Note: Descriptions are shown in the official language in which they were submitted.
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TRANSGENIC ANIMALS INCORPORATING
EXOGENOUS GRF GENES
The present invention is directed to producing
faster growing and enlarged animals and to the animals
produced thereby.
BACKGROUND OF THE INVENTION
Recombinant DNA technologies have opened up the
possibility of producing genetically-altered animalsO
An important field where research has been conducted and
where research is ongoing is the possible correction of
genetic defects, particularly with respect to eventual
correction of genetic defects in humans. For example,
it has been proposed that hemoglobinopathies might be
corrected by replacing abnormal globin genes with normal
globin genes. Preliminary to such effort, E.
Wagner et al. P.N.A.S. 78, 5016-5020 (1981) and
T. Wagner et al. P.N.A.S. 78, 6376-6380 (1981) have
inserted exogenous globin genes into mammalian embryos
and have thereby produced transgenic mammals which carry
the gene and in some cases express the gene product.
Although this work may eventually lead to gene therapy
in humans, genetic experimentation with humans or human
embryos is still considered to be some distance in the
future.
Of more immediate potential importance is the
production of transgenic, non-human animals which
exhibit specific useful attributes. For example, it is
desirable to have meat-producing animals which more
efficiently metabolize feed and grow both faster and
larger. Increased lactation in milk-producing animals
and enhanced disease-resistance are other desirable
attributes which potentially could be imparted to a
transgenic animal.
Palmiter et al., Nature 300, 611-615 (1982)
report transgenic mice that developed from embryos which
were transformed with a recombinant gene that included a
rat growth hormone-encoding DNA sequence. The mice were
found to grow to up to double the normal weight of
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similar mice and were found to have up to 1000 times the
normal levels of growth hormone (GH) in their peripheral
blood serum.
The dramatic increases in growth rate and adult
size of transgenic mice incorporating the exogenous
growth hormone gene in their genome suggests that
transgenic meat-producing animals might similarly be
produced which would also grow larger. Reproducing
strains of transgenic animals could potentially
substantially increase meat supplies and/or reduce the
cost of producing meat. To date, the use of transgenic
animals for producing meat has not been described.
One important problem with transgenic animals
carrying an exogenous GH gene is that the transgenic
females tend to be sterile, posing very substantial
problems with respect to establishing reproducible
strains and particularly in producing strains that are
homozygous for the exogenous gene. Why the fertility
problem exists has not been determined; however, the
highly elevated levels of the powerful growth hormone
may upset other endocrine systems, including those which
regulate fertility. The exogenous growth hormone as
described by Palmiter et al., Nature 300 (1982~ supra.,
unlike endogenous growth hormone, is expressed in a
variety of organs throughout the body and is, therefore,
; not subject to normal regulatory mechanisms which
control its level of production and release.
Growth hormone molecules are large, e.g.,
typically about Y21 kD, and exhibit a good deal of
structural differences from species to species~
Although growth hormone molecules frequently exhibit
cross-species hormonal activity, the different
structures of the various growth hormones may induce
undesirable immune responses in foreign species. Thus,
a transgenic animal may exhibit a significant autoimmune
response to growth hormone expressed by its exogenous
genetic material.
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Growth hormone releasing factor (GRF3 is a
hormone which acts on the pituitary to effect the
release of GH. However, R. Clark & I. Robinson, Nature
314, 281-283 (1985) reported that continuously
administered doses of GRF are ineffective in increasing
growth rates, thus suggesting that exogenous GRF, as
would be expressed by an exogenous GRF-encoding gene,
would not increase either the growth rate or the
eventual size of the animals. An exogenous GRF gene,
like an exogenous GH gene, would be expected to be
expressed in widely distributed organs, not only the
hypothalamus, and therefore would be expected to be
outside of the control of the normal endocrine
regulatory mechanisms, thereby causing the
overexpression of GRF, a result which has been shown to
be inconsistent with increased growth rate.
The present invention creates transgenic
animals which incorporate an exogenous gene encoding
growth hormone releasing factor, and surprisingly and
unexpectedly, exhibit increased growth rate and an
increase in the eventual size of the animal which is
produced. The expression of GRF in transgenic animals
carrying the GRF gene is widely distributed in various
organs, and GRF production in such animals is highly
elevated and appears to be continuous; nevertheless, in
contrast to experiments in which the exogenous GRF was
continuously introduced by infusion into the bloodstream
without enhancing growth rate, the exogenous,
gene-expressed GRF enhances the growth rate and the
eventual size of the animals.
Of significant interest to the background of
the invention are numerous publications of prior
investigations relating to regulation of mammalian gene
expression and to introduction of purified genes into
eukaryotic cells.
Specifically indicating the background of the
invention and illustrating the state of the prior art
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are the following publications: Durnam, et al.,
"Isolation and Characterization of the Mouse
Metallothionein-I Gene", P N.A.S. 77, 6511-6515 (1980);
Durnam, et al., J. Biol. Chem~ 256, 5712-5716 (1981);
Mayo, et al., J. Biol. Chem. 256 2621-2624 (1981);
Hager, et al., Nature, 291, 30-342 (1981);
Glanville, et al., Nature, 292, 267-269 (1981); and
Beach, et al., P.N.A.S. 78, 2210-2214 (1981). The
foregoing all deal with DNA sequences specifying
production of low molecular weight, metal-binding
protein found in one or more forms in most vertebrate
tissues. More particularly, the publications treat
mouse metallothionein genes as well as their
promoter/regulator DNA sequences and the responsiveness
of such promoter/regulator sequences to metals and
steroid hormones.
Additional relevant publications are:
McKnight, et alO, J. Biol. Chem. 255, 148-153 (1980);
and Palmiter, et al., J. Biol. Chem. 256, 7910-7916
(1981). Also relevant is a publication dealing with
microinjection of plasmids into germinal vesicles of
mouse oocytes or pronuclei of fertilizes mouse ova,
Brinster, et al., Science 211, 396-398 (1981).
The following publications of Evans and
co-workers dealing with growth hormone releasing factor
mRNA sequences and also dealing with the cloning of rat
growth hormone genes and their introduction into and
expression in mammalian cells are also relevant: (1)
M.M. Harpold, P.R. Dobner, R.M. Evans and F.C. Bancroft,
Construction and identification by positive
hybridization-translation of a bacterial plasmid
containing a rat growth hormone structural gene
sequence, Nucleic Acids Research 5, 2039-2053 (1978);
(2) M.M. Harpold, P.R. Dobner, R.M. Evans, F.C. Bancroft
and J.E. Darnell, Jr., The synthesis and processing of a
nuclear RNA precursor to a rat pregrowth hormone
messenger RNA, Nucleic Acids Research 6, 3133-3144
.
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135l~85
(1979); (3) H. Soreq, M. Harpold, R.M. Evans, JoEo
Darnell, Jr. and F.C. Bancroft, Rat growth hormone
gene: Intervening sequences separate the mRNA regions,
Nucleic Acids Research 6, 2471-2482 (1979); (4) Doehmer,
J., Barinaga, M., Vale, W., Rosenfeld, M.G., Verma, I.M.
and Evans, R.M.I Introduction of rat growth hormone gene
into mouse fibroblasts via a retroviral DNA vector:
Expression and regulation, P.N.A.S. 79, 2268-2272
(1982); (5) Evans, R.M., Birnberg, N.C. and Rosenfeld,
M.G., Glucocorticoid and thyroid hormone
transcriptionally regulate growth hormone gene
expression, P.N.A.S. 79, 7659-7663 (1982); (6) Mayo~
K.E., Vale, W., Rivier, J., Rosenfeld, M.G. and Evans,
R.M., Expression cloning and sequence of a cDNA encoding
human growth hormone releasing factor, Nature 306, 86-88
(1983); (7) Barinaga, M., Yamomoto, G., Rivier, C., and
Evans, R.M. Growth ho ~one releasing factor
transcriptionally regulates growth hormone expression,
Nature 306, 84-85 (1983); (8) Verma, I., Doehmer, J.,
Barinaga, M., Rosenfeld, M. and Evans, R.M., In:
Eukaryotic Viral Vectors, Y. Gluzman, ed., Cold Spring
Harbor (1982).
Also pertinent to the background of the present
invention are the publications of Illmensee, et al.,
Cell 23, 9-18 (1981) and Gordon, et al., P.N.A.S. 77,
7380-7384 (1981) which respectively treat injection of
nuclei into nucleated mouse eggs and introduction of
plasmids containing the herpes thymidine kinase gene and
SV40 (Simian virus) sequences into mice.
SUMMARY OF THE INVENTION
The present invention is directed to producing
transgenic animals which incorporate exogenous GRF in
their genome and, as a result, grow at a faster than
normal rate and achieve a greater than normal adult
size. Such transgenic animals are particularly useful
as meat-producers and/or milk-producers. To produce the
transgenic animals, a gene that encodes a GRF, for
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example, human GRF (hGRF), is isolated. To enhance
expression, a gene portion which encodes GRF is linked
to a powerful promoter sequence, such as a mammalian
methallothionein gene promoter. A fertilized egg is
obtained from an animal of the species into which the
exogenous gene is to be introduced, and the exogenous
GRF gene is introduced into fertilized eggs, e.g., by
microinjection into each egg. The injected fertilized
eggs are implanted into host female animals which give
birth to transgenic animals. Animals initially selected
for further development are those which are shown to
incorporate the foreign genetic material in their
genome. Development of the animals is followed, and
transgenic animals are selected for breeding purposes
which exhibit elevated growth rates and achieve greater
adult size. Transgenic animals which incorporate an
exogenous GRF gene, both male and female, are generally
fertile and pass on the exogenous gene to their progeny
by normal hereditary mechanisms.
IN THE DRAWINGS
FIGURE 1 Shows structure of a mouse
metallothionein-I/human growth hormone-releasing factor
fusion gene, MT-GRF. The upper portion of the figure is
a restriction map for common 6-bp cutters. BglII/SmaI
indicates the site of the fusion between the mouse MT-I
promoter and a human GRF "minigene". The lower portion
of FIGURE 1 is a schematic representation of the 2.5-kb
EcoRI-HindIII fragment of DNA used for microinjection.
Open boxes represent 5' and 3' untranslated exon
regions, and shaded boxes represent the coding exon
regions. The derivations of various parts of the fusion
gene are indicated. The fusion occurs in the 5'
untranslated regions of both genes. The large exon
labelled 3-5 was constructed by replacing part of the
human GRF gene with a human GRF cDNA, effectively
eliminati ~ intron C (2.4 kb) and intron D (3.0 kb) of
the human GRF gene. Consensus sequences involved in
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transcription initiation, translation initiation and
termination, polyadenylation, and proteolytic processing
of the GRF peptide from its precursor are shown.
FIGURES 2A-2E illustrate the stepwise
construction of the GRF "minigene" and the fusion gene
designated "MT-GRF".
FIGURE 3 is a diagram of pedigree analysis of
MT-GRF transgenic mice. Mice that carry the MT-GRF
fusion gene ~determined by DNA blotting of tail snips)
are indicated by the solid symbols. Squares represent
males and circles represent females. The animal number
is given above each symbol; the numbers below the solid
symbols indicate increased weight ratios at 9 weeks old
compared with age- and sex-matched littermates. FIG. 3a
shows the pedigree of MT-GRF mouse 762-5; FIG. 3b shows
that of MT-GRF mouse 765-2. Mouse 765-2-6 died but was
previously scored as a non-carrier.
Figure 4 is a copy of Northern Blot analysis of
messenger RNA for a control mouse depicting the presence
or absence of mRNA for MT-I and for GRF in RNAs isolated
from the six different tissues obtained from the animal.
DETAILED ~ESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS
In accordance with the present invention,
transgenic animals incorporating exogenous GRF-encoding
genes in their genome express exogenous GRF hormone and
exhibit enhanced growth rate and enlarged adult size.
Importantly, both male and female transgenic animals
carrying the exogenous GRF gene are fertile and pass on
the gene to their progeny. Production of such a
transgenic animal begins with isolating a GRF-encoding
yene. Preferably the process begins with constructing,
through gene splicing techniques, a recombinant
GRF-encoding gene with enhanced expression capabilities.
Fertilized eggs are obtained from animals of a desired
species, and the constructed GRF genes are microinjected
into the eggs, e.g., into the male pronuclei of
fertilized eggs. The microinjected eggs, some of which
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incorporate the injected GRF genes into their genomes,
are implanted into host mothers of the species from
which the eggs are obtained. Animals which develop from
the eggs and are born are initially tested for
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incorporation of the exogenous gene, e.g., with
appropriate DNA probes. As the animals which
incorporate the exogenous gene develop, they are
followed for increased expression of GRF and GRF mRNA,
increased secretion of GH, and manifestations of
increased growth. Suitable transgenic animals are bred,
and those of their progency which similarly express
exogenous GRF are used to establish new breeding lines.
A GRF gene used to produce a transgenic animals
includes a GRF-encoding DNA sequence and a promoter DNA
sequence to which the GRF-encoding sequence is operably
linked. Although the promoter sequence could be the
natural GRF promoter sequence, it is preferred to link
the GRF-encoding sequence to a stronger promoter, such
as a promoter of a mammalian metallothionein gene. This
helps to ensure expression of the exogenous G~F in the
transgenic animal and desirably promotes expression of
the exogenous GRF at significantly elevated levels,
relative to normal levels of endogenous GRF. As it is
intended that the transgenic animal produce
significantly elevated levels of GRF, the promoter
preferably helps to ensure that GRF is expressed in
widely distributed organs, not only in the hypothalamus,
and thus, a promoter is preferably linked to the
GRF-encoding sequence which is known to promote
expression in widely distributed organs.
Preferably, the promoter sequence includes or
is linked to a regulatory sequence by which expression
of the gene in the transgenic animal may be controlled
by exogenous agents, such as a metal or hormone whi~h is
fed to or inoculated into the animalO Promoter/regulator
DNA sequences suitable for use in practice of the
invention are derived from avian and mammalian cells and
include: the iron and steroid hormone-responsive
promoter/regulator sequence naturally associated with
the transferrin (conalbumin) gene of chickens; the
steroid hormone-responsive promoter/regulator sequence
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associated with ovalbumin gene in chickens; and the
metal and steroid hormone-responsive promoter/regulator
sequence of the mouse metallothionein-I or
metallothionein-II genes.
The invention, however, is not limited to
promoters with associated regulator DNA sequences.
Examples of promoter DNA sequences which are not either
meta] or steriod- or hormone-responsive which might be
employed include: liver promoters, i.e. albumin,
glycolytic enzymes, transferrin, caeruloplasmin and
alpha-2-microglobulin; histocompatibility gene
promoters, immunoglobulin gene promoters, interferon
gene promoters, heat shock gene promoters and retroviral
gene promoters.
In accordance with a preferred aspect of the
invention, the GRF-encoding sequence is a "minigene"
which is a hybrid construct of natural gene sequences
and cDNA sequences. The hybrid construct substantially
reduces the length of the gene relative to the natural
genomic sequence, which is generally so long that it is
difficult to work with. The natural human genomic
sequence encoding GRF, for example, is 10 kb long and
contains 4 introns. Although the entire sequence of the
human GRF genomic sequence is known, the long sequence
has previously been cloned only in portions, and,
therefore, the cloned sequences must be appropriately
pieced together to form the entire hGRF genomic
sequence. CDNA, on the other hand, is much shorter, by
reason of eliminating all introns from the natural
genomic sequence. cDNA, however, lacks genomic
regulatory elements, such as polyadenylation sequences.
GRF minigenes, according to the invention, are designed
to incorporate the desirable features of the natural
genomic sequence, including regulatory sequences, and
the desirable feature of shortness of the CDNA. The GRF
minigene therefore includes at least one sequence
corresponding to the natural genomic sequences and at
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least one sequence corresponding to the cDNA sequence.
Preferably, in order to include both 5' and 3'
regulatory sequences of genomic DNA, a minigene includes
a 5' sequence corresponding to the natural GRF genomic
sequence, a middle cDNA sequence and a 3' sequence
corresponding to the natural GRF genomic sequenGe.
Because eukaryotic genes generally include
introns, it is assumed that a gene will be best
recognized in a eukaryotic host cell as a eukaryotic
gene and be appropriately processed if it contains at
least one intron, therefore, preferred GRF minigenes
have fewer introns than the natural GRF gene, but retain
at least one intron.
The invention will now be described in greater
detail by way of specific examples.
EXAMPLE 1
This example relates to procedures for
preparation of a fusion GRF gene and use of the fusion
gene to produce transgenic mice. In this example, a DNA
plasmid, pMThGRF, is produced which includes a DNA
sequence encoding the hGRF structural gene which is
operatively associated with the promoter/regulator DNA
sequence of the mouse metallothionein-I (M~-I) gene.
The MT portion of the fusion gene was constructed using
p d mlpEE3.8, a plasmid in which the 2.8 kb
EcoRI-EcoRI fragment of me-lambda 26 is inserted at the
EcoRI site of pBR322, as disclosed in Durnam, et al.,
P N.A.S. 77, 6511-6515 (1980).
-
A fragment of a cloned human growth hormone
releasing factor gene, from which the 5' regulatoryreyion has been deleted, is fused to the MT-I
promoter/regulator region to form plasmid pMTGRF. More
specifically, the unique B~II site of the MT-I genomic
clone mlpEE3 8 is destroyed by digesting with BglII,
followed by filling in the sticky ends with Klenow
fragment of DNA polymerase in the presence of ATP, GTP,
CTP and TTP. This plasmid is digested with PvuII and is
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ligated to the blunt ends of a _maI-to-SmaI fragment
containing the human GRF structural gene to give MThGRF
(8.3 kb). A 4.3 kb BstEII to _maI fragment is used for
subsequent injection studies. The fusion gene is
predicted to direct transcription of an mRNA containing
68 bases contributed by MT-I, followed by the entire
human GRF sequence.
The 4.3 kb fragment fusion gene extending from
the BstEII site of MT-l to the SmaI site of GRF is
restricted from MThGRF and separated from other
fragments on an agarose gel and used for microinjection
into fertilized eggs. The male pronuclei of fertilized
eggs are microinjected with 2 picoliters containing
about 1,000 copies of this fragment, and 170 eggs are
inserted into the reproductive tracts of foster
mothers. 20 mice develop from these eggs.
When the mice are weaned, total nucleic acid is
extracted from a piece of tail and used for DNA dot
hybridization to determine which animals carry MThGRF
sequences. Using a nick-translated probe complementary
to the hGRF gene, 7 of the animals give hydridization
signals above background, and their DNA is analyzed
further by restriction enzyme digestion and Southern
blotting. This analysis shows that all 7 animals have a
~ 25 predicted intact hGRF fragment. 7-21 weeks post
-; parturition, peripheral blood serum is obtained from
each of the hybridization probe-positive mice. The
presence of human growth hormone releasing factor in the
sera is determined by radioimmunoassay using antibody
raised against human GRF. The levels of human GRF in
the mice sera are determined by radioimmunoassay to
range from about 10 to about 1,000 ng per milliliter.
GRF is normally not present in the peripheral blood of
either mice or humans in amounts approaching one
nanogram per ml concentrations.
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EXAMPLE 2
This example relates to procedures for
preparation of another fusion gene of the present
invention. In this example, a DNA plasmid, pThGRFr is
5 shown to include a DNA sequence coding for human growth
hormone releasing Eactor structural gene which is
operatively associated with the promoter/regulator DNA
sequence of the chicken transferrin gene. The fusion
gene is constructed using plasmid pl7 disclosed in
McKnight, et al., Cell 34, p. 335-341 (1983).
A fragment of a cloned GRF from which the 5'
regulatory region has been deleted is fused to a
transferrin promoter/regulator region to form plasmid
pThGRF. More specifically, the unique EcoRI site in the
first intervening sequence of the transferrin genomic
clone pl7 is destroyed by digesting with EcoRI, followed
by filling in the sticky ends with Klenow fragment of
DNA polymerase in the presence of ATP, GTP, CTP and
TTP. This plasmid is digested with PvuII and is ligated
to the blunt ends of a SmaI to _maI fragment containing
the entire human growth hormone releasing factor
structural gene to give pThGRF. A 7.3 kb _maI-to~_maI
fragment is used for subsequent injection studies which
is isolated from an agarose gel by the NaClO4 method
of Chen, et al., Anal. Biochem. 101, 339-341 (1980) .
The fusion gene is predicted to direct transcription of
an mRN~ containing 5' untranslated sequences contributed
by the transferrin gene, followed by the entire GRF gene.
The 7.3 kb fragment fusion gene extending from
the KpnI site of transferrin (-185) to the SmaI site of
GRF is restricted from pThGRF, separated from other
fragments on an agarose gel and used for injection into
eggs. The male pronuclei of fertilized eggs are
microinjected with 2 picoliters containing about 1,000
copies of this fragment, and 170 eggs are inserted into
the reproductive tracts of foster mothers as in
Example I. 20 mice develop from these eggs.
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When the mice are weaned, total nucleic acid is
extracted from a piece of tail and used for DNA dot
hybridization to determine which animals carry pThGRF
sequencesO Using a nick-translated probe complementary
to the GRF gene, 7 of the animals give hybridization
signals above background, and their DNA is analyzed
further by restriction with restriction enzymes and
Southern blotting. This analysis shows that all 7 have
a predicted intact hGRF fragment. 7-21 weeks post
parturition, peripheral blood serum is obtained from
each of the hybridization probe-positive mice. The
presence of human growth hormone releasing factor in the
sera is determined by radioimmunoassay according to the
method of Doehmer, J. et al., P.N.A.S. 79, 2268 (1982),
using antibodies raised against GRF as described in
Rivier et al., Nature 30_, p. 276 (1982). The level of
GRF in the mice sera is determined by radioimmunoassay
to range from about 10 to about 1,000 ng per milliliter.
EXAMPLE 3
This example represents a preferred embodiment
of the present invention in which the promoter sequence
is a mammalian metallothionein promoter and the
GRF-encoding portion includes segments from -the natural
genomic sequence that encodes the human GRF precursor
protein as well as sequences from corresponding cDNA,
resulting in a "minigene" which is shortened relative to
the natural genomic sequence by deleting all introns but
the second intron.
The structure of the mouse MT-I/human GRF
fusion gene construct used (referred to as MT-GRF) is
shown in Figure 1. A 770-base pair (bp) fragment of the
mouse MT-I gene, including sequences responsible for
metal-inducibility and transcription initiation, was
fused to a human GRF "minigene" which includes the
entire coding region of the GRF precursor protein. The
human GRF minigene was created by combining cDNA and
genomic clones such that the 10-kilobase (kb) human GRF
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gene, which normally includes five exons (K.E.
Mayo et al., P.N.A.S. 82, 63-67 (1985)), has been
reduced to less than 1 kb and retains a single intron of
230 bp (see Fi~ure 1) . Analysis of human GRF cDNA
clones suggests that the GRF precursor protein can
consist of either 107 or 108 amino acids, differing in
the presence or absence of serine 103 (U. Gubler et al.,
P.N.A.S. 80, 4311-4314 (1983)). DNA sequence analysis
of the human GRF gene indicates that this difference may
be explained by alternative RNA processing. The cDNA
used herein to construct the MT-GRF fusion gene encodes
only the 108-amino-acid form of the GRF precursor
protein.
More particularly, the GRF "minigene" and the
MT-GRF construct were constructed in a multistep process
as diag rammed in Figures 2A-2E.
Step A. A vector was constructed that is a
derivative of pBR322 but is deleted between the
EcoRV and PvuII sites. This was done by
digesting with these two enzymes and ligating
the resulting blunt-ends together. This
vector, therefore, lacks any BamHl and PvuII
sites, a necessity for subsequent steps.
Step B. The EcoRl-to-HindIII insert from human
GRF cDNA clone phGRF-54 (see Mayo et al.,
Nature 306, 86-88 (1983)) was ligated into the
EcoRl - HindIII sites of the vector described
in Step A. The cDNA essentially provides fused
exons 3-5 for the final product.
Step C. The BamHl-to-H_III fragment at the
3'-end of the cDNA (about 200 bp) was removed
and replaced with a BamH1 to HindII fragment of
about 1 kb from genomic clone hGRF 101 (see
- Ma~o et al., P.N .A .S. 82, 63-67 (1985)). This
~ragment provides the polyadenylation site and
3'-flanking sequences for the final product.
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Step D. An EcoRl-to-PvuII fragment at the
5'-end of the cDNA (about 100 bp3 was removed
and replaced by~ an EcoRl-to-PvuII fragment of
about 1.5 kb f rom genomic clone hGRF cos49 (see
Mayo et al., P.N.A.S. 82, 63-~7 (1985)). The
1.5 kb f ragment was removed by partially
digesting a plasmid sukclone of hGRF cos49 with
PvuII and completely digesting with EcoRl.
This fragment provides exon 2 and intron B for
the final product.
Step E. The hGRF "minigene" (the product of
Step D) is digested partially with SmaI to cut
only at the site within exon 2. This SmaI site
is made into a _~II site using synthetic
linkers. An EcoRl-to-B~II fragment of about
1 kb is removed and is replaced with a 770 bp
EcoRl-to-_glII fragment from the mouse
metallothionein-I gene (see Glanville et all.,
Nature 292, 267-269 (1981)). This fragment
provides the promoter/regulatory activity for
the final product.
To initially determine whether the MT-GRF
fusion gene could be expressed and regulated correctly,
it was introduced into cuItured mouse fibroblast cells
using CaPO4-mediated DNA transfection [F.L.
Graham et al., Virology 52, 456-467 (1973)] and
co-selection for neomycin resistance conferred by the
vector pSV2-neo [P.J. Southern et al., Molec. Appl.
Genet. ul, 327-341 (1982)]. Several stable cell lines
that were generated in this manner express a MT-GRF
fusion mRNA of the expected size whose abundance is
increased by metal treatment, and accumulate
radioassayable hGRF in the cultu re medium.
A 2.5-kb EcoRI-HindIII fragment containing the
MT-GRF fusion gene (about 1,600 molecules) was
microinjected into the male pronuclei of 350 F2 hybrid
eggs (obtained by mating C57BL/6 X SJL hybrid adults),
and the eggs were transferred into the oviducts of
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pseudopregnant recipients as described in R.L.
Brinster et al., Cell 27, 223-231 (1981). Fifty-nine
animals developed from the microinjected eggs.
At weaning, DNA was isolated from a piece of
-tail and analyzed for the fusion gene by dot-blotting
using a human GRF cDNA as probe (K.E. Mayo et al.,
Nature 306, ~6-88 (1983)). Positive animals were
re-analyzed by Southern DNA blotting and copy number
determined by comparison with a standard curve generated
from known amounts of MT-GRF plasmid DNA. Positive
animals were maintained on water containing 25 mM
ZnSO4. RNA was isolated following partial hepatectomy
and analyzed by Northern blotting using a human GRF cDNA
probe. Autoradiograms were densitometrically scanned to
determine relative RNA amounts. Plasma GRF and GH were
determined by radioimmunoassay of serum samples from
animals at 9 weeks old. The GH radioimmunoassay was
able to detect 16 ng/ml serum GH (two control animals
had 16 and 46 ng/ml GH), and the GRF radioimmunoassay
was able to detect 10 ng/ml serum GRF (two control
animals had less than 10 ng/ml GRF). The growth ratio
was determined at 9 weeks of age and represents a
comparison with age- and sex-matched littermates.
Fourteen animals carried the MT-GRF fusion
gene; these animals were maintained on water with 25 mM
ZnSO4 to enhance expression of the fusion gene. Table
1 summarizes the expression of the MT-GRF fusion gene in
these 14 transgenic mice.
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Table 1
Expression of the MT-GRF fusion gene in transgenic mice
Liver
No. of GRF
gene mRNA Plasma Plasma
5copies (relative hGRF mGH Growth
Mouse ~ amount) (ng/ml) (ng/ml) (ratio)
M760-5 1 3 24 1,051 1.35
M762-3 2 0 10 21 0.93
F762-5 ~ 63 26 141 1.33
F765-2 2 102 207 415 1.24
M800-1 1 0 10 149 1.12
F800-8 1 3 14 402 1.05
M801-3 3 0 10 38 0.97
F801-5 8 38 99 809 1.35
M801-9 1 4 20 541 1.42
F802-3 5 1 45 913 1.51
M803-4 10 118 263 1,095 1.41
F803-5 1 5 24 166 1.45
M803-6 10 16 50 302 1.24
F803-7 20 * * * 1.36
* F803-7 died prematurely, Although liver GRF mRNA has
measured, the amount could not be quantitated because of
RNA degradation in the postmortem liver. Plasma GRF and GH
levels could not be determined accurately for the same
; 25 reason.
Eleven of the 14 animals express the MT-GRF
messenger RNA in the liver, a major site of
metallothionein expression. The amount of the fusion
mRNA in the livers of these mice varies by as much as
100-fold and does not seem to be strongly correlated
with the number of copies of the fusion gene in each
animal. Animals expressing the fusion gene have
measurable levels of hGRF in their serum, and they have
increased levels of serum growth hormone. Ten of the
mice showed significant increases in growth at 9 weeks
old and were 25-50% larger than control littermates.
Examination of the data in Table 1 reveals that there is
no strong correlation between GRF levels, GH levels and
growth.
To determine whether the MT-GRF fusion gene and
the large size are heritable~ two the MT-GRF founder
animals were bred with control animals; offspring from
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these matings were analyzed for the presence of the
MT-GRF fusion gene and for their rate of growth.
Figure 3 shows two pedigrees demonstrating that both the
fusion gene and the "large" phenotype are inherited by
about 50% of the total off spring. DNA analysis showed
that all positive offspring carried the same number of
copies of the fusion gene as the corresponding parent.
Four other females were also mated and produced
litters. Thus, all six of the females tested were
fertile (two were killed before breeding) . In addition,
male MT-GRF mice 760-5 and 801-9 (see Table 1) sired
litters that included offspring carrying the fusion gene
and displaying the large phenotype, indicating both
males and females carrying the MT-GRF fusion gene are
fertile and transmit the gene. It is now known that
both the MT-GRF fusion gene and the large phenotype are
inherited by about 50% of the offspring examined in the
F2 generation of animals.
To determine whether expression of the MT-GRF
fusion gene in transgenic mice demonstrates a tissue
specificity similar to that of the endogenous MT-I gene,
both MT-I and MT-GRF RNAs we re analy zed in six tissues
f rom either a control mouse or an MT-GRE' mouse
(female 765-2-3) (see Fl, in Figure 3b). Both animals
were maintained on water with 76 mM ZnSO4 for several
weeks before killing. The indicated organs were removed
and frozen at -70C until use. Total RNA was prepared
by homogenization in guanidine isothiocyanate and
centrifugation through cesium chloride, as described in
J.M. Chirgwin et al., Biochemistry 18, 5294-5299 (1979);
5 ug of each RNA was denatured and electrophoresed on
formaldehyde/l.5% agarose gels (J. Meinkoth et al.,
Analyt. Biochem. 138, 267-284 (1984)) and the RNA
transferred to nitro-cellulose filters as described in
P. Thomas, P.N.A.S. 77, 5201-5205 (1980). Filters were
probed with either a mouse MT-I genomic clone, pSH (D.M.
Durnam et al., P.N.A.S. 77, 6511-6515 (1980)), or a
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human GRF cDNA clone, phGRF-54 (K.E. Mayo et al., Nature
306, 86-88 (1983))~ Probes were labelled by
nick-translation using [alpha-32P]dCTP. Exposure time
of the autoradiograms was 16 hours.
The control animal expressed MT-I mRNA at high
levels in liver, kidney, gut and pancreas, and at lower
levels in brain and spleen, as can be seen in Figure 4
obtained using standard Northern Blot analysis of RNAs
obtained from six different tissues. This agrees well
with the known tissue distribution of MT-I mRNA (D.M.
Durnam et al., ~. Biol. Chem. 256, 5712-5716 (1981)).
The control animal did not express detectable hGRF mRNA
in any tissue. Although the hGRF probe might detect
endogenous mouse GRF mRNA in the brain, the low
abundance of GRF mRNA even in the hypothalamus (less
than 0.01%) makes detection unlikely. The transgenic
mouse F765-2-3 showed a tissue distribution of MT-I mRNA
much like that of the control mouse with one exception;
the amount of liver MT-I mRNA was substantially reduced.
In the transgenic mouse, the MT-GRF fusion mRNA
is expressed at high levels in the liver, gut and
pancreas, at low levels in kidney and spleen, and is not
detected in the brain. Although the expression of the
fusion gene in this animal demonstrates a tissue
specificity similar to that of the mouse MT-I gene,
there are clear and reproducible differences, most
noticeably the low level of expression of MT-GRF mRNA in
the kidney. The pattern of MT-GRF gene expression has
recently been examined in a second pedigree (N801-5-9),
and it is found that, although the general pattern of
tissue specificity parallels that of mouse MT-I, there
are subtle differences particular to this animal and
distinct from the MT-GRF animal F765-2-3. Similar
results were obtained on examination of the tissue
specificity of metallothionein/growth hormone fusion
genes (R. D. Palmiter et al., Science 222, 809-814
(1983)). In this case, an extensive survey of multiple
animals showed that expression varied among different
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tissues and different animals, suggesting that factors,
such as the site of integration, probably influence
expression of the exogenous gene.
Although many tissues apparently express the
MT-GRF mRNA and presumably synthesize the GRF precursor
protein, it is not known which tissues are capable of
proteolytically processing this precursor to generate
the mature GRF hormoneO The antibody used to detect
plasma GRF would detect both the precursor and the
mature peptide and cannot be used to distinguish the
mature peptide from the precursor. Such processing must
occur to some extent, as the free N-terminus of the GRF
peptide is known to be required for biological activity
(J. Rivier, et al., Nature 300, 276-278 (1982) R.
Guillemin et al., Science 218, 585-587 (1982)).
The generation of transgenic mice which develop
from microinjected eggs and carry the MT-GRF fusion gene
have elevated levels of plasma growth hormone and grow
at a rate of 25-50% greater than that of control
littermates; this is significantly less than the
increased growth observed in transgenic mice which
develop from microinjected eggs and express growth
hormone, many of which grow to twice the normal size (R.
D. Palmiter et al., Nature 300, 611-615 (1982); R. D.
Palmiter et al., Science 222, 809-814 (1983)). However,
in the transgenic animals expressing growth hormone,
most body tissues express the gene and have the
potential to make growth hormone. In the experiments in
which animals carry exogenous GRF gene, growth hormone
is only made in the anterior pituitary and thus may be
rate-limiting.
The first generation of progeny of the
transgenic mice which develop from the microin~ected
eggs tend to grow even larger than their parents, which
may be a artifact of general weakening of the parent
mice caused by the microinjection process, which
weakening is overcome in their progeny. The increased
growth rates of the second and subsequent generations of
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progeny tend to be reflective of the increased growth
rate exhibited in the first generation of progeny.
Transgenic animals containing exogenous GRF
genes tend to vary in size ranging from somewhat larger
than the normal mice to up to about twice as large.
This contrasts with transgenic mice incorporating
exogenous GH gene which tend to generally grow to about
double normal size. Developing transgenic animals
incorporating exogenous GRF gene rather than exogenous
GH gene may therefore be advantageous in allowing a
desired size to be selected for breeding lines
Although it is an object of the invention to produce
faster growing and larger animals, too high growth rate
and/or too large eventual size may prove to be
incompatible with animal health, e.g., placing too large
a burden on the skeletal structure of larger animals.
In such cases, animals exhibiting enhanced growth rate,
but not greatly exaggerated growth rate, may be
selected. Where exaggerated growth proves to be
consistent with animal health and viability, such faster
growing animals may be bred.
An unexpected finding was that females
expressing the MT-GRF fusion gene are generally fertile,
although transgenic female mice expressing the growth
hormone gene are generally infertile (R. E. Hammer et
al., Nature 311, 65-67 (1984)). This suggests that the
enhancement of growth in the MT-GRF females is more
physiological, perhaps because the effect is mediated
through endogenous somatotropes.
The fertility of both sexes of animals
incorporating an exogenous GRF gene is considered highly
important for producing breeds of rapidly growing
transgenic animals. Any economic advantage derived by
elevated growth of an animal overproducing GH may be
negated by the infertility of a substantial portion of
the progeny, particularly a substantial portion of the
female progeny. Transgenic animals incorporating
exogenous GRF genes do not suffer this disadvantage.
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While the invention has been described in terms
of certain preferred embodiments, modifications obvious
to one with ordinary skill in the art may be made
without departing from the scope of the present
inventionO For example, although the invention has been
described in terms of fusion genes that encode human GRF
precursor, there is a substantial degree of homology
between GRF's of different vertebrate species,
particularly different mammalian species and also a
substantial degree of cross-species reactivity. This is
particularly true among mammals, but GRF's can be
cross-reactive between species of different genuses,
e.g., between mammalian and avian species. Similarity
in GRF protein structures between species is considered
important when producing transgenic animals that express
an exogenous protein from a different species, because
it is less likely that autoimmune responses will be
induced in transgenic animals expressing closely similar
exogenous proteins. Accordingly, GRF-encoding sequences
may be obtained from a wide variety of animals.
Similarly, although the invention has been
described in terms of linking the GRF-encoding sequence
to certain strong promoters, such as a promoter of a
metallothionein protein, a variety of promoters may be
used, including weaker promoters. The gene used to
transform the fertilized egg may very well include the
- natural promoter of the GRF precursor protein. Weaker
promoters may be advantageous in some instances,
particularly if transgenic animals having GRF-encoding
sequences linked to strong promoters should grow at
excessively elevated rates or to excessively elevated
size.
; Various features of the invention are emphasized
in the following claims.
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