Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FOR GENE TARGETING
1_e RELATED APPLICATION DATA.
This application claims the benefit of U.S. Provisional Application No.
60/888,529 filed on February 6, 2007, which is incorporated herein by
reference.
20 FIELD OF THE INVENTION
The present invention is related to the field of molecular biology, and
provides
methods for disrupting and modifying genes.
3. BACKGROUND
Two methods are commonly used to disrupt or "knock out" a gene in a cell:
homologous
recombination and gene trapping. Homologous recombination is usually performed
by
creating a construct which is derived from the gene in vitro using standard
recombinant
techniques. The construct is introduced into the cell by transfection,
transformation, etc.
At some frequency, the cellular machinery recombines the introduced construct
with
homologous sequences in the chromosome thereby disrupting the gene. Various
selection methods may be utilized to select or screen cells for the rare
recombination
event (Capecchi, M.R., Science, 244:1288-1292, 1989; Capecchi, M.R. et al.,
U.S. Pat.
No. 5,464,764). Typically, one gene at a time may be disrupted by homologous
recombination.
Gene trapping involves the nonspecific insertion of DNA (an insertion
element), which
carries a selectable marker, into a chromosome. If the DNA is inserted into a
gene, the
gene may be disrupted. Subsequent steps in the protocol entail analysis of the
insertion
site to determine if a gene of interest has been disrupted. Typically, many
cells
containing independent insertions will be analyzed to produce a large
collection of gene
knock-outs. The selectable marker is frequently introduced by an engineered
retrovirus
or transposon. Various selections may be employed to enrich for insertions
into genes,
e.g. promoter trapping, poly-A trapping, etc. (Zambrowicz, B. et al., U.S.
Pat. No.
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6,080,576; Tessier-Lavigne, M. et al., U.S. Pat. No. 6,248,934; Ishida Y. et
al, Nucleic
Acids Res., 27:e35, 1999; Durick, K. et al., Genome Res., 9:1019-1025, 2007).
Homologous recombination is a technique more suited to analyzing a small
number of
specific genes because of the upfront labor required to create gene specific
constructs and
the subsequent labor necessary to isolate the cells that have undergone the
correct
recombination event. Gene trapping is more suited to analyzing larger numbers
of genes
that are not determined at the outset. The randomness of the integration
process limits
the likelihood that any one gene will be disrupted unless a very large number
of
integration events are examined. The amount of effort required to disrupt all
the genes in
a cell or organism is so prohibitive at present that only a consortium of well
funded
scientists would undertake the task.
What is needed in the art is a general method for disrupting genes in a cell
that is simple
and inexpensive enough to target specific genes and reduce the cost and effort
to disrupt
all or a large number of genes in a cell or organism. The instant invention
describes such
a method.
4. 13111EF 1)ESC .IP"1 IO I) F 11i {, ICI RE S
FIG. 1 is a drawing of a preferred embodiment of a gene trap vector and the
integration of the vector into a gene.
FIG. 2 is a drawing of a preferred embodiment of a construct for targeting a
gene
by homologous recombination and the resulting recombination product.
5. SI.`MMARY
It is an object of the invention to provide methods for gene targeting. The
invention provides methods for generating and characterizing gene targeting
events by
using tags. More specifically, the method employs RNAi and other tag-specific
selections to enrich for cells that have undergone the desired targeting
event.
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6. I)F-' 'AIL!<'D fSCI II ' 'ION OF PREFE-RIRED EMBODIMENTS
Strathmann previously described a method for generating a collection of cells
wherein
many of the cells contain tagged insertion elements (Strathmann, M., U.S. Pat.
No.
6,480,791, which is hereby incorporated in its entirety). The tag is a stretch
of sequence
within the insertion element that is unique to that cell or more accurately
that clonal
population of cells (cell clone) within the collection. A collection of cell
clones is
generated for example by randomly inserting the tagged insertion elements into
the
genome so that usually any one cell (or organism) preferably will have
undergone only
one integration event. These cells can be spatially separated. For example,
mammalian
cells can be infected with a collection of tagged retroviral vectors. Each
vector may
contain a reporter gene GFP). The transfected cells (that is, the cells that
express
the reporter gene) can be spatially separated from each other and from
uninfected cells by
flow cytometry and cell sorting (see Galbraith, D.W. et al., Methods Cell
Biol., 58:315-
41, 1999), or by other means. Though this example is directed towards random
integration events, the method is equally applicable to "targeted" integration
events. For
example, insertion elements have been described that target the integration
events to
genes by providing selectable markers that lack promoters, or must be properly
spliced to
function, etc. (g Sedivy, J.M. et al., Proc. Natl. Acad. Sci. USA, 86:227-31,
1989;
Friedrich, G. et al., Genes Dev., 5:1513-23, 1991; Skarnes, W.C. et al., Proc.
Natl. Acad.
Sci. USA, 92:6592-6, 1995; Ruley, H.E. et al., U.S. Pat. No. 5,627,058, 1997;
Sands, A.
et al., PCT Pat. Pub. No. WO 98/14614, 1998).
The location of a tagged insertion element in the genome can be determined by
rescuing
one or both junctions (i.e. the genomic DNA that flanks the insertion element)
along with
the tag (the tagged junctions) by methods well known in the art (see for
example
Strathmann, M., U.S. Pat. No. 6,480,791, and references therein). By
sequencing the
rescued tag and junctions, it is possible to establish the identity of the tag
and the location
of the associated insertion element within the genome. cDNA may also be used
to rescue
some junctions, but if the insertion element resides within an intron, then
the precise
location may not be evident from cDNA sequence due to splicing. Enormous
economies
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of scale may be achieved by rescuing all the tagged junctions from a pooled
collection of
cells containing tagged insertion elements at different positions. The
sequence of all the
tags and their associated junctions may be determined simultaneously using a
massively
parallel sequencing platform such as 454 Life Sciences, Solexa, etc.,
(Margulies, M. et
al., Nature, 437:376-80, 2005). For example, one or a small number of
sequencing
primers that hybridize to a common region in the insertion element may be used
with the
454 Life Sciences' machine to sequence through the tags into the genomic DNA
at the
junctions. Consider, for example, a typical retrovirus vector (e.g. RET,
Ishida Y. et al,
Nucleic Acids Res., 27:e35, 1999; Shigeoka T. et al., Nucleic Acids Res.
33:e20, 2005)
that is used in gene trap experiments. A large collection of tagged vectors
can be easily
created using standard recombinant techniques by ligating a large collection
of
oligonucleotides of random sequence (e.g. 25-mers) into a restriction site
within the
vector so that any one vector incorporates one oligonucleotide. The sequence
of this
oligonucleotide is the tag sequence. This collection of tagged vectors may be
transfected
en masse into a packaging cell line to produce virus particles. The virus
particles are
combined with a cell line such that many cells are infected with one or no
virus particles.
Cells harboring an integrated provirus are selected using standard procedures.
The cells
may be clonally expanded, for example in individual wells of a microtiter
plate, or the
entire population may be maintained as a single culture. In this way, a
collection of cells
comprising tagged insertion elements is created. If the cells are maintained
in individual
wells, the cells or nucleic acid from the cells may be pooled prior to
rescuing the
junctions. Obviously, this step is not needed if the cells are maintained as a
single
culture. The rescued collection of tagged junctions may then be subjected to
massively
parallel sequencing to determine the identity of the tag and the location of
the tagged
insertion element. Some of the insertion elements will reside within genes in
such a way
that the function of the genes are disrupted.
Some methods for massively-parallel sequencing do not produce very long
sequencing
reads (e.g. the Genome Sequencer FLX from 454 Life Sciences and Roche Applied
Science has an average read length between 200 and 300 bases, while Solexa's
instrument can read 30-35 bases). If reads are short and a retroviral vector
is utilized to
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introduce the tagged marker, it is possible to use a poly-A trap vector and
sequence
cDNA as long as the tag is positioned just upstream of the splice-donor site.
In this way,
the tag will be positioned very near the endogenous splice-acceptor and any
intervening
retroviral sequences will be removed as part of the intron. Alternatively, the
tag may be
positioned near a restriction site and junctions may be rescued from genomic
DNA by
circularizing the DNA, which joins this restriction site to genomic DNA at
some distance
from the retroviral LTR (long terminal repeat). This method is analogous to
paired-end
sequencing protocols that have been developed by the instrument makers (see
for
example Korbel, J.O., Science 318:420-426, 2007). The Genome Sequencer FLX may
be
capable of sequencing junctions rescued from genomic DNA with a first
sequencing
primer in the retroviral LTR and then sequencing through the tag with a second
primer.
The second sequence may be determined after denaturing and removing the first
sequencing product or by simply terminating the first sequencing product with
a dideoxy
nucleotide prior to annealing the second primer to initiate the sequencing
reaction from a
second site.
While there is utility in having a collection of cells comprising tagged
insertion elements
in known locations (see for example, Mazurkiewicz P. et al., Nat. Rev. Genet.
7:929-39,
2006; Smith, V. et al, Proc. Natl. Acad. Sci USA, 92:6479-83, 1995; Ross-
Macdonald, P.
et al, Nature 402:362-3, 1999; Chun, K.T. et al., Yeast 13:233-40, 1997), a
great deal
more information can be learned by isolating a cell clone comprising one
tagged insertion
element (or a small number). If the population of cells described above was
originally
stored as cell clones in separate wells of a microtiter plate then each tag
can be associated
with a cell clone. One method for rapidly determining these associations
involves a sub-
pooling strategy, amplification of the tags and hybridization to an array of
oligonucleotides that are complementary to the tags (see Strathmann, M., U.S.
Pat. No.
6,480,791 for a complete description). If the collection of cells is
maintained as a single
culture, another method is needed to isolate from the population the specific
cell clone
comprising the tagged-insertion element of interest.
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A general method for selecting or enriching for a cell clone comprising a
tagged insertion
element (or any tagged component) exploits the mechanism of RNA Interference
(Tijsterman M. et al., Annu. Rev. Genet. 36:489-519, 2002) to degrade a
transcript which
contains the tag sequence. Consider a tagged insertion element that comprises
a
selectable marker (for example, HSV thymidine kinase, gpt, etc., see Karreman,
Nucleic
Acids Res. 10:2508-2510, 1998) such that the transcript for the selectable
marker
contains the tag sequence. Such a marker is defined as a "tagged marker". For
illustrative purposes, it is simplest to think of the product of the tagged
marker as a
protein that confers a simple property on the cell, such as resistance to a
chemical
compound. One skilled in the art will recognize the product of the tagged
marker may be
for example, a subunit of a larger protein or may not be a protein at all,
rather the product
may be for example a nucleic acid that confers a selectable property on the
cell. The
tagged insertion element is easily constructed using standard recombinant
techniques to
place the tag, for example between a promoter and the coding sequence of the
marker (5'-
untranslated) or downstream of the coding sequence but before termination
signals (3'-
untranslated). This transcript will be degraded by introducing into the cell
siRNA
molecules that target the tag sequence. In other words, siRNA specific to the
tag will
downregulate the selectable marker in the cell. If loss of the marker confers
a selectable
phenotype on the cell, then only those cells that no longer produce the marker
will
survive. Starting with a population of cells, wherein each cell expresses a
tagged marker,
one can select or enrich for cells carrying a specific tagged marker by
introducing into the
cells siRNA directed to that one tag followed by the appropriate negative
selection.
Depending on the cell type, one may also introduce double-stranded RNA, short-
hairpin
RNA (shRNA), DNA vectors that result in sequence-specific (i.e. tag-specific)
RNA
inhibition, etc. Other examples of sequence-specific RNA inhibition include
antisense
oligonucleotides, microRNA (miRNA), ribozymes, etc. In fact, any tag-specific
means to
prevent or inhibit production of the tagged marker is suited to the selection
scheme
outlined above. Suitable markers include gpt, HSV-tk, etc (Karreman, Nucleic
Acids
Res. 10:2508-2510, 1998).
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It is preferable to ensure the starting population of cells all express the
marker gene
therefore a preferred marker will also confer a positive advantage to the
cells under
different selective conditions (see for example, Karreman, Nucleic Acids Res.
10:2508-
2510, 1998; Besnard, C. et al., Mol. Cell. Biol. 7:4139-4142, 1987; Wei, K. et
al. J. Biol.
Chem. 271:3812-3816, 1996). In this way, one can select for the marker
(positive
selection) before inducing RNA inhibition and selecting against the marker
(negative
selection). The result is a lower "background" of cells that survive the
negative selection
for reasons other than RNA inhibition. For example, a population of cells
carrying a
tagged gpt marker may be grown in the presence of HAT medium. After
transfecting
siRNA to one tag (or more), the growth media is changed to remove HAT and add
6-
thioxanthine so only those cells that do not express gpt will survive
(Besnard, C. et al.,
Mol. Cell. Biol. 7:4139-4142, 1987). Of course it may be adequate, for
example, to
introduce a second, different marker gene along with the tagged marker in the
insertion
element. In this way, a positive selection may be applied through the second
marker
while the negative selection is applied through the tagged marker. The second
marker
could be driven by a second promoter or it could be driven by the same
promoter as the
first marker to form a polycistronic transcript by, for example, introducing
an internal
ribosome entry site (IRES). In the case of a polycistronic transcript, both
markers are
subject to downregulation by the introduction of an siRNA to the tag sequence.
Again,
the goal is to reduce the "background" cells that survive the negative
selection through
means other than RNA inhibition.
It will be obvious to one skilled in the art that there are many variations of
the RNAi
selection for tagged markers described above. For example, the loss of the
marker
transcript need only produce a phenotype or characteristic that is
distinguishable in some
way from expression of the transcript. For example, the marker could be GFP
(green
fluorescent protein) and cells are sorted by FACS (fluorescence-activated cell
sorting) to
separate those cells that no longer fluoresce. The marker could be a
transcription factor
that inhibits expression of a cell surface antigen. Loss of the marker leads
to expression
of the surface antigen which allows isolation of cells by for example FACS,
panning with
antibodies to the surface antigen, etc.
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More generally, one skilled in the art will recognize any means to modulate
production of
the tagged marker that depends on the sequence of the tag may be used to
select from a
population of cells comprising different tags those cells comprising a
specific tag. For
example, any tag-specific means to induce production of the tagged marker may
be
employed to select for the presence of the tagged marker. For example, triplex
forming
oligonucleotides, engineered zinc-finger binding proteins, etc. may be used as
engineered
transcription factors to modulate gene expression in a sequence (e.g. tag)
specific manner
(Visser, A.E. et al, Adv. Genet. 56:131-161, 2006; Gommans, W.M. et al, J.
Mol. Biol.
354:507-519, 2005). In this context, the "tagged marker" comprises a marker
and a tag
that are not necessarily present on the same transcript. Rather, the tag is
functionally
linked to the transcript comprising the marker by the means to modulate
production of the
marker. It will be obvious to one skilled in the art how to tag a marker to
make a tagged
marker given the means to modulate the marker. Note, the term marker can be
used to
denote a gene or the product of that gene and the meaning is obvious to the
skilled artisan
from the context. By definition, a "tag-specific selection" refers to a means
for
modulating the activity of a tagged marker based on the sequence of the tag so
that if the
sequence of a second tag is substantially different, the tagged markers
comprising the
second tag will not be so modulated. The degree to which the sequence of two
tags must
differ depends on the means for modulating the activity of the tagged marker
and is
obvious to one skilled in the art. Examples of tag-specific selections include
RNAi,
miRNA, antisense oligonucleotides, ribozymes, etc.
The examples above describe an RNAi selection method wherein both the tag and
the
marker are introduced to the cell by some means such as, for example,
transfection,
transformation, infection, etc. A similar method may be applied to a
population of cells
in which only the marker is introduced exogenously. In the latter case, the
tag is a
genomic tag as described in U.S. Pat. No. 6,480,791. The genomic tag is
determined by
proximity to the insertion element in the genome. For example, standard gene-
trap
vectors can be used to produce a population of cells with random or quasi-
random
integration sites in genes throughout the genome. The vectors result in fusion
transcripts
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between sequences present in the genome and a marker gene present in the
vector. To
select for cells in which a specific gene is "trapped", one need only induce
RNA
interference to that specific gene followed by selection for loss of the
marker. Depending
on the cell type, RNA interference may be induced by introducing to the cells
siRNA to
the gene of interest, double-stranded cRNA to the gene, shRNA, etc. The
specific gene
will be downregulated by RNA interference (if it is expressed) but so too will
be the gene
fusion transcript. Loss of the fusion transcript results in loss of the marker
which in turn
allows the cell to survive the selection.
The RNAi selection methodology described above may also be used to select or
enrich
for homologous recombination between an exogenous construct and its homologous
site
in the genome. Typically, to perform gene targeting by homologous
recombination a
marker is ligated into genomic sequences in vitro by standard recombinant
techniques.
The resulting construct is introduced into cells followed by selection for the
presence of
the marker. In many cell types, the frequency of random integration of the
construct in
the genome is much greater than the frequency of homologous recombination. In
a
manner analogous to that described above for trapped genes, one can select or
enrich for
homologous recombination events by directing RNA interference to the gene
designed to
undergo targeting by homologous recombination. The targeting construct should
be
designed to produce a transcript that encodes the marker and carries
additional sequence
from the gene of interest. The additional sequence should not be part of the
construct,
rather it is incorporated when the construct undergoes homologous
recombination. For
example, in vertebrates the marker may be designed like the marker in a poly-A
trap
vector, which has a splice donor downstream of the marker. The targeting
construct will
have genomic sequence on either side of the marker to allow recombination with
the
endogenous gene. Transcription of the marker leads to splicing with downstream
exons
that by design are not part of the targeting construct. RNA interference may
then be
targeted to the downstream exon sequences (i.e. a downstream exon comprises
the
genomic tag). If the construct integrates randomly, then downstream sequences
will not
be present in the transcript which encodes the marker. Consequently, the
marker will not
be downregulated by RNA interference and selection against the marker will for
example
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kill the cell. Only a construct which has integrated into the genome in an
orientation that
yields a fusion transcript between the marker and the downstream sequences
will be
subject to RNA interference. Only this orientation of the integrated construct
will permit
the cell for example to survive under the negative selection conditions. This
orientation
is most likely to occur as the result of homologous recombination. Therefore,
by
choosing the appropriate marker gene (e.g. gpt, HSV-tk, etc.) and using the
RNAi
selection scheme one can select or enrich for homologous recombination events.
In some
cell types, it may be necessary to include only intron sequences in the
targeting construct
or design the fusion transcript to include upstream exons to target for RNAi
so that the
effects of transitive RNAi on randomly integrated constructs may be avoided
(Sijen, T. et
al., Cell, 107:465-476, 2001).
The RNAi selection scheme as practiced with a tagged marker is a general
method for
selecting or enriching for a specific tagged cell from a population of tagged
cells. This
scheme can have utility in addition to the gene targeting applications
described above.
For instance, a certain property or phenotype may vary among tagged cells.
This
variation may be monitored in the population under some set of experimental
conditions
which could lead to the identification of only a small subset of tagged cells
of interest.
This subset may be quickly isolated from the population by applying the RNAi
selection
scheme. For example, a large collection of tagged cells are created as
described above by
using tagged insertion elements. The insertion elements are designed to
integrate at only
one location in the genome by using for example site-specific recombination.
The
population of cells is subjected to chemical mutagenesis so that a small
number of
mutations are introduced at random in each tagged cell. The population is
exposed to a
drug for some period of time and hypersensitivity to the drug is investigated.
By using
microarrays comprising oligonucleotides complementary to the tags, it is
possible to
monitor the loss from the population of certain tagged cells as described by
Mazurkiewicz et al. (Nat. Rev. Genet. 7:929-39, 2006). The cells which show
hypersensitivity may be isolated from the untreated population of cells using
the RNAi
selection scheme (i.e. siRNA directed to the appropriate tag) and investigated
further.
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70 EXAMPLES
Example 1: Gene Trapping - Identification and Isolation of a Specific Clone
A collection of gene trap vectors is made by standard recombinant techniques
as shown
in Figure 1. The vector backbone is a 3' gene trap (i.e. polyA trap) vector
described in
U.S. Pat. No. 6,080,576. The selectable marker is gpt (xanthine-guanine
phosphoribosyl
transferase). The presence of gpt can be both selected for and against (see
U.S. Pat. No.
6,689,610 for selective agents and preferred concentrations). The vectors are
identical
except for a 25 basepair sequence indicated as the "tag" in the figure. The
tags are first
synthesized as effectively random sequences and then ligated into a
restriction site in the
parent vector, which lacks a tag, to generate the collection of vectors.
This collection of vectors is then packaged into retroviral particles by
standard means as
described in U.S. Pat. No. 6,080,576 (see also Viral Vectors for Gene Therapy:
Methods
and Protocols Ed. Machida, C.A., Humana Press, New Jersey (2003); Gene
Delivery to
Mammalian Cells: Volume 2: Viral Gene Transfer Techniques Ed. Heiser, W.C.,
Humana
Press, New Jersey (2004); The Centre for Modeling Human Disease Gene Trap
resource,
http://www.cmhd.ca/genetrap/protocols.htmi). Supernatant from the packaging
cells is
added to embryonic stem cells for 16 hours and the cells are grown in the
presence of gpt
selection reagent (Millipore, Billerica, MA) according to the manufacturer's
instructions
(see also U.S. Pat. No. 5,627,033) for 10 days. Surviving cells (i.e. those
cells expressing
gpt) are isolated into 100 pools of about 1000 distinct clones per pool. Each
pool is
grown up and subjected to automated RNA isolation and reverse transcription by
standard protocols (see U.S. Pat. No. 6,080,576) to make cDNA. cDNA from each
pool
is combined to make a single pool of cDNA products from about 100,000 distinct
clones.
The tags are PCR amplified from the single pool of cDNA products using a 3'-
RACE
protocol. Two rounds of PCR with nested primers (see pl and p2 in Figure 1)
are
performed as described (ibid).
The amplified 3'-RACE PCR products containing the tags are sequenced using the
Genome Sequencer FLX System instrument sold by Roche (Indianapolis, IN) using
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protocols supplied by the manufacturer. The sequence information indicates
where the
gene trap vector has inserted in the genome and the unique tag associated with
each
insertion site.
A specific cell clone is isolated using the unique tag (Tag l) associated with
the clone.
First, a PCR primer is designed to hybridize to Tagl in the orientation shown
in Figure 1
(see pTl). PCR is performed with pTl and the gene specific primer, pGl (see
Figure 1),
on the cDNA isolated from each of the 100 pools of clones described above. The
presence of an amplification product identifies the pool to which the specific
cell clone
belongs.
The specific clone is isolated from the identified pool of about 1000 clones
by using the
RNAi selection method. An siRNA targeted to the tag sequence (siRNA-T, in
Figure 1)
is synthesized (Qiagen, Valencia, CA) and introduced by transfection into the
appropriate
pool of 1000 clones using the HiPerFect Transfection Reagent (Qiagen,
Valencia, CA)
according to the manufacturer's instructions. After 2 days the cells are again
transfected
with siRNA as above and transferred to fresh media supplemented with 100 .tM 6-
thioxanthine to select for the loss of gpt. After three days, the surviving
cells are
transferred to fresh media and grown in the absence of selective pressure for
three days.
Finally the cells are transferred to media supplemented with gpt selection
reagent to
eliminate any cells that survived 6-thioxanthine treatment by losing the gpt
gene (by for
example chromosome loss or mutation of the gene). The resulting cells are
highly
enriched for the cell clone carrying the specific tag, Tag I.
Alternatively, the RNAi selection procedure as described above is performed
with siRNA
targeted to the gene in which the gene trap vector resides. In this case, a
genomic tag is
utilized for the procedure and siRNA-G (see Figure 1) is introduced into the
pooled cells
by transfection.
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Example 2: Homologous Recombination - Selection for the Correct Recombinant
Events
Capecchi and Thomas describe the disruption of the INT-2 gene in mouse ES
cells by
homologous recombination with an introduced construct (U.S. Pat No.
5,464,764). The
construct shown in Figure 2 is made using standard recombinant techniques. The
construct contains the gpt gene from Example 1 above flanked on both sides by
sequences derived from the INT-2 gene so that the last exon (30 in Figure 2)
is
truncated(see Example 1 and Figures 5A, 5B & 5C in U.S. Pat No. 5,464,764; and
Mansour, S.L. & Martin, G.R., EMBO, 7:2035-2041, 1988). The purified construct
is
introduced into mouse ES cells by transfection as described (see U.S. Pat No.
5,464,764).
The transfected cells are grown in the presence gpt selection reagent as
described above
to select for the expression of gpt. Most of the surviving cells are due to
random
integration of the construct into the genome. Only a small percentage of the
cells have
incorporated gpt by homologous recombination with the endogenous INT-2 gene.
These
rare recombination events are selected using the RNAi selection scheme
described above.
The siRNA shown in Figure 2, siRNA-INT, is designed to target a portion of the
INT-2
transcript that is not present in the construct shown in Figure 2. This siRNA
is
introduced by transfection and cells are selected for the loss of gpt function
as described
above in Example 1. After this negative selection is performed, the surviving
cells are
again subjected to a positive selection for gpt function also described in
Example 1. The
cells that survive this procedure are highly enriched for the integration of
the construct
into the INT-2 gene by homologous recombination.
INCORPORATION BY REFERENCE
The contents of all cited references (including literature references,
patents, and
patent applications) that may be cited throughout this application are hereby
expressly
incorporated by reference.
9. EQUIVALENTS
The invention may be embodied in other specific forms without departing from
the spirit
or essential characteristics thereof. The foregoing embodiments are therefore
to be
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considered in all respects illustrative rather than limiting of the invention
described
herein. Scope of the invention is thus indicated by the appended claims rather
than by the
foregoing description, and all changes that come within the meaning and range
of
equivalency of the claims are therefore intended to be embraced herein.
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