification system (Kwoh, et al., Proc. Natl. Acad. Sci.
USA, 86:1173-77 (1989)),
Q-Beta Replicase (Lizardi et al., BiolTechnology, 6:1197 (1988)), or any other
nucleic acid
amplification method, followed by the detection of the amplified molecules
using techniques well
known to those of skill in the art. These detection schemes are especially
useful for the detection of
nucleic acid molecules if such molecules are present in very low numbers.
In one embodiment of such a detection scheme, a cDNA molecule is obtained from
an RNA
molecule of interest (e.g., by reverse transcription of the RNA molecule into
cDNA). Cell types or
tissues from which such RNA may be isolated include any tissue in which wild
type fingerprint gene
is known to be expressed, including, but not limited, to monocytes,
endothelium, and/or smooth
muscle. A sequence within the cDNA is then used as the template for a nucleic
acid amplification
reaction, such as a PCR amplification reaction, or the like. The nucleic acid
reagents used as
synthesis initiation reagents (e.g., primers) in the reverse transcription and
nucleic acid amplification
steps of this method may be chosen from among the gene nucleic acid reagents
described herein. The
preferred lengths of such nucleic acid reagents are at least 15-30
nucleotides. For detection of the
amplified product; the nucleic acid amplification may be performed using
radioactively or non-
radioactively labeled nucleotides. Alternatively, enough amplified product may
be made such that the
product may be visualized by standard ethidium bromide staining or by
utilizing any other suitable
nucleic acid staining method.
Antibodies directed against wild type or mutant gene peptides may also be used
as disease
diagnostics and prognostics. Such diagnostic methods, may be used to detect
abnormalities in the
level of gene protein expression, or abnormalities in the structure and/or
tissue, cellular, or subcellular
location of fingerprint gene protein. Structural differences may include, for
example, differences in
the size, electronegativity, or antigenicity of the mutant fingerprint gene
protein relative to the normal
fingerprint gene protein.
Protein from the tissue or cell type to be analyzed may easily be detected or
isolated using
techniques which are well known to those of skill in the art, including but
not limited to western blot
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analysis. For a detailed explanation of methods for carrying out western blot
analysis. (See, e.g.
Sambrook, et al. (1989) supra, at Chapter 18). The protein detection and
isolation methods employed
herein may also be such as those described in Harlow and Lane, for example,
(Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
New York (1988)).
Preferred diagnostic methods for the detection of wild type or mutant gene
peptide molecules
may involve, for example, immunoassays wherein fingerprint gene peptides are
detected by their
interaction with an anti-fingerprint gene-specific peptide antibody.
For example, antibodies, or fragments of antibodies useful in the present
invention may be
used to quantitatively or qualitatively detect the presence of wild type or
mutant gene peptides. This
can be accomplished, for example, by immunofluorescence techniques employing a
fluorescently
labeled antibody (see below) coupled with light microscopic, flow cytometric,
or fluorimetric
detection. Such techniques are especially preferred if the fingerprint gene
peptides are expressed on
the cell surface.
The antibodies (or fragments thereof) useful in the present invention may,
additionally, be
employed histologically, as in immunofluorescence or immunoelectron
microscopy, for ih situ
detection of fingerprint gene peptides. 1h situ detection may be accomplished
by removing a
histological specimen from a patient, and applying thereto a labeled antibody
of the present invention.
The antibody (or fragment) is preferably applied by overlaying the labeled
antibody (or fragment)
onto a biological sample. Through the use of such a procedure, it is possible
to determine not only the
presence of the fingerprint gene peptides, but also their distribution in the
examined tissue. Using the
present invention, those of ordinary skill will readily perceive that any of a
wide variety of
histological methods (such as staining procedures) can be modified in order to
achieve such ire situ
detection.
Immunoassays for wild type, mutant, or expanded fingerprint gene peptides
typically
comprise incubating a biological sample, such as a biological fluid, a tissue
extract, freshly harvested
cells, or cells which have been incubated in tissue culture, in the presence
of a detectably labeled
antibody capable of identifying fingerprint gene peptides, and detecting the
bound antibody by any of
a number of techniques well known in the art.
The biological sample may be brought in contact with and immobilized onto a
solid phase
support or carrier such as nitrocellulose, or other solid support which is
capable of immobilizing cells,
cell particles or soluble proteins. The support may then be washed with
suitable buffers followed by
treatment with the detectably labeled gene-specific antibody. The solid phase
support may then be
washed with the buffer a second time to remove unbound antibody. The amount of
bound label on
solid support may then be detected by conventional means.
The terms "solid phase support or carrier" are intended to encompass any
support capable of
binding an antigen or an antibody. Well-known supports or carriers include
glass, polystyrene,
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polypropylene, polyethylene, dextran, nylon, amylases, natural and modified
celluloses,
polyacrylamides, gabbros, and magnetite. The nature of the carrier can be
either soluble to some
extent or insoluble for the purposes of the present invention. The support
material may have virtually
any possible structural configuration so long as the coupled molecule is
capable of binding to an
antigen or antibody. Thus, the support configuration may be spherical, as in a
bead, or cylindrical, as
in the inside surface of a test tube, or the external surface of a rod.
Alternatively, the surface may be
flat such as a sheet, test strip, etc. Preferred supports include polystyrene
beads. Those skilled in the
art will know many other suitable carriers for binding antibody or antigen, or
will be able to ascertain
the same by use of routine experimentation.
The binding activity of a given lot of anti-wild type or -mutant fingerprint
gene peptide
antibody may be determined according to well known methods. Those skilled in
the art will be able
to determine operative and optimal assay conditions for each determination by
employing routine
experimentation.
One of the ways in which the gene peptide-specific antibody can be detectably
labeled is by
linking the same to an enzyme and using it in an enzyme immunoassay (EIA)
(Voller, Ric Clin Lab,
8:289-98 (I978) ["The Enzyme Linked Immunosorbent Assay (ELISA)", Diagnostic
Horizons 2:1-7,
1978, Microbiological Associates Quarterly Publication, Walkersville, Md.];
Voller, et al., J. Clin.
Patlaol., 31:507-20 (1978); Butler, Meth. Erazymol., 73:482-523 (1981); Maggio
(ed.), Enzyme
Immunoassay, CRC Press, Boca Raton, Fla. (1980); Ishikawa, et al., (eds.)
Enzyme hnmunoassay,
Igaku-Shoin, Tokyo (1981)). The enzyme which is bound to the antibody will
react with an
appropriate substrate, preferably a chromogenic substrate, in such a manner as
to produce a chemical
moiety which can be detected, for example, by spectrophotometric, fluorimetric
or by visual means.
Enzymes which can be used to detectably label the antibody include, but axe
not limited to, malate
dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast
alcohol dehydrogenase;
alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish
peroxidase, alkaline
phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease,
urease, catalase,
glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The
detection can be
accomplished by colorimetric methods which employ a chromogenic substrate for
the enzyme.
Detection may also be accomplished by visual comparison of the extent of
enzymatic reaction of a
substrate in comparison with similarly prepared standards.
Detection may also be accomplished using any of a variety of other
immunoassays. For
example, by radioactively labeling the antibodies or antibody fragments, it is
possible to detect
fingerprint gene wild type, mutant, or expanded peptides through the use of a
radioimmunoassay
(RIA) (see, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh
Training Course on
Radioligand Assay Techniques, The Endocrine Society, March, 1986). The
radioactive isotope can be
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detected by such means as the use of a gamma counter or a scintillation
counter or by autoradi-
ography.
It is also possible to label the antibody with a fluorescent compound. When
the fluorescently
labeled antibody is exposed to light of the proper wave length, its presence
can then be detected due to
fluorescence. Among the most commonly used fluorescent labeling compounds are
fluorescein
isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-
phthaldehyde and
fluorescamine.
The antibody can also be detectably labeled using fluorescence emitting metals
such as
152Eu, or others of the lanthanide series. These metals can be attached to the
antibody using such
metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or
ethylenediamine-tetraacetic
acid (EDTA).
The antibody also can be detectably labeled by coupling it to a
chemiluminescent compound.
The presence of the chemiluminescent-tagged antibody is then determined by
detecting the presence
of luminescence that arises during the course of a chemical reaction. Examples
of particularly useful
chemiluminescent labeling compounds are luminol, isoluminol, theromatic
acridinium ester,
imidazole, acridinium salt and oxalate ester.
Likewise, a bioluminescent compound may be used to label the antibody of the
present
invention. Bioluminescence is a type of chemiluminescence found in biological
systems in, which a
catalytic protein increases the efficiency of the chemiluminescent reaction.
The presence of a
bioluminescent protein is determined by detecting the presence of
luminescence. Important
bioluminescent compounds for purposes of labeling are luciferin, luciferase
and aequorin.
Throughout this application, various publications, patents and published
patent applications
are referred to by an identifying citation. The disclosures of these
publications, patents and published
patent specifications referenced in this application are hereby incorporated
by reference into the
present disclosure to more fully describe the state of the art to which this
invention pertains.
The following examples are intended only to illustrate the present invention
and should in no
way be construed as limiting the subject invention.
Examples
Example 1: Generation and Analysis of Mice Comprising GPCR Gene Disruptions
Targeting Construct for Platelet Activatifag Factor Receptor Gefae. To
investigate the role
of genes encoding GPCRs, particularly, platelet activating factor receptor
genes, disruptions in genes
comprising the sequence set forth in SEQ ID N0:1 were produced by homologous
recombination.
More particularly, as shown in Figure 2A-2B, a specific targeting construct
having the ability to
disrupt or modify genes, specifically comprising SEQ ID NO:1 was created using
as the targeting
arms (homologous sequences) in the construct, the sequences identified herein
as SEQ ID N0:3 and
SEQ ID N0:4.
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Trahsgereic Mice. The targeting construct was introduced into ES cells by
electroporation and
chimeric mice were generated. The ES cells were derived from 129/Sv-+P+Mgf-
SLJ/J mouse
substrain. Fl mice were generated by breeding with C57BL/6 females. The
resultant F1N0 hetero-
zygotes were intercrossed to produce F2N0 homozygotes, or were backcrossed to
C57BL/6 mice to
generate F1N1 heterozygotes. F2N1 homozygous mutant mice were produced by
intercrossing F1N1
heterozygous males and females.
Phenotypic Analysis. The transgenic mice were analyzed for phenotypic changes.
The
homozygous mice demonstrated at least one of the following behavior
phenotypes:
Homozygous mice spent significantly more time in the central region on the
open field test
than the wild-type mice as shown in Figure 4 and the data presented in Table 1
below. This indicates
that the homozygous mice may have less anxiety in comparison to the wild-type
mice.
TABLE 1- OPEN FIELD TEST: TIME SPENT IN CENTRAL REGION
GenotypeF N FamilySubFamily' Gene Time Std.'
Count
Generation, Generation Name , (sec)Err.:
.
platelet
+/+ 2 ; 0 GPCR O~han activating 4 3
' ; 15 81
69
GPCR .
receptor
homolog
platelet
+/+ 2 1 GPCR O~han activating 6 9
34 20
36
GPCR . .
receptor
homolog
platelet
-/- 2 0 GPCR O~han ' activating6 6
; 18 06
35
GPCR . .
receptor
homolog
platelet
-/- 2 1 GPCR G~han a activating13 11
: 49.07 :
4
GPCR ' receptor .
homolog
Homozygous mice had significantly longer latencies to fan or lick their hind
paw on the hot
plate test as shown in Figure 3 and the data presented in Table 2 below. More
particularly, the
homozygous mice displayed an increased response latency to lick or fan their
hind paw on the hot
plate test, indicating that the homozygous mice may have a higher pain
threshold in comparison to the
wild-type mice.
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TABLE Y TIME KING
2 - TO
HOT HIND_PAW
PLATE LIC
TEST:
LATENC
GenotypeF N FamilySubFamilyGene Time Std.Count
' i ; ;
~ G
i
v enerat Name (sec)Err.
on
on :~ Generat
~ platelet
+/+ ' 2 0 GPCR O~han activating15.551.316
. ' '
GPCR receptor
homolog
platelet
+/+ 2 1 GPCR O~han ; activating'12.061.66'12
'
GPCR receptor
homolog
platelet
-/- ' 2 ' 0 GPCR Orphan activating19.504 8
: v 74
':
GPCR receptor .
homolog
platelet
-/- ' 2 1 , GPCR Orphan activating23,414.5011
' .
GPCR receptor
homolog
Example 2: Generation and Analysis of Mice Comprising GPCR Gene Disruptions
Targeting Construct for PAF Receptor Gehe. To investigate the role of genes
encoding
GPCRs, particularly, PAF receptor genes, disruptions in genes comprising the
sequence set forth in
SEQ 1D N0:4 were produced by homologous recombination. More particularly, as
shown in Figure
6A-6B, a specific targeting construct having the ability to disrupt or modify
genes, specifically
comprising SEQ ID NO:4 was created using as the targeting arms (homologous
sequences) in the
construct, the sequences identified herein as SEQ 1D NO:S and SEQ ID N0:6.
Example 3: Generation and Analysis of Mice Comprising LPRS Gene Disruptions
Targeting Construct. To investigate the role of LPRS, disruptions in genes
comprising the
sequence set forth in SEQ ID N0:7 were produced by homologous recombination.
More particularly,
as shown in Figure 9A-9C, a specific targeting construct having the ability to
disrupt or modify genes,
specifically comprising SEQ ID N0:7 was created using as the targeting arms
(homologous
sequences) in the construct, the sequences identified herein as SEQ ID N0:9
and SEQ ID NO:10.
Transgercic Mice. The targeting construct was introduced into ES cells derived
from the
129lOlaHsd mouse substrain by electroporation to generate chimeric mice. Fl
mice were generated
by breeding with C57BL/6 females. The resultant F1N0 heterozygotes were
intercrossed to produce
F2N0 homozygotes, or were backcrossed to C57BL/6 mice to generate F1N1
heterozygotes. F2N1
homozygous mutant mice were produced by intercrossing F1N1 heterozygous males
and females.
Phenotypic Analysis. The transgenic mice were analyzed for phenotypic changes.
The
homozygous mice demonstrated eye abnormalities, including retinal
regeneration.
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Specifically, histopathology studies demonstrated that the eyes of the
homozygous mice
suffered from retinal degeneration, including bilateral retinal regeneration.
In each homozygous
mutant, at least one of the following retinal changes were present: retinal
folds; thinning and
vacuolation of the pigment epithelium layer; degeneration of photoreceptors;
thinning, disorganiza-
tion, and pyknosis of the outer nuclear layer; thinning and disorganization of
the outer plexiform
layer, including juxtaposition of the photoreceptor nuclei and the bipolar
cell or inner nuclear layer;
disorganization of the inner nuclear layer; thinning of the inner plexiform
layer; loss of ganglion cell
nuclei, especially large ganglion cells; and, gliosis of the nerve fiber
layer. The changes were general-
ly more prominent in the outer layers of the retina (photoreceptor layers) and
least pronounced in the
inner layers (inner nuclear layer, inner plexiform layer, ganglion cell layer,
and nerve fiber layer).
BehaviorAzzalysis: The homozygous mice demonstrated at least one of the
following
behaviorphenotypes:
The homozygous mice spent significantly less time in the central region in the
open field test,
indicating possible increased anxiety as compared to wild-type mice. This is
shown in Figure 10.
The homozygous mice displayed a decrease in total distance traveled in the
open field test.
The homozygous mutants were significantly hypoactive, in that they moved about
and explored the
open field less than wild-type mice. This is shown in Figure 11.
Example 4: Generation and Analysis of Mice Comprising Cerberus Gene
Disruptions
Targeting Cozzstruct. To investigate the role of cerberus genes, disruptions
in genes
comprising the sequence set forth in SEQ m NO:11 were produced by homologous
recombination.
More particularly, as shown in Figure 13A-13B, a specific targeting construct
having the ability to
disrupt or modify genes, specifically comprising SEQ ID N0:11 was created
using as the targeting
arms (homologous sequences) in the construct, the sequences identified herein
as SEQ >D N0:13 and
SEQ m N0:14.
Trafzsgezzic Mice. The targeting construct °twas introduced into ES
cells derived from the
129/SvJ x 129/Sv-CP mouse by electroporation to generate chimeric mice. Fl
mice were generated
by breeding with C57BL/6 females. The resultant F1N0 heterozygotes were
intercrossed to produce
F2N0 homozygotes, or were backcrossed to C57BL/6 mice to generate F1N1
heterozygotes. F2N1
homozygous mutant mice were produced by intercrossing F1N1 heterozygous males
and females.
Phenotypic Azzalysis. The transgenic mice were analyzed for phenotypic
changes. The
homozygous mice at least one of the following behavioral phenotypes:
The homozygous mice displayed several differences during the open field test.
Homozygous
mice displayed a decrease in their average velocity during episodes of
movement, and total distance
traveled, indicating decreased activity (e.g., hypoactivity). In addition,
homozygous mutants
displayed an increase in the number of fecal boli deposited during the ten-
minute test, suggesting
increased anxiety or nervousness.
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The homozygous mice displayed a difference during the tail suspension test.
Homozygous
mice were more active during the six-minute test resulting in a decrease in
immobility time,
suggesting that the homozygote mice exhibit a decreased susceptibility to
depression (anti-depressive
behavior phenotype).
In summary, homozygous mutant mice displayed at least one of the following
behaviors:
1) A decrease in average velocity of movement during open field testing
compared.to wild-
type mice;
2) A decrease in total distance traveled during open field testing compared to
wild-type
mice (see Table 3 below and Figure 14);
IO 3) An increase in the number of fecal boli during open field testing
compared to wild-type
mice; and
4) A decrease in total time immobile during the tail suspension test (see
Table 4 below and
Figure 15).
TABLE - OPEN FIELD TEST: ISTANCE RAVELED
3 TOTAL D T
GenotypeF N Family SubFamilyGene Distance Std. Count
Generation Generatio n Name
(cm) Err.
Growth '
cerberus
+/+ 2 0 Factor Cerberus543
60 131
34 9
~~bitor ~ .. 1
.
.
Growth '
cerberus
+/+ 2 1 Factor Cerberus934.27 98 25 10
Inhibitor ~ . 1
Growth
cerberus
-/- 2 ' 0 Factor Cerberus476.52 135.97 ' 9
Inhibitor' 1
Growth
cerberus
-/- 2 ; 1 Factor Cerberus596
52 120 70 10
Inhibitor 1 ,
.
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TABLE EST: TAL MMOBI LE
4 - TO TIME
TAIL I
SUSPENSION
T
GenotypeF N Family SubFamilyGene TimeStd. Count
(,Generation n Name (sec)Err.
~ Generatio '
Growth cerberus'
+l+ 2 0 Factor Cerberus 130.8921.839
Inhibitor 1
Growth cerberus
+/+ 2 1 Factor Cerberus! 143.4823.0010
'
Inhibitor 1
Growth
cerberus
-/- 2 0 Factor Cerberus 119.76: 9
14.99
Inhibitor' 1
Growth ~~~
cerberus
-/- 2 1 Factor Cerberus 81 3 10
' 13 16
33
Inhibitor 1 . .
Example 5: Generation and Analysis of Mice Comprising Brainiac Gene
Disruptions
Targetiyzg Construct. To investigate the role of brainiac genes, disruptions
in genes
comprising the sequence set forth in SEQ ID N0:17 were produced by homologous
recombination.
More particularly, a specific targeting construct having the ability to
disrupt or modify genes,
specifically comprising SEQ ID N0:17 was created using as the targeting arms
(homologous
sequences) in the construct, the sequences identified herein as SEQ 1D N0:15
and SEQ ID N0:16.
Trarzsgenic Miee. The targeting construct was introduced into ES cells derived
from
129lOlaHsd mouse substrain by electroporation to generate chimeric mice. F1
mice were generated
by breeding with C57BL/6 females. The resultant F1N0 heterozygotes were
intercrossed to produce
F2N0 homozygotes, or were backcrossed to C57BL16 mice to generate F1N1
heterozygotes. F2N1
homozygous mutant mice were produced by intercrossing F1N1 heterozygous males
and females.
As is apparent to one of skill in the art, various modifications of the above
embodiments can
be made without departing from the spirit and scope of this invention. These
modifications and
variations are within the scope of this invention.
