Copyright  2000 by the Genetics Society of America
Mechanisms Involved in Targeted Gene Replacement in Mammalian Cells
Julang Li and Mark D. Baker
Department of Molecular Biology and Genetics and Department of Pathobiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada
Manuscript received April 14, 2000
Accepted for publication June 26, 2000
ABSTRACT
The “ends-out” or omega (⍀)-form gene replacement vector is used routinely to perform targeted
genome modification in a variety of species and has the potential to be an effective vehicle for gene
therapy. However, in mammalian cells, the frequency of this reaction is low and the mechanism unknown.
Understanding molecular features associated with gene replacement is important and may lead to an
increase in the efficiency of the process. In this study, we investigated gene replacement in mammalian
cells using a powerful assay system that permits efficient recovery of the product(s) of individual recombination events at the haploid, chromosomal ␮-␦ locus in a murine hybridoma cell line. The results showed
that (i) heteroduplex DNA (hDNA) is formed during mammalian gene replacement; (ii) mismatches in
hDNA are usually efficiently repaired before DNA replication and cell division; (iii) the gene replacement
reaction occurs with fidelity; (iv) the presence of multiple markers in one homologous flanking arm in
the replacement vector did not affect the efficiency of gene replacement; and (v) in comparison to a
genomic fragment bearing contiguous homology to the chromosomal target, gene targeting was only
slightly inhibited by internal heterology (pSV2neo sequences) in the replacement vector.
G
ENE targeting studies commonly utilize the omega
(⍀)-form or “ends-out” gene replacement vector
to make predetermined alterations in chromosomal
genes (Waldman 1992; Bertling 1995). Gene replacement vectors usually consist of a dominant selectable
drug resistance gene flanked on both sides by homology
to the desired genomic locus. Correct homologous recombination replaces endogenous chromosomal sequences with those in the transferred DNA, including
the selectable marker. Drug-resistant cells are usually
selected in batch culture whereupon targeted cells are
identified by appropriate assays such as screening by
PCR or Southern analysis. In spite of its routine use
as a tool for targeted genome alteration, mechanisms
associated with the mammalian gene replacement reaction are not well understood.
Previously, we reported a gene targeting system that
detects homologous recombination between transferred vector DNA and the haploid, chromosomal immunoglobulin heavy chain locus in mouse hybridoma
cells (Baker et al. 1988). In the present study, this system
was modified and exploited to investigate mechanisms
of mammalian gene replacement. A gene replacement
vector was constructed that contained the selectable neo
gene flanked by homology to the constant region of
the immunoglobulin heavy chain ␮- and ␦-locus in the
hybridoma cells (the C␮ and C␦ region, respectively).
Corresponding author: Mark D. Baker, Department of Pathobiology,
Ontario Veterinary College, University of Guelph, Guelph, Ontario
N1G 2W1, Canada. E-mail: [email protected]
Genetics 156: 809–821 (October 2000)
The SV40 early region enhancer was removed from the
vector-borne neo gene creating an “enhancer-trap” gene
replacement vector. As shown previously (Bautista and
Shulman 1993; Ng and Baker 1998), similar enhancertrap vectors enrich for hybridoma cells targeted at the
chromosomal ␮-locus. This occurs because the ␮-locus
supplies the enhancer (or equivalent) activity required
for expression of the enhancerless neo gene in the targeted vector, whereas most random sites of vector integration in the hybridoma genome do not. To address
recombination mechanisms, the vector-borne C␮ region was marked with six restriction enzyme sites distinguishable from the corresponding sites in the chromosomal C␮ region. To isolate independent recombinants,
immediately following electroporation, the hybridoma
cells were segregated to individual wells of tissue culture
plates and placed under G418 selection as described
previously (Ng and Baker 1999a; Li and Baker 2000a).
Use of the enhancer-trap gene targeting vector in association with the segregation of the hybridoma cells immediately following gene transfer has three novel advantages in the study of mammalian recombination
mechanisms: (i) it ensures that each G418R recombinant
arises from a single cell deposited in the culture well;
(ii) it results in retention of the G418R product(s) of
individual gene replacement events for molecular analysis; and (iii) it permits targeted cells to be identified
without selection bias favoring recovery of a functional
recombinant ␮-gene. Using this system, we have identified and characterized several independent gene replacement events. The results reveal important new insights into the mammalian gene replacement process.
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J. Li and M. D. Baker
MATERIALS AND METHODS
Recipient hybridoma cell line: The haploid, chromosomal
immunoglobulin ␮-␦ heavy chain locus in the igm482 hybridoma cell line was used as the target for gene replacement
(Figure 1). The hybridoma cell line igm482 was isolated from
the wild-type Sp6 hybridoma cell line (subclone Sp6/HL)
that makes cytolytic, polymeric IgM(␬-chain) specific for the
hapten trinitrophenyl (TNP; Köhler and Shulman 1980;
Köhler et al. 1982). The distinguishing feature of the igm482
hybridoma cell line is that it bears a 2-bp deletion in the third
constant region exon of the TNP-specific chromosomal
␮-gene (C␮3). The 2-bp C␮3 deletion destroys an XmnI restriction enzyme site normally present in the wild-type Sp6/HL
C␮3 exon, creating in its place a TfiI site (position 4 in the
igm482 C␮ region shown in Figure 1). The igm482 mutation
results in the synthesis of a truncated ␮-chain lacking the C␮4
domain that is assembled into a monomeric form of IgM.
Unlike the normal TNP-specific IgM synthesized by the wildtype Sp6/HL hybridoma cell line, the mutant IgM made by
the igm482 cells is unable to activate complement-dependent
lysis of TNP-coupled sheep red cells (TNP-SRC). As described
below, the different properties of the IgM made by the mutant
igm482 and wild-type Sp6/HL hybridoma cell lines were exploited in the analysis of the recombinant hybridoma cell
lines. Aside from the different C␮3 exons, the chromosomal
␮-␦ region in the mutant igm482 and wild-type Sp6/HL hybridoma cell lines is otherwise isogenic. The methods used for
hybridoma cell culture have been described (Köhler and
Shulman 1980; Köhler et al. 1982).
Gene replacement vectors: The 13.1-kb ⍀-form, enhancertrap vector, pC␮M1-6C␦ (Figure 1) was used in the gene replacement studies. The backbone of this vector is derived from
pSV2neo (Southern and Berg 1981) from which the 372bp NsiI/NdeI fragment encompassing the SV40 early region
enhancer sequence responsible for neo gene expression was
removed. On the left and right flanking sides of the enhancerdeleted pSV2neo are genomic DNA segments from the wildtype Sp6/HL hybridoma cell line consisting of a 4.2-kb Bst1107/
XbaI C␮ region fragment and a 3.5-kb SpeI/SacI C␦ region
fragment, respectively. The position of these DNA segments
with respect to the chromosomal ␮-␦ region in the mutant
igm482 hybridoma cell line is indicated in Figure 1. The C␮
and C␦ homology regions share a 63-bp overlap between the
SpeI and XbaI sites located 3⬘ of C␮. The vector-borne C␮ and
C␦ homology regions are isogenic with those of the igm482
hybridoma cell line except for the following modifications.
As described previously (Ng and Baker 1999a), site-directed
mutagenesis was used to create the novel vector-borne C␮
region KpnI, EcoRV, DraI, AatII, and ScaI sites located at positions 150 bp, 557 bp, 1117 bp, 1601 bp, and 2041 bp, respectively, relative to the Bst1107 half site that marks the beginning
of the vector-borne C␮ region (nucleotide position 0 bp).
The vector-borne sites replace the endogenous C␮ region
AvaII, SacI, AflII, EarI, and NheI sites located at genomic positions 93 bp, 503 bp, 1053 bp, 1537 bp, and 1968 bp, respectively, according to the numbering in Goldberg et al. (1981).
In addition, as indicated above, the 2-bp mutant igm482 deletion creates a TfiI site in the C␮3 exon as opposed to the wildtype XmnI site in the corresponding position in the vectorborne C␮ region (position 1506 bp). Therefore, the C␮ region
in pC␮M1-6C␦ bears six diagnostic markers that distinguish it
from the corresponding endogenous sites in the recipient
igm482 hybridoma cell line. Originally, the vector-borne markers were constructed in the C␮ region of the gene targeting
sequence insertion vector pC␮En⫺M1-6 by site-directed mutagenesis with all sites being verified by DNA sequencing (Ng
and Baker 1999a). For this study, the markers were moved
into the pC␮M1-6C␦ gene replacement vector by subcloning
appropriate C␮ region segments.
To investigate potential effects of the C␮ region markers
in pC␮M1-6C␦ on the efficiency of gene replacement, the vector
pC␮WTC␦ was constructed. This was accomplished by swapping
a 1.9-kb Bst1107/DraIII fragment containing the marked C␮
region in pC␮M1-6C␦ with the corresponding segment from the
genomic, wild-type Sp6/HL C␮ region. With the exception
of the 2-bp mutant igm482 C␮3 deletion, the ␮-genes of the
wild-type Sp6/HL and mutant igm482 hybridoma cell lines
are the same. Thus, for illustrative simplicity, the position of
the 1.9-kb Bst1107/DraIII wild-type C␮ segment is presented in
Figure 1 in the corresponding position of the mutant igm482
chromosomal C␮ region. Restriction enzyme sites that were
convenient for this C␮ fragment exchange did not remove
the ScaI polymorphism (position 2041 bp in the vector-borne
C␮ region), but otherwise the C␮ region in pC␮WTC␦ was wild
type.
To examine possible effects associated with the heterology
created by the 5.4-kb enhancer-trap pSV2neo sequences in
pC␮M1-6C␦ and pC␮WTC␦, an 8.2-kb Bst1107/NdeI fragment of
C␮-C␦ genomic DNA (Figure 1) was isolated from cloned,
genomic DNA of the wild-type Sp6/HL hybridoma and tested
in the gene targeting reaction. Again, for illustrative purposes,
the position of this segment in the mutant igm482 chromosomal ␮-gene is shown in Figure 1.
DNA transfer and isolation of independent G418R transformants: The vector pC␮M1-6C␦ (8.7 pmol) was transferred to
2 ⫻ 107 recipient igm482 hybridoma cells by electroporation
as described (Baker et al. 1988). Trypan blue staining revealed
that typically ⵑ50% of the hybridoma cells survived electroporation. Independent G418R transformants were isolated by a
plating procedure described previously (Ng and Baker 1999a)
that ensures each transformant is derived from a single G418R
cell deposited in the culture well and that the product(s) of
each gene replacement event are retained in the culture well
for analysis.
In other studies, the frequency of gene targeting was measured in a different way. In separate experiments, 8.7 pmol
of either pC␮M1-6C␦, pC␮WTC␦ or the genomic Bst1107/NdeI
C␮-C␦ region fragment were transferred to 2 ⫻ 107 recipient
igm482 hybridoma cells by electroporation (Baker et al. 1988).
After ⵑ48 hr, the frequency of gene targeting was determined
by measuring plaque-forming cells (PFC) in a sensitive, TNPspecific plaque assay as described previously (Baker et al.
1988).
Identification and analysis of targeted, G418R recombinants:
Genomic DNA was prepared from individual G418R transformants generated following transfer of pC␮M1-6C␦ by the
method of Gross-Bellard et al. (1973). Individual DNAs were
screened by a specific PCR assay utilizing the primer pair
AB9703/AB9745 that binds outside the vector-borne C␮ region of homology as described previously (Ng and Baker
1999a; Li and Baker 2000a). As shown in Figure 1, this primer
pair generates a specific 4.8-kb PCR product from the C␮
region of targeted recombinants. Hybridoma cells identified
in this first screening were further characterized by Southern
analysis for the diagnostic restriction enzyme fragment sizes
predicted by the targeting event (Figure 1). For Southern
analysis, restriction enzymes were purchased from New England Biolabs Inc. (Beverly, MA), Amersham Pharmacia Biotech Inc. (Baie d’Urfé, Québec), and Canadian Life Technologies Inc. (Burlington, Ontario) and used in accordance with
the manufacturer’s specifications. Gel electrophoresis, transfer of DNA onto nitrocellulose membrane, 32P-labeled probe
preparation, and hybridization were all performed according
to standard procedures (Sambrook et al. 1989).
For analysis of C␮ region genetic markers in the recombi-
Mechanisms Involved in Targeted Gene Replacement
nants, the 4.8-kb PCR product was tested for its resistance or
sensitivity to digestion with each of the diagnostic restriction
enzymes indicated in Figure 1. As described earlier (Ng and
Baker 1999a), each enzyme produces diagnostic fragment
sizes that can be resolved by standard gel electrophoresis, thus
permitting assignment of the various genetic markers to the
correct positions.
Analysis of IgM production by the various hybridoma cell
lines was accomplished by testing culture supernatants for the
presence of either mutant monomeric or wild-type polymeric
TNP-specific IgM by hemagglutination and complementdependent lysis assays of TNP-SRC as described (Köhler and
Shulman 1980).
RESULTS
Description of experimental system: The single copy
of the chromosomal immunoglobulin ␮-␦ region in the
murine hybridoma cell line, igm482, serves as the target
for recombination with the 13.1-kb ⍀-form or “endsout” enhancer-trap, gene replacement vector, pC␮M1-6C␦
(Figure 1). In pC␮M1-6C␦, the C␮ and C␦ arms of homology used to effect gene replacement are 4.2 kb and 3.5
kb, respectively, and are separated by pSV2neo sequences. The enhancer-trap vector enriches significantly for gene targeting events at the chromosomal ␮-␦
locus (Bautista and Shulman 1993; Ng and Baker
1998), permitting recovery of independent recombinants by a plating procedure described previously (Ng
and Baker 1999a; Li and Baker 2000a). The plating
procedure ensures that each G418R recombinant arises
from a single cell and that the G418R product(s) of gene
replacement are retained for molecular analysis. To examine recombination mechanisms, the C␮ region of
pC␮M1-6C␦ was marked with six restriction enzyme site
polymorphisms, permitting it to be distinguished from
the corresponding markers in the mutant igm482 chromosomal C␮ region. The various restriction enzyme
site markers, along with their positions relative to the
Bst1107 site that marks the beginning of the C␮ region
(position 0 bp), are indicated in Figure 1.
Isolation of gene replacement events: A total of 163
independent G418R colonies were obtained following
electroporation of recipient mutant igm482 hybridoma
cells with the enhancer-trap pC␮M1-6C␦ vector. As hybridoma cell survival averaged ⵑ50% following electroporation, the frequency of G418R transformants/cell was
ⵑ163/(1 ⫻ 107) ⫽ ⵑ1.63 ⫻ 10⫺5. To identify putative cases in which pC␮M1-6C␦ had interacted with the
haploid chromosomal ␮-␦ locus, the G418R transformants were screened by PCR using the primer pair
AB9703/AB9745, which as shown previously (Ng and
Baker 1999a) generate a 4.8-kb product specific for the
recombinant C␮ region (Figure 1). No PCR product
was detected in 143 of the G418R transformants but
in 20 the correct 4.8-kb PCR product was observed.
Southern analysis using C␮- and neo-specific probe fragments was performed to verify correct gene replacement
in these cell lines. Hybridization with C␮ probe F re-
811
vealed that in 16 of the 20 G418R transformants, the
endogenous 25.0-kb HpaI ␮-␦ fragment was replaced
with the 14.4-kb HpaI fragment indicative of correct
gene replacement. Further verification of correct gene
replacement in these hybridoma cell lines was obtained
by hybridization of ScaI-digested genomic DNA with neo
probe G where the correct 12.6-kb ScaI fragment expected for gene replacement (Figure 1) was observed.
Representative examples of correctly targeted recombinants, as determined by the HpaI and ScaI digests, are
presented in Figure 2, A and B, respectively. In recombinant 25 (Figure 2B), an additional neo-hybridizing band
of 6.1 kb is visible, suggesting the possibility that, in
addition to gene replacement, a rare random vector
integration event might also have occurred. The remaining 4 of the 20 recombinants retained the 25.0-kb
HpaI fragment (data not shown), suggesting that the
endogenous ␮-␦ locus had not been modified by gene
replacement. However, in two of these hybridoma cell
lines, the 14.4-kb HpaI fragment was also visible. It is
possible that these represent cases in which the pC␮M1-6C␦
vector interacted with the chromosomal target and acquired endogenous sequences including the HpaI site
5⬘ of C␮ (Figure 1), but then ejected from the target
locus to integrate elsewhere. These cell lines are still
under investigation. Although unlikely, the possibility
was considered that the replacement vector may have
circularized, suffered a break in either the C␮ or C␦
region, and then undergone single reciprocal crossover
within the corresponding region of the chromosome,
resulting in targeted vector insertion. In the event crossover occurred between vector-borne and chromosomal
C␮ regions, HpaI fragments of 14.4 kb and 23.6 kb
would be detected with C␮-specific probe F whereas
crossover between C␦ regions would generate a single
22.1-kb HpaI fragment (predicted fragment sizes not
illustrated). However, as is evident from Figure 2A, none
of the observed fragment sizes support recombinant
generation by this mechanism. In summary, 16/163
(ⵑ10%) of the G418R transformants were identified as
being correct gene replacement events. Thus, on the
basis of the ⵑ50% survival of the igm482 hybridoma
cells following electroporation (ⵑ1 ⫻ 107 cells), the
absolute frequency of gene replacement was ⵑ16/(1 ⫻
107) ⫽ ⵑ1.6 ⫻ 10⫺6 events/cell.
Two features of the isolation procedure are important
to re-emphasize. First, plating the hybridoma cells immediately after electroporation makes it highly likely
that those destined to become G418R transformants are
segregated to individual culture wells before integration
of the transferred DNA into the chromosome. Second,
recombinants were recovered from among 3333 wells
plated and are expected to follow the Poisson distribution. Accordingly, the probability that the recombinants
in a well actually derived from more than one independent recombinant is ⵑ0.002. Thus, the G418R product(s) arising from each independent homologous re-
812
J. Li and M. D. Baker
Figure 1.—Gene replacement at the ␮-␦ locus. The structure of the haploid, chromosomal immunoglobulin heavy
chain ␮-␦ locus in the recipient
murine hybridoma cell line,
igm482, is presented along with
the chromosomal ␮-␦ structure
in a recombinant hybridoma cell
line generated following gene
replacement with the vector,
pC␮M1-6C␦. The chromosomal
and vector-borne C␮ regions are
distinguishable by the indicated
six pairs of restriction enzyme site
polymorphisms at positions numbered relative to the Bst1107 site
that defines the beginning of the
vector-borne C␮ region of homology (position 0 bp). Markers
diagnostic of the vector-borne
C␮ region are denoted in boldface type while the corresponding markers from the chromosomal C␮ region are indicated
in roman type. As gene replacement has the potential to generate different combinations of
chromosomal and/or vectorborne markers, the six corresponding positions in the recombinant C␮ region are designated by a question mark (?).
The primer pair AB9703/
AB9745 bind outside the C␮ region of homology in the replacement vector at positions described previously (Ng and
Baker 1999a; Li and Baker
2000a). They generate a specific
4.8-kb PCR product from
the recombinant C␮ region as shown. Probe fragments: C␮-specific probe fragment F is an 870-bp XbaI/BamHI fragment while probe
G is a 762-bp PvuII fragment from the neo gene. Abbreviations: C␮, ␮-gene constant region; C␦, ␦ gene constant region; VHTNP,
TNP-specific heavy chain variable region; neo, neomycin phosphotransferase gene. The thick line represents the vector, pSV2neo
(Southern and Berg 1981). The figure is not drawn to scale.
combination event are retained for analysis in a single
culture well.
Determination of C␮ region marker patterns: For
each recombinant, the specific 4.8-kb C␮ region PCR
product (Figure 1) was tested for its sensitivity or resistance to cleavage with restriction enzymes specific for
either chromosomal or vector-borne C␮ region markers. As indicated earlier (Ng and Baker 1999a), the
various restriction enzymes generate diagnostic fragment sizes that can be conveniently analyzed by standard
gel electrophoresis. This analysis was performed on each
of the 16 recombinants (data not shown) and the complete set of results is summarized in Figure 3.
In 9/16 (56%) of the recombinants (2, 3, 12, 21, 29,
71, 75, 88, and 103), a chromosomal marker was present
in every C␮ region position. In recombinants 6, 25,
30, and 32, the C␮ region positions were completely
sensitive to cleavage with restriction enzymes specific
for either the chromosomal or vector-borne marker. Unlike the other recombinants, one or more C␮ region positions in recombinants 5, 19, and 64 exhibited a mixed
cleavage pattern with restriction enzymes diagnostic of
either the chromosomal or vector-borne marker, suggesting that these recombinants were sectored (heterogeneous) cultures. As indicated above, the Poisson analysis showed that each recombinant was highly likely to
have been derived from a single cell deposited in the
culture well. Thus, the presence of a sectored site(s) in
these recombinants can be explained in the following
way. During gene replacement, heteroduplex DNA
(hDNA) may form between vector-borne and chromosomal sequences. In the event mismatches are left unrepaired prior to DNA replication and division of the
individual recombinant cell in the culture well, genetically distinct molecules are generated that segregate
to different daughter cells. Therefore, each sectored
Mechanisms Involved in Targeted Gene Replacement
813
Figure 2.—Southern analysis
of gene targeting events. Genomic DNA from the representative recombinants was digested
with (A) HpaI or (B) ScaI electrophoresed through a 0.7%
agarose gel and blotted to nitrocellulose. The HpaI blot (A)
was hybridized with C␮-specific
probe fragment F. As a control
for probe specificity, genomic
DNA was included from the recipient igm482 hybridoma cell
line in which the chromosomal
C␮-C␦ region is present on a
25.0-kb HpaI fragment (refer
also to Figure 1). The ScaI blot
(B) was hybridized with neo
probe G. As a control for the
specificity of probe G, genomic
DNA from the hybridoma cell
line (49/9) was included. As described previously (Ng and Baker 1999a), this cell line bears a single targeted copy of a pSV2neoderived sequence insertion vector and, consequently, probe G is expected to detect a single 20.6-kb ScaI fragment. The sizes of
fragments of interest are presented on the left of each blot while relevant DNA marker bands are indicated on the right.
recombinant very likely originated from the failure to
completely repair hDNA.
Recombinants 19 and 64 each bore a single mixed
C␮ region site. In the case of recombinant 19, the mixed
site was at position 6 while for recombinant 64, it was at
position 5. The gel analysis revealed that approximately
one-half the PCR product was sensitive to digestion with
the chromosomal and vector-borne restriction enzymes
specific for these sites. This suggested that each recombinant was composed of two distinct cell populations in
equal frequency that differed at these sites. Recombinant 5 was mixed for five of the six C␮ region sites and
thus it was necessary to determine the linkage relationship between the markers. To accomplish this, recombinant 5 was cloned at 0.1 cell/well and 12 independent
G418R subclones were isolated. The C␮ region marker
patterns in the recombinant 5 subclones are expected to
reflect the configuration present in the original hDNA
intermediate. Thus, restriction enzyme digestion of the
C␮ region PCR product was performed for each recombinant 5 subclone. As shown in Figure 3, this analysis
revealed that recombinant 5 was composed of not two
but four distinct subclone types in approximately equal
frequency.
Fidelity of the gene replacement reaction: The
igm482 hybridoma cell line was isolated as a mutant of
the wild-type Sp6/HL hybridoma cell line and bears a
2-bp deletion in the C␮3 exon (Köhler et al. 1982). The
2-bp igm482 deletion destroys an XmnI site normally
present in the wild-type C␮3 exon, creating in its place
a novel TfiI site. In addition, it results in production of
a truncated ␮-protein that when incorporated into the
TNP-specific IgM synthesized by the hybridoma cells
renders it distinguishable from the normal TNP-specific
IgM of the wild-type Sp6/HL hybridoma cell line on
the basis of lysis and agglutination of TNP-SRC (Köhler
and Shulman 1980).
Depending on the outcome of gene replacement,
recombinants are expected to bear either the mutant
igm482 TfiI or the wild-type XmnI restriction enzyme
site in exon C␮3 and to synthesize the mutant igm482
or wild-type IgM, respectively. As all other markers are
located in C␮ introns they are not expected to affect
␮-chain synthesis. In this study, recombinants were isolated on the basis of a predicted change in ␮-gene structure rather than function. Therefore, an unbiased indication of the fidelity of the gene replacement reaction
can be obtained by examining IgM production in the
recombinants.
Recombinants 5, 25, 30, and 32 bear the wild-type
(vector-borne) XmnI site in the C␮3 exon (Figure 3).
As shown in Table 1, the IgM produced by these recombinants has the same pattern of complement-dependent
lysis and agglutination of TNP-SRC as that of the wildtype Sp6/HL hybridoma cell line. In the remaining
recombinants, the TfiI site is present in exon C␮3 and,
like the mutant igm482 hybridoma, the IgM made by
these cells cannot lyse TNP-SRC and can only agglutinate TNP-SRC in the presence of anti-␮-serum, a distinguishing feature of the IgM made by mutant igm482
hybridoma (Köhler and Shulman 1980). Therefore,
the results in Table 1 are fully in accord with recombinants possessing a functional C␮ region in which the
C␮3 exon bears either the mutant igm482 or wild-type
sequence. These results, together with the C␮ region
marker analysis in Figure 3, support a gene replacement
mechanism that proceeds with fidelity.
C␮ region markers do not affect the efficiency of
gene replacement: To investigate whether the various
C␮ region markers affected the efficiency of gene re-
814
J. Li and M. D. Baker
TABLE 1
Analysis of TNP-specific IgM production
Hemagglutination of
TNP-SRC
Figure 3.—Marker patterns in the chromosomal C␮ region
of the recombinants. For clarity, the diagram focuses on only
the six C␮ region marker positions in the recombinant hybridoma cell lines as determined from restriction enzyme digestion
of the C␮ region PCR products. Markers denoting the vectorborne C␮ region are designated in boldface and by a solid
circle, while the corresponding markers in the chromosomal
C␮ region are indicated in roman type and by an open circle.
The C␮ region positions that were heterogeneous (sectored)
are denoted by a half-solid circle. Each recombinant was generated from an independent gene replacement event. Recombinant 5 was sectored for five positions in the C␮ region and
consisted of four distinct recombinant cell lines in the indicated frequencies. As detailed in the text, the four recombinant cell lines arose from four distinct genotypes determined
by the marker heterogeneity at C␮ position 6 (checkered
circle).
placement, the marked C␮ region in pC␮M1-6C␦ was exchanged for the wild-type C␮ region generating the
replacement vector, pC␮WTC␦. Available restriction enzyme sites for fragment exchange did not remove the
ScaI polymorphism at position 6 in the vector-borne C␮
region. However, with the exception of this site, the C␮
region in pC␮WTC␦ was otherwise wild type. As an assay,
we took advantage of the fact that recombination between transferred DNA bearing a wild-type C␮ region
and the mutant igm482 chromosomal ␮-gene can regenerate a wild-type ␮-gene sequence and restore normal
IgM production in the recombinants, permitting them
to be detected as PFC in a sensitive TNP-specific plaque
assay (Baker et al. 1988). As shown in Table 2, the mean
absolute frequency of TNP-specific PFC generated with
pC␮WTC␦ was 0.76 ⫻ 10⫺6 events/cell whereas with
pC␮M1-6C␦, it was 0.77 ⫻ 10⫺6 events/cell. According to
Hybridoma
Without
anti-␮ serum
With
anti-␮ serum
Hemolysis
of TNP-SRC
Controls
Sp6/HL
igm482
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2
3
5a
6
12
19
21
25
29
30
32
64
71
75
88
103
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⫺
⫺
⫾
⫺
⫺
⫺
⫺
⫹
⫺
⫹
⫹
⫺
⫺
⫺
⫺
⫺
a
In the sectored recombinant 5, an equal proportion of
subclones bear either the wild-type or mutant igm482 C␮3
exon as indicated by mixed results of the hemagglutination
and hemolysis assays.
a two-sampled t-test, the means are not significantly
different (P ⫽ 0.95). Thus, the C␮ region markers in
pC␮M1-6C␦ do not adversely affect the frequency of gene
replacement. Also, the absolute frequency of TNP-specific PFC generated with pC␮M1-6C␦ in the plaque assay
was similar to an independent determination of this
frequency derived from the fraction of recombinants
making normal TNP-specific IgM reported in Table 1,
multiplied by the absolute frequency of gene replacement as determined from the Southern analysis of
G418R colonies recovered in the 96-well tissue culture
plates [i.e., (fraction of recombinants making normal
TNP-specific IgM/total recombinants) ⫻ the absolute
frequency of recombination ⫽ (4/16) ⫻ (1.6 ⫻ 10⫺6) ⫽
0.4 ⫻ 10⫺6 events/cell]. This suggests that a common
gene replacement mechanism is involved in generating
recombinants in the two assay procedures.
Gene replacement in the absence of the vector-borne
neo gene: Further experiments were conducted to determine whether the heterology created by the pSV2neo
vector backbone in pC␮M1-6C␦ reduced the frequency
of gene targeting. To answer this question, an 8.2-kb
Bst1107/NdeI fragment spanning the ␮-␦ region of the
wild-type Sp6/HL hybridoma cell line was isolated from
cloned genomic DNA. As shown in Figure 1, this geno-
Mechanisms Involved in Targeted Gene Replacement
TABLE 2
Influence of C␮ region genetic markers on the
efficiency of gene replacement: Frequency of
generating TNP-specific PFC (⫻10⫺6)
pC␮M1-6C␦
pC␮WTC␦
0.70
0.78
0.75
0.83
x ⫽ 0.77
0.58
0.83
0.73
0.90
0.76
mic fragment contained the C␮-C␦ homology region
present in pC␮M1-6C␦ but as an uninterrupted segment.
The genomic fragment was transferred to recipient
igm482 hybridoma cells by electroporation and the
frequency of generating TNP-specific PFC was compared to that obtained with pC␮M1-6C␦. As shown in
Table 3, the frequency of TNP-specific PFC obtained
with pC␮M1-6C␦ was 0.73 ⫻ 10⫺6 events/cell, a value similar to that obtained with pC␮WTC␦ reported in Table 2.
A slightly higher frequency of generating TNP-specific
PFC (approximately twofold) was obtained with the genomic C␮-C␦ fragment, which, according to a two-sampled t-test, was significant (P ⫽ 0.003). Thus, the results
suggested that the 5.4 kb of pSV2neo sequences in the
gene replacement vectors had a marginal inhibitory effect on the frequency of gene replacement.
DISCUSSION
In this study, we investigated targeted gene replacement at the haploid, chromosomal immunoglobulin
␮-␦ locus in a murine hybridoma cell line using the
⍀-form enhancer-trap vector, pC␮M1-6C␦. Six novel restriction enzyme sites replaced the corresponding endogenous sites in the vector-borne C␮ region. These
restriction enzyme site polymorphisms served as genetic
markers permitting the contribution of vector-borne
and chromosomal C␮ region sequences in the recombinant products to be distinguished. Independent recombinants were isolated and identified by a powerful assay
TABLE 3
Influence of pSV2neo heterology on the efficiency
of gene replacement: Frequency of generating
TNP-specific PFC (⫻10⫺6)
pC␮M1-6C␦
C␮-C␦ fragment
0.60
0.80
0.83
0.68
x ⫽ 0.73
1.88
1.22
1.44
1.38
1.48
815
system in which the G418R product(s) of each individual
homologous recombination event was retained for molecular analysis. In addition, independent recombinants
were isolated without any selection bias favoring recovery of the functional product(s) of recombination.
A common feature of the 16 recombinants analyzed
in this study was the loss of one or more vector-borne
markers from the beginning of the C␮ region and their
replacement with the corresponding chromosomal sequence. In all recombinants, the KpnI site 150 bp from
the start of the vector-borne C␮ region was replaced
with the chromosomal AvaII site. In 13/16 recombinants, more extensive loss of vector-borne markers occurred. In recombinants 19 and 64, chromosomal markers replaced vector-borne markers up to and including
the XmnI site at position 1506 bp, while in recombinant
25, vector-borne markers were replaced up to the DraI
site at position 1117 bp. However, in a significant fraction of the recombinants (9/16 or 56%), all vectorborne markers were replaced with chromosomal markers. In principle, vector-borne markers might have been
removed by degradation from the end of the replacement vector with the deleted information being replaced by DNA synthesis using the homologous chromosomal sequence as template. While previous studies have
shown that degradation can occur at DNA ends during
mammalian gene transfer it does not appear to be extensive (Shulman et al. 1990; Hasty et al. 1992; Jiang et
al. 1992; Pfeiffer et al. 1994; Richard et al. 1997; Elliot
et al. 1998). Further, our previous gene targeting studies
using insertion (O)-type vectors also suggested that
DNA ends created by the vector-borne double-strand
break were usually subject to only slight degradation
(Ng and Baker 1999a; Li and Baker 2000a,b). Thus,
although the above studies are consistent with the possibility that degradation might have removed some vectorborne markers near the beginning of the C␮ region,
they are not consistent with the extensive amount of
degradation (exceeding 2041 bp) required to remove
all vector-borne markers in the major class consisting
of recombinants 2, 3, 12, 21, 29, 71, 75, 88, and 103.
Extensive terminal degradation is also inconsistent with
the retention of vector-borne markers near the beginning of the C␮ region in recombinants 5, 6, 30, and 32.
An alternate explanation for the replacement of vector-borne with chromosomal markers is that of mismatch repair (MMR) of hDNA. What is the evidence
for hDNA formation in the recombinants? The strongest
evidence is the sectoring observed in recombinants 5,
19, and 64. In yeast and fungi, sectoring is indicative of
a region of hDNA that did not undergo complete MMR
prior to DNA replication and division in the single cell
undergoing recombination and that can be detected in
both meiosis and mitosis (Petes et al. 1991). In this
study, the pattern of sectoring observed in recombinant
5 suggested that hDNA formation was extensive beginning prior to the second C␮ marker position (557 bp)
816
J. Li and M. D. Baker
and spanning all remaining 3⬘ marker positions over a
distance of at least 1484 bp. Unexpectedly, recombinant
5 was composed of not two but four distinct genotypes
in approximately the same frequency. A proposed mechanism for the generation of this unusual recombinant
is presented below. In recombinant 19, hDNA spanned
at least the last two marker positions for a minimum of
440 bp, while in recombinant 64 the evidence suggested
that, at a minimum, hDNA encompassed the fifth C␮
marker at position 1601 bp. Although only three sectored recombinants were found, it is unlikely that this
was due to any failure to recover hybridoma cells bearing
hDNA. That is, the isolation procedure involved hybridoma cells being segregated to the culture wells within
ⵑ1 hr following electroporation with the replacement
vector. This short interval would have provided insufficient time for the hybridoma cells to complete the process of gene replacement and undergo cell division
prior to their deposition in the culture well, events that
would have obscured evidence of hDNA.
While the position of sectored sites provided the best
evidence for hDNA, other evidence was indirect and
based on the pattern of MMR. In recombinant 6, it was
concluded that hDNA spanned a minimum of 1484 bp
encompassing C␮ region markers between the second
and sixth positions inclusive (557–2041 bp). Within this
region of hDNA, MMR occurred in patches: some mismatches were converted to the chromosomal sequence
whereas others underwent restoration to the vectorborne sequence. Patchiness in MMR was also revealed in
recombinant 19 where, in the hDNA tract that spanned
marker positions 5 and 6, marker 5 was restored to
the vector-borne sequence while marker 6 remained
unrepaired. It was argued above that, in the major class
of recombinants (2, 3, 12, 21, 29, 71, 75, 88, and 103),
replacement of the majority of vector-borne with chromosomal markers could not easily be accounted for by
a mechanism involving DNA end degradation. If so,
then much of the gene conversion observed in this
recombinant class can also be explained on the basis
of MMR of hDNA. If, as suggested from the data above,
hDNA does play an important role in the mammalian
gene replacement reaction, then the observation that
most C␮ region positions in the recombinants contained either a chromosomal or vector-borne marker
suggests that, in individual hybridoma cells undergoing
gene replacement, mismatches in hDNA are usually efficiently repaired prior to DNA replication and cell division.
The finding that recombinant 5 was sectored in five
of the six C␮ positions was very surprising because sectoring was not as frequent in the other recombinants.
Another very unusual feature was that this recombinant
consisted of four distinct genotypes rather than the two
expected if hDNA were generated by a single gene replacement event in which markers had escaped MMR.
As indicated in the results section, according to the
Poisson distribution the probability that the recombinants in a well actually derived from more than one
independent recombinant is ⵑ0.002. This makes it
highly unlikely that two gene replacement events occurred in separate hybridoma cells in the culture well.
Further, such an occurrence would require unrepaired
hDNA to persist in both cells and for markers to be
present in the reciprocal linkage pattern observed. A
proposed mechanism that more readily accounts for
the C␮ region marker pattern in recombinant 5 is depicted in Figure 4. As shown in Figure 4A, a gene replacement event occurs in the ␮-␦ locus of a single
hybridoma cell with hDNA being generated across the
C␮ region. The vector-borne KpnI site may have been
replaced with the chromosomal AvaII site by repair synthesis. The C␮ region hDNA escapes MMR and, following DNA replication, two sister chromatids are generated that differ in this region (Figure 4B). A sister
chromatid exchange event then occurs, reversing the
linkage of the last pair of markers at position 6 [i.e., the
chromosomal NheI site (open circle) and the vectorborne ScaI site (solid circle) generating the recombinant in Figure 4C]. Following this, the cell divides to
form two daughter cells bearing unrepaired hDNA (Figure 4D). The hDNA remains unrepaired and, following
a second division, four genotypically distinct subclones
types are formed in equal proportion (Figure 4E). If
this interpretation is correct, recombinant 5 is a very
interesting hybridoma cell line. The properties of multiple recombination and unrepaired hDNA are similar
to some MMR deficiencies in the mouse; for example,
inactivation of the mouse Msh2 gene results in a mismatch repair deficiency, hyper-recombination, and predisposition to cancer (de Wind et al. 1995). Recombinant 5 is under further investigation in our laboratory.
In this study, recombinants were isolated and identified by procedures not normally utilized in studies of
mammalian homologous recombination, that is, by
methods that did not require the recombination products to be functional. This provided the opportunity
to investigate whether mammalian gene replacement
occurs with fidelity. According to our analysis of diagnostic C␮ region markers and ␮-chain production in the
recombinants, the overall process appears faithful.
These results agree with the majority of recombinants
examined in our previous studies of targeted vector
integration (Baker et al. 1988; Baker and Shulman
1988; Ng and Baker 1999a,b) and with the results of
other gene targeting studies (Zheng et al. 1991; Thomas
et al. 1992). Also, sequencing studies have suggested
that intrachromosomal homologous recombination in
mammalian cells occurs with fidelity (Stachelek and
Liskay 1988). It is known that illegitimate integration of
transfected DNA at random positions in the mammalian
genome can be associated with rearrangements near
the integration site (Roth and Wilson 1988). However,
at least for targeted vector integration, this does not
Mechanisms Involved in Targeted Gene Replacement
817
Figure 4.—Proposed mechanism for the generation of recombinant 5. As indicated in results,
the mitotically sectored recombinant 5 bears four distinct genotypes. The following pathway is
proposed to explain this unusual
mixed genotype. For further details, refer to the text.
seem to be a problem because the site of vector linearization is usually restored in the vast majority of recombinants in which this has been examined (Smithies et al.
1985; Baker et al. 1988; Baker and Shulman 1988; Ng
and Baker 1998, 1999a; Li and Baker 2000a,b). Of
further relevance to the issue of whether the genome
of targeted cells contains unwanted genetic changes is
the observation that gene targeting is usually not associated with illegitimate integration of vector DNA, a potentially mutagenic event (Baker et al. 1988; Baker and
Shulman 1988; Bollag et al. 1989; Waldman 1992,
1995; Bertling 1995; Ng and Baker 1998, 1999a,b; Li
and Baker 2000a,b). Nevertheless, some studies suggest
that gene targeting might be error prone (Thomas and
Capecchi 1986; Doetschman et al. 1988; Brinster et
al. 1989) and, more specifically, that gene replacement
might be less accurate than targeted vector integration
(Hasty et al. 1991). The precise mechanisms involved
in generating these unwanted genetic alterations are not
known although, in one case (Thomas and Capecchi
1986), it was pointed out that the changes might have
reflected peculiarities associated with the recombining
sequence. It has since been learned that the efficiency
of gene targeting is similar whether insertion or replacement vectors are utilized (Thomas and Capecchi 1987;
Deng and Capecchi 1992). It has also been shown that
the fidelity of gene targeting is reduced when the vector
bears DNA that is nonisogenic to the target (Deng and
Capecchi 1992; Te Riele et al. 1992). In view of the
hDNA that is formed during gene replacement, as
shown here, and during targeted vector integration, as
shown previously (Ng and Baker 1999a,b; Li and Baker
2000a,b), it is possible that a low error rate might be
associated with MMR processing or perhaps with repair
synthesis of double-stranded gaps, as suggested previously (Strathern et al. 1995). Another critical factor
is the length of homology that can affect both the efficiency (Shulman et al. 1990; Deng and Capecchi 1992)
and accuracy of gene targeting (Thomas et al. 1992).
For example, the accuracy of gene replacement can be
reduced when the amount of target-homologous DNA
on one arm falls below ⵑ1 kb (Thomas et al. 1992) and
this can generate recombinants bearing one homologous and one nonhomologous junction by a mechanism
involving one-sided invasion (Belmaaza et al. 1990;
Berenstein et al. 1992). Thus, one or more of the above
factors may have contributed to the mutations observed
in the earlier gene targeting studies (Thomas and
Capecchi 1987; Doetschman et al. 1988; Brinster et
al. 1989; Hasty et al. 1991). A conclusion from most
studies and one that appears to be supported by the
present work is that gene targeting, like other forms of
homologous recombination is largely a faithful process.
The issue of fidelity in gene targeting is important from
818
J. Li and M. D. Baker
the basic viewpoint of understanding mechanisms of
homologous recombination. It is also important from
the practical standpoint of its use in directed genome
modification because, ideally, gene targeting would involve precise alteration of a chromosomal gene in the
absence of extraneous changes.
To determine whether the C␮ region markers in
pC␮M1-6C␦ had any effect on gene targeting we compared the efficiency of generating TNP-specific PFC between pC␮M1-6C␦ and the vector pC␮WTC␦ in which the
C␮ region was wild type. No significant difference in
the gene targeting frequency was observed for the two
vectors, suggesting that the C␮ markers did not reduce
the recombination frequency. A similar result was also
obtained in gene targeting studies using enhancer-trap
insertion vectors (Ng and Baker 1999a). Previous studies have suggested that mismatches between two recombining sequences can reduce the efficiency of homologous recombination although usually greater than a few
percentage points mismatch is required (Waldman and
Liskay 1987; Deng and Capecchi 1992; Te Riele et al.
1992). The genetic markers in this study contribute
⬍1% heterology to the vector-borne C␮ region. Further,
with the exception of the markers at positions 4 and 5,
the remaining adjacent markers are separated by a few
hundred base pairs of sequence homology, which is
greater than the ⵑ134–232 bp of uninterrupted homology required for efficient mammalian intrachromosomal recombination (Waldman and Liskay 1988). Out
of concern that the 5.4 kb of pSV2neo vector sequences
in pC␮M1-6C␦ (and pC␮WTC␦) might have exerted a
strong inhibitory effect on the frequency of gene replacement, we compared the frequency of generating
TNP-specific PFC with pC␮M1-6C␦ to that obtained with
an isolated fragment of genomic DNA in which the
C␮-C␦ homology region was contiguous. The results
revealed only a slight (approximately twofold) increase
in gene targeting frequency with the genomic C␮-C␦
fragment.
The possibility was considered that gene replacement
as measured by the TNP-specific plaque assay might not
occur by the same mechanism that generated the G418R
recombinants identified by Southern analysis of transformants arising in the 96-well tissue culture plate assay.
That is, generation of a TNP-specific PFC would require
only that the mutant igm482 chromosomal C␮3 exon
be corrected to the wild-type sequence present in the
C␮ region of the replacement vector or the genomic
C␮-C␦ fragment and not necessarily that the transfected
DNA be incorporated into the chromosome such as
occurs in the gene replacement reaction. However, the
similarity in the absolute frequency of TNP-specific PFC
generated with pC␮M1-6C␦ and, for the same vector, the
absolute frequency of G418R recombinants making normal TNP-specific IgM recovered from the 96-well tissue
culture plates suggests that the two assay systems recover
recombinants that are probably generated by a common
gene replacement mechanism.
Of the 20 recombinants initially identified in the PCR
screening of the G418R transformants, 16 bore the expected structure for correct replacement targeting at
the endogenous ␮-␦ locus. However, 4 of the 20 cell
lines had an intact endogenous target site but were
PCR positive probably as a consequence of the vector
acquiring sequences from the target locus prior to random integration. This phenomenon is likely the same
or similar to the gene conversion-like process described
previously (Adair et al. 1989; Ellis and Bernstein
1989), which is consistent with one-sided invasion (Belmaaza et al. 1990). In this study, the frequency of this
event might be an underestimate as the one-sided invasion process would not necessarily always extend to the
primer AB9703 binding site and give a positive PCR
signal. The generation of hybridoma cell lines bearing
correct replacement events, as well as those in which the
transferred vector appears to have interacted transiently
with the target locus, suggests that the two ends of the
vector are behaving independently during the recombination process. This predicts another class of recombinants which, while not observed in this study, have been
described previously: those in which one end of the
replacement vector undergoes homologous recombination with the target locus while the other end undergoes
illegitimate recombination nearby (Berenstein et al.
1992; Dellaire et al. 1997).
Several features of the gene replacement reaction
documented in this study are similar to those obtained
in a previous study of targeted vector integration utilizing an enhancer-trap insertion (O-type) vector in which
the C␮ region of homology contained the same genetic
markers (Ng and Baker 1999a). That is, the absolute
frequencies of gene targeting are similar (ⵑ10⫺6 events/
cell), hDNA formation can be extensive in both cases,
small heterologies had no measurable effect on the gene
targeting frequency, and MMR tended to occur prior
to DNA replication in both circumstances. The latter
result suggests that the hybridoma cell lines are normally
MMR proficient at least for these simple mismatches
and that the repair of hDNA is not influenced by the
way in which it was generated during replacement or
integrative forms of gene targeting. Although targeted
vector integration is consistent with the double-strandbreak repair (DSBR) model of recombination (Valancius and Smithies 1991; Ng and Baker 1999a; Li and
Baker 2000a,b), gene replacement is not expected to
occur by standard DSBR. Thus, in theory, the two modes
of gene targeting could have been quite different. However, as it turns out, the results suggest that the two
modes of gene targeting are similar in some respects.
These similarities between gene replacement and targeted vector insertion in mammalian cells appear to
contrast with data for the comparable events in the yeast
Saccharomyces cerevisiae. In S. cerevisiae, targeted vector
Mechanisms Involved in Targeted Gene Replacement
insertion is also consistent with the DSBR model of
recombination (Orr-Weaver et al. 1981; Szostak et al.
1983), whereas assimilation of a single strand of incoming DNA into the chromosome is suggested to be an
important mechanism of gene replacement (Leung et
al. 1997). Extensive hDNA is formed during gene replacement in yeast, but during targeted vector insertion,
formation of hDNA around the double-strand break
might be limited to perhaps a few hundred base pairs
(Orr-Weaver et al. 1988; Sweetser et al. 1994). Also,
the efficiency of gene replacement and targeted vector
insertion may differ in yeast (Leung et al. 1997).
In principle, the mammalian gene replacement reaction is consistent with three mechanisms. One mechanism involves assimilation of a single strand of the vector
into the chromosome, a process that would generate
hDNA across the entire region. A second mechanism
involves two independent crossover events confined to
the homologous ends of the replacement vector. No
hDNA is formed and internal double-stranded DNA in
the vector is inserted into the chromosome. A third
mechanism also postulates two crossing-over events, but
in this case associated with extensive hDNA formation.
Regarding how vector ends pair with their homologous
chromosomal sequence, the marker patterns in recombinants 5, 6, 30, and 32 suggest that, in the case of the
C␮ region, pairing initiates near the beginning of the
homology region. Pairing between vector-borne and
chromosomal strands near the beginning of the C␮
region would allow for potential crossover near the DNA
end and thus readily explain the marker pattern in
recombinants 30 and 32. In addition, it would provide
the opportunity for hDNA formation to begin near the
start of the C␮ region and span internal sites as suggested from the marker patterns in at least recombinants 5 and 6.
As indicated above, Leung et al. (1997) favored strand
assimilation to explain replacement of a chromosomal
allele in S. cerevisiae by a single, contiguous 2-kb homologous DNA fragment released by HO endonuclease cleavage from a different position in the yeast genome. In
contrast, a crossover-at-ends model was proposed to explain gene replacement in mammalian cells (Deng et
al. 1993) and in S. cerevisiae (Negritto et al. 1997). In
the study by Deng et al. (1993), the conclusion was based
on the ⱖ50% frequency of cotransfer of a selectable
neo marker and a nonselectable ClaI marker located 3
kb away. However, this result would also appear consistent with two crossing-over events if, as shown in the
present study, hDNA formation was extensive and encompassed this site whereupon MMR occurred in the
direction of the vector-borne ClaI marker. In the investigation by Negritto et al. (1997), a DNA fragment bearing a region of 17% mismatch to the chromosomal
target was released by HO-endonuclease cleavage from
a single-copy plasmid in the nucleus of S. cerevisiae. One
of the main pieces of evidence supporting crossover at
819
the fragment ends was the inhibition of gene replacement by mismatches, but only when they were positioned near the terminus of the fragment. However, in
their article, Leung et al. (1997) argued that these results might also be expected if replacement occurred
by strand assimilation.
The results of the present study tend to suggest against
strand assimilation as the mechanism. Assimilation of a
single strand of the pC␮M1-6C␦ or pC␮WTC␦ gene replacement vectors into the chromosome would be expected
to be strongly impeded by the 5.4 kb of heterologous
pSV2neo sequences. In the event such strand assimilation occurred at all, a large looped-out region would
form in the hDNA intermediate and to generate G418R
recombinants, MMR would have to favor the pSV2neo
sequences. Alternatively, DNA replication and cell division would permit survival of only one of the two daughter cells. This would preclude hDNA, contrary to what
was observed in some recombinants in this study. In
contrast, strand assimilation is expected to be much
more efficient with the genomic C␮-C␦ fragment because it shares nearly perfect contiguous sequence homology to the chromosomal target. Another reason to
anticipate a more efficient gene replacement reaction
with the isolated genomic fragment are studies of gene
replacement in yeast that suggest that heterologies in
the transforming DNA (such as the pSV2neo sequences
in the replacement vectors used in this study) are frequently removed by the MMR system in favor of the
sequences residing in the chromosome (Leung et al.
1997). It was surprising then that our results revealed
gene targeting with the C␮-C␦ fragment to be only
slightly (approximately twofold) more efficient than
with the replacement vectors. In considering the above
information, it seems more likely that gene replacement
with either vector DNA or contiguous genomic segments in mammalian cells involves two crossovers that
are associated with hDNA formation.
It might be possible to distinguish conclusively between the various gene replacement mechanisms. What
is required are genetic markers that are poorly repairable by the mammalian MMR machinery such that,
when they are included in homologous flanking DNA
on both sides of the selectable marker, evidence for
hDNA will frequently be preserved. Recently, we have
reported that a small palindrome genetic marker when
encompassed within hDNA formed in vivo during homologous recombination in the hybridoma cells avoids
MMR generating sectored recombinants at high frequency (Li and Baker 2000a). Our current work aims
to exploit the palindrome genetic marker to elucidate
the mechanism of mammalian gene replacement.
We thank Philip Ng for helpful comments at the inception of this
work and Erin Wever and Leah Read for excellent technical assistance.
This research was supported by a Post-Doctoral Fellowship from the
Medical Research Society (MRC) of Canada to J.L. and an MRC
Operating Grant (MT-14416) to M.D.B.
820
J. Li and M. D. Baker
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