U.S. patent application number 10/209615 was filed with the patent office on 2003-05-01 for method for chromosomal rearrangement by consecutive gene targeting of two recombination substrates to the deletion endpoints.
This patent application is currently assigned to BAYLOR. Invention is credited to Bradley, Allan, Liu, Pentao, Ramirez-Solis, Ramiro, Su, Hong, Zheng, Binhai.
Application Number | 20030084467 10/209615 |
Document ID | / |
Family ID | 21799651 |
Filed Date | 2003-05-01 |
United States Patent
Application |
20030084467 |
Kind Code |
A1 |
Bradley, Allan ; et
al. |
May 1, 2003 |
Method for chromosomal rearrangement by consecutive gene targeting
of two recombination substrates to the deletion endpoints
Abstract
The present invention involves the creation of defined
chromosomal deficiencies, inversions and duplications using Cre
recombinase in ES cells transmitted into the mouse germ line. These
chromosomal reconstructions can extend up to 3-4 cM. Chromosomal
rearrangements are the major cause of inherited human disease and
fetal loss. Additionally, translocations and deletions are
recognized as major genetic changes that are causally involved in
neoplasia. Chromosomal variants such as deletions and inversions
are exploited commonly as genetic tools in organisms such as
Drosophila. Mice with defined regions of segmental haploidy are
useful for genetic screening and allow accurate models of human
chromosomal disease to be generated.
Inventors: |
Bradley, Allan; (Houston,
TX) ; Ramirez-Solis, Ramiro; (Missouri City, TX)
; Liu, Pentao; (Frederick, MD) ; Su, Hong;
(Houston, TX) ; Zheng, Binhai; (Houston,
TX) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI, LLP
1301 MCKINNEY
SUITE 5100
HOUSTON
TX
77010-3095
US
|
Assignee: |
BAYLOR
Houston
TX
|
Family ID: |
21799651 |
Appl. No.: |
10/209615 |
Filed: |
July 30, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10209615 |
Jul 30, 2002 |
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09552626 |
Apr 19, 2000 |
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6461818 |
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09552626 |
Apr 19, 2000 |
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08883616 |
Jun 26, 1997 |
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6077667 |
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60020620 |
Jun 26, 1996 |
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Current U.S.
Class: |
800/18 ;
800/21 |
Current CPC
Class: |
C12N 2800/30 20130101;
A01K 2217/05 20130101; A01K 2227/105 20130101; A01K 2207/15
20130101; A01K 2217/072 20130101; A01K 2217/075 20130101; C12N
15/102 20130101; C12N 15/902 20130101; A01K 67/0276 20130101; C12N
15/8509 20130101; A01K 67/0275 20130101; A01K 2267/03 20130101;
A01K 2217/00 20130101; A01K 2267/0306 20130101 |
Class at
Publication: |
800/18 ;
800/21 |
International
Class: |
A01K 067/027 |
Goverment Interests
[0001] The present invention was made utilizing funds of the United
States Government. The U.S. Government is entitled to certain
rights under this invention.
Claims
We claim:
21. A method for deleting a selected region of genetic material in
mice comprising the steps of: inserting a first selection cassette
at a 5' end of said selected region using conventional gene
targeting methods, said first selection cassette comprising a first
selectable marker coding sequence, a first loxP recombination site,
and a first portion of a second selectable marker coding sequence;
selecting embryonic stem cells expressing said first selectable
marker coding sequence; inserting a second selection cassette at a
3' end of said selected region using conventional gene targeting
methods, said second selection cassette comprising a third
selectable marker coding sequence, a second loxP recombination
site, and a remaining portion of said second selectable marker
coding sequence; selecting embryonic stem cells expressing said
third selectable marker coding sequence; expressing Cre recombinase
to produce recombination between said first and second loxP sites;
selecting embryonic stem cells expressing said second selectable
marker coding sequence, wherein the selected region of genetic
material is deleted; injecting selected embryonic stem cells
expressing said second marker into a recipient blastocyst; and
implanting said blastocyst into a foster mother.
22. The method of claim 21 wherein said first selectable marker
coding sequence is a puromycin resistance gene, said second
selectable marker coding sequence is an Hprt gene, and said third
selectable marker coding sequence is a neomycin resistance
gene.
23. A method for creating inversions of a selected region of
genetic material in mice comprising the steps of: inserting a first
selection cassette at a 5' end of said selected region using
conventional gene targeting methods, said first selection cassette
comprising a first selectable marker coding sequence, a first loxP
recombination site, and a first portion of a second selectable
marker coding sequence; selecting embryonic stem cells expressing
said first selectable marker coding sequence; inserting a second
selection cassette at a 3' end of said selected region using
conventional gene targeting methods, said second selection cassette
comprising a third selectable marker coding sequence, a second loxP
recombination site, and a remaining portion of said second
selectable marker coding sequence; selecting embryonic stem cells
expressing said third selectable marker coding sequence; expressing
Cre recombinase to produce recombination between said first and
second loxP sites; selecting embryonic stem cells expressing said
second selectable marker coding sequence, wherein the selected
region of genetic material is inverted; injecting selected
embryonic stem cells expressing said second marker into a recipient
blastocyst; and implanting said blastocyst into a foster
mother.
24. A method for creating a defined chromosomal deficiency,
deletion, inversion or duplication in mice comprising the steps of:
identifying a desired region of a chromosome of interest to be
targeted; inserting two native sequences at each endpoint of said
region of said chromosome of interest using a first and a second
targeting vector, each comprised of one or more selectable marker
coding sequences and a loxP site and an hprt fragment coding
sequence; transiently expressing Cre recombinase to produce
recombination between each of two said loxP sites; whereby upon
chromosomal rearrangement induced by said Cre recombinase, a
functional Hprt expression cassette is reconstructed; selecting
embryonic stem cells expressing Hprt, wherein the region of said
chromosome of interest is deficient, deleted, inverted, or
duplicated; injecting selected embryonic stem cells expressing Hprt
into a recipient blastocyst; and implanting said blastocyst into a
foster mother.
25. A transgenic mouse produced by the method of claim 23.
Description
BACKGROUND OF THE INVENTION
[0002] 1. Technical Field of the Invention
[0003] The present invention involves the creation of defined
chromosomal deficiencies, inversions and duplications using Cre
recombinase in embryonic stem cells and transmitted into the mouse
germ line. In the present invention, these chromosomal
reconstructions can extend up to 3-4 cM. Chromosomal rearrangements
are the major cause of inherited human disease and fetal loss.
Further, chromosomal translocations and deletions are recognized as
major genetic changes that are causally involved in neoplasia.
Chromosomal variants such as deletions and inversions are exploited
commonly as genetic tools in diploid organisms such as Drosophila.
In diploid organisms, such deficiencies are exploited in genetic
screens because a small portion of the genome is functionally
hemizygous. Thus, a mutation which would normally be recessive and
masked by the wildtype allele in a diploid context will be dominant
and detectable in the haploid state. In the mouse, deficiencies
have not, up to now, been available generally; thus, screens for
recessive mutations are nonexistent or particularly cumbersome.
However, the present invention provides methods to engineer mice
and cell lines with defined regions of segmental haploidy. Such
mice are useful for genetic screening and provide accurate models
of human chromosomal diseases.
[0004] 2. The Prior Art
[0005] Inherited chromosomal rearrangements such as inversions,
duplications and deficiencies are responsible for a significant
fraction of human congenital disease. Chromosomal changes also
occur somatically and are associated with neoplastic disease.
Defining the causal genetic alteration in a region of the genome
associated with chromosomal rearrangements can be relatively
straightforward if the affected gene lies in the breakpoint of an
inversion or translocation. However, in cases of duplications and
deficiencies, the specific genetic lesion(s) associated with
pathological chromosomal changes are much harder to identify.
Still, the generation of animal models that accurately recapitulate
the genetic lesion would facilitate the study of disease and could
be very helpful in the efforts to dissect specific gene-function
relationships in multigene syndromes.
[0006] In diploid organisms such as Drosophila, chromosomal
deficiencies are commonly exploited in genetic screens because a
small portion of the genome is functionally hemizygous. Thus, a
mutation which would be recessive and masked by the wildtype allele
in the diploid context will be dominant and therefore readily
detectable in the haploid state. In the mouse, deficiencies are not
available generally. Despite the limited number of deficiencies
available in the mouse, the potential for the detailed analysis of
a genetic interval using these deficiencies has been demonstrated
clearly. See Holdener-Kenny, et al., BioEssays, 14:831-39 (1992),
which is hereby incorporated by reference.
[0007] Deficiencies that are available currently in the mouse
genome were generated at random using ionizing irradiation.
Although conventional gene targeting technology in embryonic stem
(ES) cells can generate virtually any type of mutation, including
deletions of up to 20 kb, it has not been possible to delete
substantially larger fragments by using standard methodology.
Likewise, the technology required to construct large inversions and
duplications has not been established.
[0008] One mechanism by which chromosomes may be engineered is by
the use of Cre recombinase. Cre recombinase has been used in
mammalian cell lines and in vivo to delete or invert sequences
between the 34 base pair recognition sequences, loxP sites, placed
a few kb apart on the same chromosome. The recombination is
initiated by Cre proteins which bind to 13-bp inverted regions in
the loxP sites and promote synapses or joining of a pair of sites.
Next, the Cre proteins catalyze strand exchange between the pair of
sites within an asymmetric 8-bp central spacer sequence by
concerted cleavage and rejoining reactions, involving a transient
DNA-protein covalent linkage. Smith, et al., Nature Genetics,
9:376-385 (1995); Gu, et al., Science, 265:103-06 (1994) and Sauer,
Nucl. Acids Res., 17:147-61 (1989) (both of these references are
hereby incorporated by reference). Additionally, recombinases have
been used to induce mitotic recombination between homologous and
non-homologous chromosomes in Drosophila, plants and mammalian
cells. Embryonic stem cell technology has become a powerful tool
for defining the function of mammalian genes, but mainly has been
restricted to the mutation of single genes. Replacement vectors
have been used to construct deletions of up to 19 kb; however,
utilizing the same strategy to construct larger deletions (>60
kb) has not been successful. In the present invention, the
generation and direct selection of deletions, duplications and
inversions, ranging from 90 kb to 3-4 cM, in ES cells is
demonstrated.
SUMMARY OF THE INVENTION
[0009] The method of the present invention is based on consecutive
gene targeting of two recombination substrates to the deletion
endpoints and the subsequent induction of recombination mediated by
the Cre recombinase. This method generates a positive selectable
marker allowing for the direct selection of clones with the desired
chromosome structures. Despite the multitude of steps involved in
generating these rearrangements in ES cells, deletion and
duplication alleles have been transmitted into the mouse
genome.
[0010] One object of the present invention is a method for causing
a large-scale chromosomal rearrangement by first deleting a portion
of genetic material.
[0011] An additional object of the present invention is a targeting
vector system capable of inserting into two endpoint regions
constraining a desired chromosomal deletion.
[0012] Thus in accomplishing the foregoing objects, there is
provided in accordance with one aspect of the present invention a
method for deleting a selected region of genetic material in cells
comprising the steps of: inserting a first selection cassette at a
5' end of said selected region using conventional gene targeting
methods, said first selection cassette comprising a first
selectable marker, a first loxP recombination site, and a first
portion of a second selectable marker; selecting cells expressing
said first selectable marker; inserting a second selection cassette
at a 3' end of said selected region using conventional gene
targeting methods, said second selection cassette comprising a
third selectable marker, a second loxP recombination site, and a
remaining portion of said second selectable marker; selecting cells
expressing said third selectable marker; expressing Cre recombinase
to produce recombination between said first and second loxP sites;
and selecting cells expressing said second selectable marker.
[0013] Specific embodiments of the above method can include a
puromycin resistance gene as the first selectable marker, a
functional Hprt gene as the second selectable marker, and a
neomycin resistance gene as the third selectable marker. Numerous
other selectable markers will work, their presence in the
particular deletion strategy is merely to aid cell selection. In
other preferred embodiments, the first selectable marker is a
puromycin resistance gene. In still other preferred embodiments,
the second selectable marker is a functional Hprt gene. And in
still other preferred embodiments, the third selectable marker is a
neomycin resistance gene.
[0014] In still other preferred embodiments, the cells referred to
above are embryonic stem cells, though significant, they need not
be stem cells. In other preferred embodiments, the cells are
embryonic stem cells, and said cells develop into mice. And in yet
other preferred embodiments, the cells are embryonic stem cells,
and said cells are maintained as cell lines.
[0015] In yet another preferred embodiment, a viral vector is used
to replace either or both native sequences of DNA. In one
embodiment, this virus is a retrovirus. In yet another embodiment,
the viral vector referred to above has a provirus structure
comprising a cassette in turn comprising: an hprt.DELTA.5'
cassette, a loxP site, and a puromycin resistance gene.
[0016] In yet another particularly preferred embodiment, the method
for deleting a portion of chromosomal material in cells wherein the
targeting vectors are a first targeting vector for replacing said
first native sequence of DNA at said 5' end, comprising: a genomic
insert cloned into the vector of about 7.5 kb; a tyrosinase
minigene; a Neo.sup.r gene; a 5' hprt fragment; and a loxP site
embedded into said hprt fragment; and a second targeting vector for
replacing said second native sequence of DNA at said 3' end,
comprising: a genomic insert cloned into the vector of about 8.5
kb; a K14-Agouti gene; a Puro.sup.r gene; a 3' hprt fragment; and a
loxP site embedded into said hprt fragment.
[0017] In one particularly preferred embodiment of the second
aspect of the present invention, there is provided a replacement
vector system comprising a first targeting vector for replacing
said first native sequence of DNA at said 5' end, comprising a
genomic insert cloned into the vector of about 7.5 kb; a tyrosinase
minigene; a Neo.sup.r gene; a 5' hprt fragment; and a loxP site
embedded into said hprt fragment; and a second targeting vector for
replacing said second native sequence of DNA at said 3' end,
comprising: a genomic insert cloned into the vector of about 8.5
kb; a K14-Agouti gene; a Puro.sup.r gene; a 3' hprt fragment; and a
loxP site embedded into said hprt fragment.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1: 1A: depicts the Hprt-loxP minigene cassette; 1B:
depicts the hprt.DELTA.5' and hprt.DELTA.3' recombination
substrates; 1C: outlines the general strategy for Cre-induced,
targeted genomic rearrangements illustrated at the HoxB
cluster--only the intrachromosomal pathway is shown; 1D:
demonstrates alternative orientations (A or B) of the recombination
substrates at the E2DH and Gastrin loci; 1E: shows chromosomal
alterations induced by Cre recombinase for the different
orientations of the minigenes in cis and trans.
[0019] FIG. 2: Shows the results of a Southern blot analysis of the
chromosomal engineering technology used to delete the HoxB cluster.
A-C demonstrates the interpretation of, and D-F shows the actual
Southern blot data from wildtype (wt), double targeted (dt) or HAT
resistant ES cell clones (lanes 1 and 2). M1 and M2 are HindIII-cut
and BstEII-cut lambda DNA molecular weight markers, respectively.
Hoxb-1 is located in a 7.2 kb NheI fragment detected with probe a
(2A and 2D; this allele is present in all of the lanes). Targeting
of the hprt.DELTA.3'-neo cassette to the Hoxb-1 locus generates a
novel 10.2 kb NheI restriction fragment detected with probe a (2B
and 2D dt). Hoxb-9 is located on a 16 kb NheI restriction fragment
detected with probe b (2A and 2E; this allele is present in all of
the lanes). Targeting of the hprt.DELTA.5'-puro to the Hoxb-9 gene
generates a novel 20.4 kb NheI restriction fragment detected with
probe b (2B and 2E dt). Cre-induced recombination brings together
the hprt.DELTA.5' and hprt.DELTA.3 ' and produces a NheI 18.2 kb
deletion-specific junction fragment detected by both probes a and b
(2C, 2D 1 and 2). Probe c, located in the deletion region, shows a
dosage difference in Panel 2F, 1 and 2, compared to the wt and dt
lanes. Probe a is a 0.7 kb RsaI fragment located approximately 3 kb
downstream of Hoxb-1 exon 2; probe b is a 1 kb RsaI fragment
located approximately 5 kb upstream of Hoxb-1 exon 1. P and N
represent the puromycin and neomycin selection cassettes.
[0020] FIG. 3: 3A: depicts mouse chromosome 11, and the loci used
as endpoints for the chromosome engineering are illustrated. 3B:
shows NheI restriction sites (N) and fragment lengths around the
E2DH and Gastrin loci. 3C: shows chromosome 11 with both the E2DH
and Gastrin loci targeted with the hprt.DELTA.5' and hprt.DELTA.3'
vectors, respectively. For clarity, only the cis configuration and
the A orientation are shown. 3D: shows the structure of the
deletion, duplication and inversion alleles. The sizes of the
diagnostic restriction fragments and probes used to detect these
alleles are indicated. The deletion, duplication and inversion
alleles are derived from the double targeted chromosome in the AA,
BB and AB configurations, respectively. 3E: shows the results of
Southern blots which confirm the structure of the recombinant
chromosomes. The probes used with each blot (a, b, c or d) are
indicated beneath each panel and on the diagrams of the various
alleles. The lanes are coded as follows: wildtype (wt), double
targeted (dt), deletion (del), duplication (dup) and inversion
(inv).
[0021] FIG. 4: G.sub.2 trans recombination between homologous
chromosomes. 4A: depicts individual sister chromatids from
chromosome homologues still joined at the centromere. One
chromosome is illustrated with hprt.DELTA.5', the neomycin
resistance gene (N) used for targeting. The other homologue was
targeted with hprt.DELTA.3' cassette linked to the puromycin
resistance gene (P). Cre-induced recombination between loxP sites
on sister chromatids from different homologues is illustrated by an
X. B: illustrates the recombinant structure of the sister
chromatids. The individual chromatids are numbered 1-4. Chromatid 3
carries the reconstructed Hprt minigene and the deletion. 4C: shows
the results of HAT selection for chromatid 3 which will segregate
with either chromatid 1 or 2 which carries only the neo or both the
neo and puro cassettes, respectively. The chromatid 2+3 segregant
carries a duplication and deletion (genetically balanced) and is
indistinguishable from the G.sub.1 inter-chromosomal product. The
chromatid 1+3 product carries the deletion and the original single
targeted chromosome; this can only have arisen via the G.sub.2
pathway.
[0022] FIG. 5: Gene dosage analysis and segregation of the deletion
and duplication chromosomes through the mouse germ line are
demonstrated: Lane 1: wildtype allele (AB2.2 ES cell line); Lane 2:
ES cell clone with the duplication and deletion (genetically
balanced); Lanes 3 to 6: transmission and segregation of the
duplication and deletion alleles in the progeny of a chimeric male
constructed from the cells shown in lane 2. Lanes 3 and 4 show mice
with the deletion, lanes 5 and 6 show mice with the duplication;
the increase or decrease in intensity of the 9.0 kb fragment
relative to the 2.0 kb control fragment is consistent with junction
fragment analysis of the inheritance of the duplication or deletion
alleles from these mice (data not shown). Lanes 7 and 8 show mice
from heterozygous mating homozygous for the duplication allele
which is evident from the increased intensity of the 9.0 kb
fragment.
[0023] FIG. 6: Deletion of two 3-4 cM intervals on mouse chromosome
11 is shown. 6A depicts mouse chromosome 11. The shaded bars
indicate the intervals which are to be deleted. Hox B, E2DH and
Wnt3 are the loci which serve as the deletion endpoints. SBC
(Sporadic Breast Cancer) loci are indicated by the black bars.
These loci are the putative location of tumor suppressor genes
based on the analysis of loss of heterozygosity in breast cancer.
6B depicts a double-targeted chromosome which has been targeted
with the hprt.DELTA.3' cassettes to the Hoxb9 and E2DH genes or to
the E2DH and Wnt3 genes. The A orientation E2DH-targeted clones
were used with the HoxB-E2DH deletion and the B orientation clones
were used with the E2DH-Wnt3 deletion. Only the orientations of the
hprt.DELTA.5' cassette which give the deletion products are
illustrated. The vertical bars represent NheI sites; the sizes of
the fragments are indicated and the probes are indicated by shaded
boxes labelled c and d. 6C shows the structure of deletion alleles.
Diagnostic NheI fragments for the deletion are indicated. 6D
reveals the Southern analysis that confirms the structure of the
alleles in the wildtype (wt), double-targeted (dt) and deletion
(dt) clones. The deletion-specific junction fragments are indicated
by the arrows.
[0024] FIG. 7: Schematic representation of a provirus structure,
containing hprt.DELTA.5' minigene cassette, a loxP site, and a
puromycin resistance gene, for use as a vector in one embodiment of
the present invention. This particular vector would insert the loxP
site at the 5' end of the chromosome.
[0025] FIG. 8: A 3' anchor library that contains the expression
cassette 3' hprt, puromycin resistance gene, and k14-agouti
gene.
[0026] FIG. 9: A 5' anchor library that contains the expression
cassettes 5' hprt, neomycin resistance gene, and tyrosinase
gene.
[0027] FIG. 10: Map of an exemplary 5' endpoint targeting vector
automatically excised out of a phage clone isolated from the 5'
anchor library.
[0028] FIG. 11: Map of an exemplary 3' endpoint targeting vector
automatically excised out of a phage clone isolated from the 3'
anchor library.
[0029] FIG. 12: Map of pG12WT (Wildtype 3' hprt cassette plasmid
for making chromosomal rearrangements). The sequence is identical
to pG12 except that the mutation in 3' hprt has been fixed.
[0030] FIG. 13: Map of a portion of the mouse chromosome 11 showing
the general composition of the selection cassettes positioned at
the chromosome endpoints, and the position of the Ore-induced
deletion interval, E.sub.2DH-D11Mit199.
[0031] FIG. 14: Map of a portion of the mouse chromosome 11 showing
the general composition of the selection cassettes positioned at
the chromosome endpoints, and the position of the Cre-induced
deletion interval, E.sub.2DH-D11Mit69.
DETAILED DESCRIPTION OF THE INVENTION
[0032] It will be apparent readily to one skilled in the art that
various substitutions and modifications may be made to the
invention disclosed herein without departing from the scope and
spirit of the invention.
[0033] As used herein, the term "chromosome engineering" means
creating chromosome inversions, duplications or deletions.
[0034] As used herein, the term "chromosome deficiency" means a
lack of a chromosome or portion of a chromosome.
[0035] As used herein, the term "chromosome inversion" means
reversal of a part of a chromosome so that the genes within that
part are in reverse order.
[0036] As used herein, the term "chromosome duplication" means an
extra, duplicate chromosome or part of a chromosome.
[0037] As used herein, the term "ES cell" stands for embryonic stem
cells: cells which are derived from early mouse embryos that can be
maintained in an undifferentiated state, but, upon return to the
environment of the early embryo, can contribute to all types of
cells in the resulting chimera.
[0038] As used herein, the term "Cre-induced recombination" means
catalysis of both intramolecular and intermolecular recombination
by the Cre protein. The Cre protein is a 38 kD protein that
recombines DNA between specific, 34 bp sequences called loxP
sites.
[0039] As used herein, the term "interchromosomal recombination"
means recombination between different chromosomes.
[0040] As used herein, the term "intrachromosomal recombination"
means recombination between regions on the same chromosome.
[0041] As used herein, the term "HoxB" refers to a specific gene
cluster having the same physical distance as a P1 phage and having
known structure and orientation.
[0042] As used herein, the term "Hoxb-9" refers to a specific gene
in the HoxB gene cluster.
[0043] As used herein, the term "Hoxb-1" means the 3'-most gene of
the HoxB gene cluster.
[0044] As used herein, the term "hprt" means hypoxanthine
phosphoribosyltransferase.
[0045] As used herein, the term "loxP site" means the specific 34
bp sequence recognized by Cre recombinase.
[0046] As used herein, the term "G418 resistance" means having a
gene which confers resistance to G418.
[0047] As used herein, the term "neo resistance" means having a
gene which confers resistance to G418.
[0048] As used herein, the term "puromycin resistance" means having
a gene which confers resistance to puromycin.
[0049] As used herein, the term "HAT resistance" means cells
resistant to media containing hypoxanthine, aminopterin and
thymine. Only cells expressing the Hypoxanthine phosphoribosyl
transferase (Hprt) and thymidine kinase genes will grow in HAT
medium.
[0050] As used herein, the term "targeting deletion" means a
planned deletion created by targeting an area for recombination by
insertion of a loxP or other recombination site.
[0051] As used herein, the term "germ line transmission" refers to
a chimeric animal capable of transmitting a particular trait to its
offspring.
[0052] As used herein, the term "hemizygous" means genes present
only once in a genotype.
[0053] As used herein, the term "heterozygous" refers to the state
of an organism having two different alleles at a given locus on
homologous chromosomes.
[0054] As used herein, the term "homozygous" refers to the state of
an organism having the same two alleles at a given locus on
homologous chromosomes.
[0055] As used herein, the term "Gastrin" locus means a gene on
mouse chromosome 11 which encodes a peptide involved in stimulating
acid secretion in the stomach. See Fuller et al., Molec Endocrinol.
1:306-11 (1987).
[0056] As used herein, the term "E2DH" locus, also known as 17HSD,
is a gene whose product is involved in steroid biosynthesis. This
dehydrogenase converts estrone to estradiol. See The et al., Molec.
Endocrinol. 3:1301-06 (1989).
[0057] As used herein, the term "Wnt3" locus is a gene which
encodes a member of the wnt family of growth factors. Wnt3 is a
target for activation by MMTV which causes mammary tumors. Roelink
et al., PNAS 87:4519 (1990).
[0058] As used herein, the term "selected region" refers to that
particular region of the chromosome targeted for manipulation
(i.e., deletion, inversion, duplication).
[0059] In one embodiment of the present invention, a method is
disclosed and claimed for deleting a selected region of genetic
material in cells comprising the steps of: inserting a first
selection cassette at a 5' end of said selected region using
conventional gene targeting methods, said first selection cassette
comprising a first selectable marker, a first loxP recombination
site, and a first portion of a second selectable marker; selecting
cells expressing said first selectable marker; inserting a second
selection cassette at a 3' end of said selected region using
conventional gene targeting methods, said second selection cassette
comprising a third selectable marker, a second loxP recombination
site, and a remaining portion of said second selectable marker;
selecting cells expressing said third selectable marker; expressing
Cre recombinase to produce recombination between said first and
second loxP sites; and selecting cells expressing said second
selectable marker.
[0060] In particularly preferred embodiments of the present
invention, the method referred to above uses a first selectable
marker, a puromycin resistance gene, said second selectable marker
is an Hprt gene, and said third selectable marker is a neomycin
resistance gene.
[0061] In another particularly preferred embodiment, the first
selectable marker is a puromycin resistance gene. In yet another
particularly preferred embodiment, the second selectable marker is
a functional Hprt gene. In still another preferred embodiment, the
third selectable marker is a neomycin resistance gene. In another
preferred embodiment, the cells are embryonic stem cells. In still
another preferred embodiment, the cells are embryonic stem cells,
and said cells develop into mice. In still another preferred
embodiment, the cells are embryonic stem cells, and said cells are
maintained as cell lines. In another embodiment of the present
invention, Cre is transiently expressed Cre. In other embodiments,
it is expressed either inducibly or constitutively.
[0062] In a second general embodiment of the present invention, a
method is disclosed and claimed for deleting a selected region of
genetic material in cells comprising the steps of: inserting a
first selection cassette at a 5' end of said selected region using
either conventional targeting methods or a viral vector, said first
selection cassette comprising a first selectable marker, a first
loxP recombination site, and a first portion of a second selectable
marker; selecting cells expressing said first selectable marker;
inserting a second selection cassette at a 3' end of said selected
region using conventional gene targeting methods or a viral vector,
said second selection cassette comprising a third selectable
marker, a second loxP recombination site, and a remaining portion
of said second selectable marker; selecting cells expressing said
third selectable marker; expressing transiently Cre recombinase to
produce recombination between said first and second loxP sites; and
selecting cells expressing said second selectable marker.
[0063] In one particularly preferred embodiment, the viral vector
is a retrovirus. In yet another particularly preferred embodiment,
the viral vector has a provirus structure comprising a cassette in
turn comprising an hprt.DELTA.5' cassette, a loxP site, and a
puromycin resistance gene. In yet another particularly preferred
embodiment, the viral vector has a provirus structure comprising a
cassette in turn comprising an hprt.DELTA.5' cassette, a loxP site,
and a neomycin resistance gene. In still another particularly
preferred embodiment, the targeting or viral vectors are a first
vector for inserting said first native sequence of DNA at said 5'
end, comprising: a genomic insert cloned into the vector of about
7.5 kb; a tyrosinase minigene; a Neo.sup.r gene; a 5' hprt
fragment; and a loxP site embedded into said hprt fragment; and a
second vector for inserting said second native sequence of DNA at
said 3' end, comprising: a genomic insert cloned into the vector of
about 8.5 kb; a K14-Agouti gene; a Puro.sup.r gene; a 3' hprt
fragment; and a loxP site embedded into said hprt fragment.
[0064] In a third general embodiment of the present invention, a
replacement vector system, is disclosed and claimed comprising: a
first vector for inserting said first native sequence of DNA at
said 5' end, comprising: a genomic insert cloned into the vector of
about 7.5 kb; a tyrosinase minigene; a Neo.sup.r gene; a 5' hprt
fragment; and a loxP site embedded into said hprt fragment; and a
second vector for inserting said second native sequence of DNA at
said 3' end, comprising: a genomic insert cloned into the vector of
about 8.5 kb; a K14-Agouti gene; a Puro.sup.r gene; a 3' hprt
fragment; and a loxP site embedded into said hprt fragment.
[0065] In a fourth general embodiment of the present invention,
there is disclosed and claimed a method for creating defined
chromosomal deficiencies, deletions, and duplications comprising
the steps of: identifying a desired region of a chromosome of
interest to be deleted; inserting two native sequences at each
endpoint of said region of said chromosome of interest using a
first and a second targeting vector, each comprised of one or more
selectable markers and a loxP site and an hprt fragment;
transiently expressing Cre recombinase to produce recombination
between each of two said loxP sites; whereby upon chromosomal
rearrangement induced by said Cre recombinase, a functional Hprt
expression cassette is reconstructed.
[0066] Other and further embodiments, features and advantages will
be apparent and the invention more readily understood from a
reading of the following Examples and by reference to the
accompanying drawings forming a part thereof, wherein the examples
of the presently preferred embodiments of the invention are given
for the purposes of disclosure.
EXAMPLE A
General Strategies
[0067] The various chromosomal rearrangements described herein are
designed with strong positive selection for the desired chromosomal
change. Very generally, this was accomplished by targeting
consecutively complementary, overlapping but non-functional
hprt-loxP expression cassettes to the endpoints of a chromosomal
interval. Cre expression (either transiently, inducibly, or
constitutively) in these double-targeted ES cells induces loxP
recombination resulting in chromosomal rearrangements specific to
the relative orientation of the loxP sites. Since the loxP sites
are imbedded in the hprt minigene fragments, the chromosomal
rearrangement will also reconstruct a functional hprt expression
cassette, therefore facilitating direct positive selection for the
clones with these alterations.
[0068] The use of mouse ES (embryonic stem) cells is preferable,
though not required, to execute the method the present invention.
Use of these cells would, of course, allow large-scale chromosome
manipulation to be introduced into a germ line, which would in turn
facilitate enhanced functional study of the mouse genome.
[0069] The loxP sites were introduced by conventional gene
targeting protocols or by viral vectors into the endpoints of the
region which was to be rearranged. Or, one endpoint can be
introduced by conventional methods, and the other introduced by a
viral vector. To maximize the ability to select for the rare ES
cell clones in which Cre expression had successfully induced
recombination between loxP sites, the individual loxP sites
targeted to the endpoints of the chromosomal rearrangement were
imbedded in two complementary but non-functional fragments of an
Hprt minigene cassette. Recombination between the loxP sites would
restore the activity of this cassette, facilitating the direct
selection in HAT media of only those recombinant ES cells with the
desired chromosomal structure (FIG. 1A and B).
[0070] In one particularly preferred embodiment of the present
invention, the complementary recombination/selection substrates
consist of overlapping, but incomplete, pieces of an Hprt minigene
with a loxP site in the intron. These minigene fragments are linked
to different positive selection cassettes which are required for
selection during gene targeting. The 5' fragment of the loxP-Hprt
minigene is linked to a neomycin resistance gene (hprt.DELTA.3'
cassette), while the 3' fragment is linked to a puromycin
resistance gene (hprt.DELTA.5' cassette). Cre-induced recombination
between the loxP sites generates a fully-functional Hprt minigene
which provides resistance to HAT selection in Hprt-deficient cells.
The positive selectable markers are positioned so that following
recombination, they are lost from the deleted chromosome. All of
the clones that survive selection have the desired chromosomal
structure. A similar positive selection system for detecting a
chromosomal translocation has recently been reported by Smith et
al., Nature Genetics 9:376-385 (1995). In addition, genes such as
K14-agouti and tyrosinase genes can be preferably inserted into the
vectors for use as color-coat markers, to aid in selecting the
members of the population for which the chromosomal insert was
successful. Albino mice lack the tyrosinase gene, so reinsertion of
that gene is manifest by black mice in a population of white mice.
Similarly, the k14-agouti gene gives yellow color to the tips of
the coat hairs against a black background (i.e., it makes black
mice appear brown).
[0071] Initially, a small deletion (90 kb) was constructed which
encompasses the HoxB locus since the gene order and orientation was
known. Subsequently, much larger chromosomal alterations were
generated. For the latter alterations, knowledge of the
transcriptional direction of the genes which serve as the
rearrangement endpoints was not available. Consequently, it was
necessary to generate ES cell lines with the four possible
configurations of the hprt minigene fragments. Because the
transcriptional direction of the genes relative to the centromere
was also unknown, it was not possible to predict which combination
of orientations would give a deletion; however, clones with
deletions are readily distinguished in culture from the clones with
other classes of recombinant chromosomes because in addition to
becoming HAT resistant, both of the positive selection markers are
lost. The generation of a deletion reveals the relative
transcriptional direction of the two deletion endpoints, and if the
proximal-distal map positions are known (which was the case in
these experiments), further deletions from the same endpoint are
greatly simplified.
[0072] The frequency of recombination between the loxP sites when
they were on the same chromosome varied from 6.times.10.sup.-7 to
5.times.10.sup.-5, but a direct relationship between the distance
and the frequency was not apparent. The frequency of recombination
was, however, significantly lower than those reported when the loxP
sites are a few kb apart; see, Gu, et al., Cell 73:1155-64 (1993),
verifying that selection is required to isolate these clones. These
frequencies are derived by the transient transfection of Cre in ES
cells, but might be higher under conditions of constitutive
expression of Cre, for example, in a specific lineage in a
transgenic mouse. The frequency of recombination was reduced by one
to two orders of magnitude when the loxP sites were integrated in
trans compared to the cis configuration. This is consistent with
the knowledge that individual chromosomes occupy discrete,
non-overlapping domains in an interphase nucleus.
[0073] HAT-resistant clones derived from the trans configuration of
the double-targeted clones oriented to give deletion products were
not expected to become G418 or puro sensitive. But approximately
half of the HAT-resistant clones segregated the puro cassette while
all retained the neo cassette. This segregation pattern is
consistent with inter-sister-chromatid recombination (see FIG. 4).
Although the number of clones with the trans configuration was
relatively small, the equal ratio of puro+neo to neo-only
segregants suggests that G.sub.2 recombination is the predominant
pathway used in this case. The rescue of hprt negative daughter
cells by metabolic cooperation also suggests that a substantial
fraction of the HAT-resistant clones derived from the cis
double-targeted clones may have been generated by the
sister-chromatid pathway.
[0074] The correlation of the induced chromosomal rearrangements
with the orientation of the vectors has revealed physical mapping
information in this region of mouse chromosome 11. For instance,
the genes described in the following Examples, Gastrin, E2DH, Wnt 3
and the Hox B cluster, are all transcribed in the
centromere-to-telomere direction. The E2DH-HoxB deletion has shown
that the HoxB cluster is oriented with the Hoxb-9 gene nearest to
the centromere.
EXAMPLE B
Deletion and Duplication of 90 kb Containing the HoxB Cluster
[0075] The HoxB cluster provides an excellent substrate for the
deletion strategy since the cluster is about the same physical
distance as a P1 phage and the structure and orientation of the
individual genes is known. See, Rubock, et al., PNAS USA 87:4751-55
(1990). Moreover, a deletion allele of HoxB is very useful for
detailed genetic analysis of this region.
[0076] To generate a HoxB deletion allele, the strategy outlined in
FIG. 1 was followed. The hprt.DELTA.3' cassette was used to
construct a targeting vector for Hoxb-1, the most 3' gene of the
cluster, and targeted clones were identified (FIG. 2). An ES clone
with the Hoxb-1 targeted allele (FIG. 2B) was expanded and
transfected with the Hoxb-9 targeting vector containing the
complementary hprt.DELTA.5' cassette. The minigene fragments were
oriented in the targeting vectors so that, after targeting, they
would be in the correct order and orientation with the positive
selection cassettes (neo and puro) located between the loxP sites
(FIG. 2C). Double-targeted clones were identified (FIG. 2C), and
half of these clones would be expected to have both targeted
alleles on the same chromosome (cis) and half should have the
targeted alleles on different homologues (trans).
[0077] To induce the recombination between the loxP sites, several
independent double-targeted (Hoxb-1 and Hoxb-9) clones were
expanded and transiently transfected with a Cre expression cassette
and placed under HAT selection. Control transfections without Cre
did not yield any HAT-resistant clones. What follows is a more
detailed description of the method employed in this example.
[0078] As depicted in FIG. 1A, the PGKHprt minigene was modified by
the insertion of a loxP site from pBS64 (HindIII-EcoRI, Klenow
blunt) into the unique XbaI site (Klenow blunt) in the hprt intron.
Insertion of the loxP site did not disrupt the cassette's HAT
resistance function (not shown). The loxP-Hprt cassette was divided
into two overlapping pieces: hprt.DELTA.3', which contains the PGK
promoter, the hprt exons 1 and 2, the loxP-intron, hprt exons 3-6,
and the SV40 poly A signal. In FIG. 1A, hprt.DELTA.5' and
hprt.DELTA.3' have a 2 kb overlap including the loxP site;
independently, hprt.DELTA.5' and hprt.DELTA.3' do not provide HAT
resistance, but they do when co-electroporated. Hprt.DELTA.5' and
hprt.DELTA.3' were ligated to positively selectable cassettes
(hprt.DELTA.3' to the pol II neo gene and hprt.DELTA.5' to a
PGK-puromycin resistance gene); in both cases, the positive markers
replaced the deleted part of the loxP-Hprt cassette, ensuring that,
upon recombination, they are separated from the reconstituted
cassette. FIG. 1C depicts the general strategy for making deletions
consisting of 3 steps: Step 1: conventional replacement-type gene
targeting used to replace the Hoxb-1 gene with the
hprt.DELTA.3'-neo cassette; Step 2: ES cells identified as
correctly targeted are used as a substrate to insert the
hprt.DELTA.5'-puro cassette into the endogenous Hoxb-9 gene by
conventional replacement-style targeting (the cis configuration is
illustrated here); and Step 3: transient expression of Cre induces
recombination between the loxP sites which reconstructs a
functional hprt minigene. In FIG. 1 C4, the intra-chromosomal
recombination pathway is illustrated, and in FIG. 1 C5, cells with
the recombinant (deleted) chromosome to be positively selected in
HAT media and a chromosomal ring are generated by the
intra-chromosomal pathway. This is believed to be unstable and lost
during the growth of the colony.
[0079] The targeting vector for Hoxb-1 consists of a 3.5 kb
BgIII-NcoI fragment (5' homologous arm); the Hoxb-1 coding sequence
(1.7 kb Nco-BgIII) was replaced by the hprt.DELTA.3'-neo cassette
and a 2 kb BgIII-PvuII fragment (3' homologous arm). The vector was
linearized with SalI and a 10 .mu.g was electroporated into
hprt-negative AB2.2 ES cells. g418 selection was applied 24 hours
after the electroporation and resistant clones were arrayed in 96
well plates, and targeted clones were detected by Southern
analysis. A single targeted clone out of 384 clones analyzed was
identified with the predicted structure of the targeted allele
using probes 5' and 3' of the Hoxb-1 gene. This clone was expanded
and transfected with the Hoxb-9 targeting vector. The targeting
vector for Hoxb-9 consisted of a 6.2 kb HindIII fragment of
homology which included exon 1. The hprt.DELTA.5'-puro cassette was
cloned into the unique SalI site in exon 1. The orientation of the
cassette was such that, when targeted, the loxP sites in the
hprt.DELTA. cassettes would be in the same orientation. The vector
was linearized and 10 .mu.g of vector was electroporated into clone
#298 AB2.2 cells and plated on SNLP (puro resistant SNL76/7 cells).
Puromycin selection (5 .mu.gml) was applied 24 hours after
electroporation. Resistant clones were arrayed in 96 well plates
and screened for targeted clones by Southern analysis.
Double-targeted clones were detected at a frequency of 6%. Multiple
independent double-targeted clones were expanded and independently
transiently transfected (by electroporation) with 20 mg of a
supercoiled Cre expression cassette pOG231. HAT selection was
applied 48 hours after the electroporation. HAT-resistant clones
were arrayed and analyzed by Southern Blot analysis.
[0080] As evidenced by Table 1, two classes of double-targeted
clones could be distinguished by this assay.
1 TABLE 1 Frequency (10.sup.-7) Deletion Interval Distance I II
Inversion Hoxb9 - Hoxb1 90 kb 153 0.5 ND Hoxb9 - E2DH 3-4 cM 6 1 43
Gastrin - E2DH 1 Mb 470 1.8 334 E2DH - Wnt3 3-4 cM 30 4 19
[0081] Table 1 reports the frequency of Cre-mediated recombination
as a function of distance between the loxP sites. Deletion
frequencies are illustrated for both Class I and Class II clones
while inversion frequencies are only illustrated for Class I
clones.
[0082] One class (Type I) yielded HAT-resistant recombinants at
frequencies averaging 1.times.10.sup.-5 per treated cell, while a
second class (Type II) yielded HAT-resistant clones at a much lower
frequency.
[0083] Table 2 shows the frequency of Cre-mediated recombination as
a function of distance between the loxP sites.
2TABLE 2 Example B RECOMBINATION FREQUENCY ON THE MOUSE CHROMOSOME
11 Deletion Inversion Duplication Interval Cis Trans Cis Trans Cis
Trans Gastrin-E.sub.2DH (1 Megabase) 476 1 355 0 166 2
E.sub.2DH-D11MIT199 (2 cM) 102 3 293 0 N/A N/A HoxB-E.sub.2DH (3-4
cM) 2 0 43 0 N/A N/A E.sub.2DH-Wnt3 (3-4 cM) 36 4 19 0 N/A N/A
E.sub.2DH-D11MIT69 (22 cM) 0 0 3 0 N/A N/A (1 .times. 10.sup.7 ES
cells were electroporated with 20 ug pOG 231 (Cre expression
plasmid) and were selected with HAT medium.)
[0084] Table 2 is similar to Table 1, except that the former shows
an additional deletion frequency for an additional interval
(E.sub.2DH-D11Mit69); it also shows duplication frequency; and
finally it shows additionally deletion, inversion, and duplication
frequencies for both cis- and trans-.
EXAMPLE C
Cis and Trans Recombination
[0085] Two types of clones might correspond to the cis or trans
configuration of the loxP sites. The HAT-resistant clones derived
at a high frequency from Type I clones might be products in
intrachromosomal recombination or sister chromatid exchange (loxP
sites in cis). Type II clones might require interchromosomal or
inter-sister-chromatid recombination between homologous chromosomes
(loxP sites in trans), which may occur relatively infrequently.
These different pathways were distinguished by analyzing the
markers in recombinant HAT-resistant clones.
[0086] Deletion of the HoxB cluster from a chromosome double
targeted in cis would be accompanied by the loss of the neo and
puro cassettes. These are either segregated (sister-chromatid
pathway) or a ring is formed which is presumed to be unstable (FIG.
1). The loss of both markers could be documented in most of the
HAT-resistant clones derived from Type I double-targeted clones,
consistent with the hypothesized cis configuration of the Type I
clones.
[0087] As anticipated, the consecutive targeting events and the
Cre-induced recombination event results in the formation of novel
restriction fragments (FIG. 2). External probes identify the novel
junction fragments using an NheI digest since there is not an NheI
site present in the loxP-hprt cassette. An internal probe confirms
the loss of 90 kb of sequence by dosage difference between the
wildtype and the deletion clones (FIGS. 2 E and F).
EXAMPLE D
E2DH-Gastrin 1 Mb Deletion
[0088] One of the goals behind the methods of the present invention
is to construct chromosomal deletions so that regions of the genome
can be tested for tumor suppressor activity. Many candidate regions
have been identified from loss of heterozygosity (LOH) studies. One
well-defined region in human breast cancer maps close to the
Gastrin locus on human chromosome 17 q close to BRCA1. Miki, et
al., Science 266:66-71 (1994). This putative sporadic tumor
suppressor locus maps in a conserved linkage group on mouse
chromosome 11 between Gastrin and the E2DH locus. The generation of
hemizygous mice with a deficiency that encompasses this locus
functionally tests if this region contains a sporadic breast cancer
gene that is involved in mammary neoplasia.
[0089] The large size of the regions which contain putative
sporadic tumor suppressor loci complicates substantially the use of
deletion strategy. In the absence of a YAC cloning contig which
spans the relevant genetic interval, the gene order and
orientations were not known. This is an important consideration
since both the order and the orientation of the Hprt minigene
fragments will determine the type of chromosomal rearrangement that
is required to reconstruct a functional Hprt cassette. The possible
orientations are illustrated in FIG. 1D. The recombinant
chromosomes include deletions, duplications, inversions, and di-
and acentric chromosomes (FIG. 1E). These rearranged chromosomes
can be distinguished on Southern blots by the appearance of novel
junction fragments, but the most rapid identification of the clones
with deletions can be obtained from selection using neomycin and
puromycin resistance cassettes which have been configured to lie
between the loxP sites in the to-be-deleted interval (see FIG.
1).
[0090] To construct ES cell lines with large deletions between the
Gastrin and the E2DH locus (containing SBC I) in the absence of a
priori knowledge of the gene order and orientation, all four
possible arrangements of the hprt minigene fragments were made and
tested, only one of which will generate deletions. Two targeting
vectors were constructed for each deletion endpoint representing
the two possible orientations of the hprt minigenes. The
hprt.DELTA.3' cassette was targeted to the E2DH locus with the
alternative minigene orientations (A or B). Targeted clones
representing both the A and B orientations were, in turn,
transfected with the targeting vectors representing the different
orientations of the hprt.DELTA.5' cassette (A or B) at the Gastrin
locus. Multiple independent, targeted clones were isolated
representing the four different minigene configurations to ensure
that clones with the cis and the trans configurations were likely
to be represented. Each of these clones was expanded, transfected
with a Cre expression cassette and plated under HAT selection. What
follows is a more detailed description of the method employed in
this example.
[0091] Overlapping .lambda. phage containing the mouse E2DH locus
were isolated from a mouse 129Sv/Ev genomic library using a human
E2DH cDNA probe. Unlike the duplicated human E2DH locus, the mouse
locus is present at a single copy. .lambda. phage containing the
mouse Gastrin locus were isolated from the same library using a PCR
fragment from the rat Gastrin cDNA. The mouse E2DH gene and Gastrin
genes had not been mapped; to confirm that these genes mapped to
mouse chromosome 11, a hamster/mouse hybrid cell line in which the
mouse chromosome 11 is the only mouse genetic material was
hybridized to probes specific for the mouse Gastrin and E2DH
loci.
[0092] Standard gene replacement targeting vectors were constructed
from these genomic clones. E2DH vector: a total of 8.0 kb of
homology was used. The XhoI-XbaI 5.5 kb fragment containing the
entire E2DH coding sequence was replaced with the hprt.DELTA.3'
minigene cassette in both orientations. In the Gastrin vector, a
total of 7.5 kb of homology was used. The 3.5 kb XhoI-NheI fragment
containing the Gastrin coding region was replaced with the
hprt.DELTA.5' cassette in both orientations.
[0093] The two vectors were separately transfected into AB2.2 ES
cells. G418 resistant clones were obtained for each vector. Clones
were gridded onto 96 well plates and screened for targeted clones.
Targeted clones were identified at a ratio of 1/25 for the A
orientation vector and 1/25 for the B orientation vector. The two
types of targeted ES cells were assayed for totipotency by
generating chimeras which tested for germ line transmission.
Totipotent E2DH-targeted ES cell lines were identified for both the
A and the B orientation and these were transfected with the vectors
which target the Gastrin locus. Puromycin resistant clones were
arrayed on 96 well plates and screened for targeted clones. All
four classes of double targeted clones were obtained. For
simplicity, this figure only shows the double targeted ES cell
having the hprt.DELTA.5' and hprt.DELTA.3' cassettes in the A
orientation and in cis.
[0094] The double targeted ES cell clones were transfected with the
Cre expression plasmid as previously described. HAT.sup.r colonies
were recovered and sibselected to test for puromycin and G418
resistance. Individual clones were expanded and analyzed for
junction fragments using multiple probes. Each blot was hybridized
with two probes, one from the E2DH locus and the other from the
Gastrin locus. The frequency of obtaining HAT resistant colonies
from the different clones is summarized in Table 3.
3 TABLE 3 Clones m HAT.sup.r Category Class tested (10.sup.7) G418
Puro AA I 4 470 S S II 2 1 R R/S AB I 5 344 R R II 1 0 -- -- BA I 3
377 R R II 2 0 -- -- BB I 3 166 R R II 6 18 R/S R
[0095] Table 3 reveals the frequency of the Cre-mediated
recombination and retention of the markers in recombinant clones.
All of the data is derived from the E2DH-Gastrin double targeted
clones. The categories of clones are illustrated in FIG. 1D, and
the expected products are described in FIG. 1E. Class I
double-targeted clones give a high frequency of HAT-resistant
recombinants, while Class II clones give a low frequency of
HAT-resistant clones. Retrospective analysis has revealed that the
class I clones and Class II clones have the targeted genes in cis
and trans. S and R refer to resistance or sensitivity to G418 or
puromycin as assayed by selection. Both resistant and sensitive
clones were recovered.
[0096] HAT-resistant clones were recovered from each of the four
alternative split minigene configurations. The individual clones
within a specific orientation group could be placed into one of two
classes, based on the frequency with which HAT-resistant clones
could be recovered (Table 3). Selection analysis identified the AA
class I HAT-resistant clones as those that had lost the neomycin
and puromycin resistance genes; these clones are the most likely to
have the desired deletion. Since the AA class I clones gave the
deletion product, this allows predictions to be made on the likely
products of the alternative configurations: BB gives duplications,
and AB or BA should give inversions. These predictions have been
confirmed by detailed molecular analysis summarized in FIG. 3. In
particular, the juxtaposition of the hprt minigene fragments which
were previously positioned approximately 1 Mb apart in the genome
results in unique junction fragments that are specific for the
different types of rearrangement.
[0097] The AA type II clones yield HAT-resistant recombinants at a
low frequency. It was hypothesized that these clones represented
the cases where the deletion selection cassettes had integrated in
trans. Interchromosomal recombination would result in both the neo
and puro resistance genes being located on one chromosome, while
the reconstructed hprt minigene would be on the homologue. Thus it
would be anticipated that all of the positive selection markers
would be retained in such a cell. Sibselection identified two
classes of HAT-resistant clones which were represented at
approximately equal frequencies. One type only retained the neo
cassette, and a second type retained both the neo and the puromycin
resistance cassettes (Table 1A). The segregation of the puro
resistance gene from the neo cassette is explained readily if
Cre-induced recombination between sister chromatids (FIG. 4)
occurred. This occurrence was confirmed by the molecular analysis
of these clones. While the clones with the duplicated and deleted
chromosomes can be generated by either interchromosomal or
non-sister chromatid exchange, the clones which only carry the neo
cassette can only have arisen by the non-sister chromatid
recombination pathway. These clones have been confirmed to carry
both the deletion chromosome and the non-recombinant chromosome
with only the E2DH targeted locus (FIG. 4).
[0098] The clones with the deletion on one chromosome and the
duplication on the other are genetically balanced. Therefor these
clones were considered to be the best candidates for germ line
transmission. Four independent clones were injected into
blastocysts, representing clones descended from both the A and B
orientation of the E2DH targeted allele. Alleles from three of
these clones were transmitted into the germ line, despite three
cycles of subcloning and expansion. Segregation of the deletion and
duplication alleles from a chimeric male is illustrated by gene
dosage analysis (FIG. 5). Mice which are hemizygous for this
deletion (1 copy) are fully viable. Mice which are heterozygous for
the duplication (3 copies) or homozygous (4 copies) are also fully
viable (FIG. 5) and fertile.
EXAMPLE E
Deletion of Two 34 Centimorgan Regions on Chronosome 11
[0099] Given the apparent insensitivity of the Cre-induced
recombination to the distance between the loxP-hprt substrates, two
additional experiments were performed to investigate if Cre could
delete a larger fragment. Two 3-4 cM intervals were chosen,
proximal or distal to the E2DH locus on mouse chromosome 11. This
region is syntectic with a region on human chromosome 17q where
loss of heterozygosity studies have identified several distinct
regions that are likely to contain tumor suppressor genes which are
mutated in 30-70% of sporadic breast cancer. These regions have
been termed SBCI, SBCII and SBCII (FIG. 6A).
[0100] Since the E2DH-Gastrin deletion had revealed the orientation
of the E2DH locus, one of the A orientation E2DH-targeted clones
was selected for the proximal deletion and a B orientation
E2DH-targeted clone was selected for the distal deletion. Targeting
vectors (two orientations) were constructed for the HoxB locus
(Hoxb-9) and for Wnt3 (see Roelink, et al., PNAS USA 87:4519-23
(1990)). Double-targeted clones were generated, transfected with
the Cre expression cassette and HAT-resistant clones were selected.
One vector orientation yielded G418 and puro sensitive clones which
were hypothesized to have a 3-4 cM deletion, while the other
orientation yielded HAT-resistant clones which all retain the neo
and puro cassettes. This latter category of clones were confirmed
to be inversions of the 3-4 cM interval by molecular analysis. The
molecular analysis of the G418 and puro sensitive clones identified
two classes of clones that occur with approximately equal
frequency. The first type of clone was consistent with a simple
deletion event, illustrated for the Hoxb9-E2DH deletion (FIG. 6D).
The other class of clone exhibited the expected deletion junction
fragment, but also retained a junction fragment that is diagnostic
for the targeted chromosome, but should have been lost during the
deletion event (illustrated for the E2DH-Wnt3 deletion in FIG. 6D).
The retention of this junction fragment and the acquisition of the
expected deletion-specific fragment in about half the clones can be
explained by two different recombination pathways. The pure clones
are believed to be products of an inter-chromosomal pathway (FIG.
1C), while those clones that retain the primary targeted allele may
reflect a sister-chromatid exchange. While the duplicated
chromosome should be segregated to a daughter cell and does not
carry the reconstructed hprt minigene, extensive metabolic
co-operation between the hprt.sup.+ and hprt.sup.31 ES cells in a
colony facilitate cross rescue and substantial contribution of the
hprt.sup.31 daughter cells to the HAT-resistant clones. What
follows is a more detailed description of the method employed in
this example.
[0101] E2DH and Hoxb9 vectors have been described previously. Wnt3
genomic clones were isolated from a 129Sv/Ev genomic library using
a cDNA probe. Conventional replacement targeting vectors containing
7.0 kb of homology were constructed. The hprt.DELTA.5' cassette
replaces a 2.1 fragment (contains exons 3 and 4) of the Wnt3 gene.
The Hoxb9 vectors and the Wnt3 vectors were independently targeted
into the E2DH targeted cell lines. An "A" orientation clone was
used for the Hoxb9 targeting vectors. Transfected cells were
selected in puromycin and targeted clones were identified as
previously described. Multiple independent double targeted clones
were transfected with the Cre recombinase. HAT-selection was used
to isolate recombinant clones which were sib-selected and tested by
Southern analysis for the predicted junction fragments.
EXAMPLE F
Using a Virus Rather than Targeting to Effect the Recombination
[0102] Rather than relying on traditional targeting techniques,
either or both of the desired deletion endpoints can be added to
the genome by means of a retrovirus. In this example, a viral
vector is used to insert the endpoint at the 5' end only. FIG. 7 is
a schematic representation of a provirus structure suitable for
this use, which is comprised of an hprt.DELTA.5' minicassette, a
loxP site and a puromycin resistance gene. Hence, the provirus
structure is similar to the non-viral targeting vectors described
in the previous examples.
[0103] In this example, only the 5' endpoint was inserted using the
viral vector depicted in FIG. 7, though in other embodiments, both
endpoints can be added using viral vectors. Chromosomal deletions
were induced using the methods substantially as described in the
previous examples. Hence, insertions are made at the two endpoints
framing the desired chromosomal deletion. The insertions are
preferably made one at a time, and involve replacing a first native
sequence (i.e., the first endpoint) on the chromosome of interest
with a first selection cassette. This selection cassette consists
of three elements: a first selectable marker, a loxP site located
in the hprt minigene intron, and a first portion of a second
selectable marker, preferably a non-functional fragment of an Hprt
minigene cassette. The first selectable marker is preferably a
neomycin resistance gene (hprt.DELTA.5' cassette) or a puromycin
resistance cassette (hprt.DELTA.3' cassette). The cells expressing
the marker (either the neomycin resistance gene or the puromycin
resistance gene) are then selected. Next, the process is
essentially repeated for second endpoint on the chromosome of
interest. Thus, the cells selected possess both loxP sites framing
the desired portion of the chromosome to be deleted. The difference
in this step is that the hprt minigene fragment--also
non-functional--is the complementary portion to that inserted into
the first endpoint. Third, the selected cells are contacted with
Cre, which may be expressed in one of the three ways described
above, which induces recombination between the loxP sites. This
recombination generates a fully functional Hprt minigene. This
minigene provides resistance to HAT selection in hprt deficient
cells. Additionally, the positive selectable markers are positioned
so that following recombination, they are lost from the deleted
chromosome. Therefore, the methods for inducing the deletion
described in this example are nearly identical to those for
practicing the method using a non-viral vector. Inherent
differences in method and technique that result from using a viral
versus non-viral vector are well-known to the skilled artisan,
hence a detailed description of any embodiment of the method of the
present invention involving non-viral vectors could be easily
adapted by the skilled artisan using a viral vector. The
recombination efficiencies obtained from using the viral vector
(described in FIG. 7) at the 5' endpoint are shown below in Table
4.
4TABLE 4 # cell out of 10.sup.7 cre electroporated ES cells
HAT.sup.r Puro.sup.r NO Drug Rec Efficiency plate #3 275 1.62
.times. 10.sup.5 3.9 .times. 10.sup.5 1.7 .times. 10.sup.-3 plate
#4 388 2.54 .times. 10.sup.5 3.9 .times. 10.sup.5 1.5 .times.
10.sup.-3 Average 331 2.09 .times. 10.sup.5 3.9 .times. 10.sup.5
1.6 .times. 10.sup.-3
EXAMPLE G
Frequency of Cre-induced Deletion Between E.sub.2DH and D11 Mit199
Using an Improved Targeting Vector
[0104] This example illustrates the enhanced Cre-induced deletion
frequency using a different targeting vector compared with that
used in the previous examples. The details of the protocol are
substantially as described in the previous examples. Details of
this "improved vector" compared with the vector used in the
previous examples are shown in FIGS. 10 through 12 (the original
vector is shown by comparison in FIG. 12). FIG. 10 shows a map of
an exemplary 5' endpoint targeting vector automatically excised out
of a phage clone isolated from the 5' anchor library. The 5' anchor
library is shown in FIG. 9; this anchor library contains the
expression cassettes 5' hprt, neomycin resistance gene, and
tyrosinase gene. FIG. 11 shows a map of an exemplary 3' endpoint
targeting vector automatically excised out of a phage clone
isolated from the 3' anchor library. The 3' anchor library is shown
in FIG. 8; this anchor library contains the expression cassette 3'
hprt, puromycin resistance gene, and k14-agouti gene. FIG. 12 shows
a map of pG12WT (Wildtype 3' hprt cassette plasmid for making
chromosomal rearrangements). At the bottom of FIG. 12, a comparison
of the sequence used to generate the data in Examples B-E versus
the sequence used to generate the data in this and following
examples is shown. As evidenced by FIG. 12, the two sequences are
identical to pG12 except that the mutation in 3' hprt of the
wildtype cassette plasmid has been fixed.
[0105] FIG. 13 shows the portion of the mouse chromosome 11 at
which the deletion strategy is directed; FIG. 13 also shows the
general composition of the selection cassettes positioned at the
chromosome endpoints, and the position of the Cre-induced deletion
interval, E.sub.2DH-D11Mit199.
[0106] Table 4 shows the frequency of Cre-induced deletion between
E.sub.2DH and D11Mit199, which can be compared with Tables 1A, 1B,
and 4. The frequency shown is the number of HAT-resistant colonies
per Cre-electroporated cell. The numbers are obtained by averaging
data from at least two experiments with at least two cell lines
(except for the new vector in the trans configuration). A
comparison of the data presented in Table 4 with those in Tables 1,
2, and 3 reveal that the cassette shown in FIG. 11 mediates
recombination approximately 10.sup.3 more efficiently than the
cassette used to generate the data in Tables 1, 2, and 4.
EXAMPLE H
Frequency of Cre-induced Deletion Between E.sub.2DH and D11 Mit69
Using an Improved Targeting Vector
[0107] Similar to Example H, this example also illustrates the
enhanced Cre-induced deletion frequency using a different targeting
vector compared with that used in the previous examples. Example H
illustrates the Cre-induced deletion frequency between E.sub.2DH
and D11Mit199--a distance of about 2 CM. By contrast, Example I
illustrates the Cre-induced deletion frequency between E.sub.2DH
and D11Mit69--a distance of about 22 CM). The details of the
protocol are substantially as described in the previous examples.
Details of this "improved vector" compared with the vector used in
the previous examples are shown in FIGS. 10 through 12 (the
original vector is shown by comparison in FIG. 12). FIG. 10 shows a
map of an exemplary 5' endpoint targeting vector automatically
excised out of a phage clone isolated from the 5' anchor library.
The 5' anchor library is shown in FIG. 9; this anchor library
contains the expression cassettes 5' hprt, neomycin resistance
gene, and tyrosinase gene. FIG. 11 shows a map of pG12WT (Wildtype
3' hprt cassette plasmid for making chromosomal rearrangements).
The sequence is identical to pG12 except that the mutation in 3'
hprt has been fixed.
[0108] FIG. 14 shows the portion of the mouse chromosome 11 at
which the deletion strategy is directed; FIG. 14 also shows the
general composition of the selection cassettes positioned at the
chromosome endpoints, and the position of the Cre-induced deletion
interval, E.sub.2DH-D11Mit69. Table 6 shows the frequency of
Cre-induced deletion between E.sub.2DH and D11Mit69, which can be
compared with Tables 1A, 1B, and 4.
5TABLE 6 Example H Markers Frequency HAT.sup.r, Puro.sup.s,
Neo.sup.2 5.8 .+-. 3.3 .times. 10.sup.-6 (n = 5)/2 .times.
10.sup.-5 (n = 1) HAT.sup.r, Puro.sup.r, Neo.sup.r 1.1 .+-. 0.4
.times. 10.sup.-5 (n = 10)/3 .times. 10.sup.-5 (n = 1)
[0109] Two frequencies are reported in each row, reflecting two
separate trials. The "frequency" of Cre-induced loxP recombination
is expressed as the number of HAT.sup.r colonies per
Cre-electroporated cell. The number of independent doubly targeted
cell lines is denoted by "n=."
6TABLE 5 Example G 3' hprt cassette Cis Trans Old (mutant) 2 .+-.
0.5 .times. 10.sup.-5 6.5 .+-. 3.2 .times. 10.sup.-7 New (wildtype)
2.3 .+-. 1.3 .times. 10.sup.-2 1.5 .times. 10.sup.-4
* * * * *