Proc. Nati. Acad. Sci. USAVol. 87, pp. 4680-4684, June 1990Genetics

Introduction of specific point mutations into RNA polymerase II bygene targeting in mouse embryonic stem cells: Evidence for a DNAmismatch repair mechanism

(site-specific mutagenesis in mammalian cells/homologous recombination/a-amanitin resistance)

CAROL MIERNICKI STEEG*, JAMES ELLIS*t, AND ALAN BERNSTEIN*tt

*Division of Molecular and Developmental Biology, Mount Sinai Hospital Research Institute, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada;and tDepartment of Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada

Communicated by Paul Berg, April 6, 1990

ABSTRACT We have introduced two specific point muta-tions, located 20 base pairs apart, into the endogenous murinegene that encodes the largest subunit of RNA polymerase II(RPII21S). The first mutation conferred resistance to themushroom toxin a-amanitin (amar), and the second mutationgenerated a restriction fragment length polymorphism withoutaltering the protein sequence. Targeted amar clones weregenerated at a frequency of 1 in 30 totipotent embryonic stemcells that expressed stably integrated DNA vectors after elec-troporation. Thirty to 40% of these clones had acquired bothmutations, whereas, surprisingly, the remaining clones hadacquired the specific amar point mutation but lacked therestriction fragment length polymorphism. We suggest that thelatter clones were generated by independent DNA mismatchrepair rather than by double crossover or gene conversion.These results demonstrate that it is possible to introducespecific point mutations into an endogenous gene in embryonicstem cells. Thus it should be possible to introduce single basesubstitutions into other cellular genes, including nonselectablegenes, by optimizing the efficiency of gene transfer and/or thesensitivity of screening for targeted clones.

The creation of specific alterations in mammalian genes attheir native chromosomal loci has been made possible bygene targeting, or recombination between introduced DNAand a homologous region in the host cell chromosome (1-3).By using gene targeting in totipotent embryonic stem (ES)cells, predetermined modifications have been introduced intothe mouse germ line (4-7). Gene targeting in ES cells thusprovides a general approach to assess the role of a particulargene in the whole animal. In addition, it permits the gener-ation of mouse models of human genetic disease (8).To date, gene targeting has been used to effect the specific

disruption (5, 6, 9-16) or correction (3, 4, 17-20) of a numberofchromosomal loci in mammalian cells. However, the utilityand versatility of this technology would be greatly increasedif specific point mutations could be efficiently introduced intothe genes of ES cells. Such subtle alterations in coding andregulatory regions would allow detailed study of gene struc-ture and function. Here, we report the introduction by genetargeting of two specific point mutations, located 20 basepairs (bp) apart, into the gene that encodes the largest subunitof murine RNA polymerase II (RPII215). In contrast toearlier targeting strategies, in which the introduced DNAgenerally contained a large internal region of nonhomology(such as the bacterial neo gene), our vector DNA containedonly 2 bp of nonhomology in several kilobases (kb) of DNAhomologous to the chromosomal target locus. ES cell clonescontaining both point mutations were generated in three

experiments. However, contrary to our expectations, the twoclosely linked point mutations frequently segregated in thetargeted clones, suggesting that the targeting mechanism wasnot double crossover or gene conversion. Instead, we pro-pose that DNA mismatch repair was responsible for theintroduction of a single point mutation into the RPII215locus. The efficiency and directionality ofthis repair pathwayin mammalian cells vary, depending upon the particularnucleotide pair mismatch (21). Thus, ifDNA mismatch repairplays a role in the targeted site-specific mutagenesis of othergenes, these parameters may determine the efficiency withwhich specific point mutations can be introduced into thegenes of mammalian cells.

MATERIALS AND METHODSPlasmids and Construction of the Targeting Vector. Plasmid

pMClNeo-PolyA (9) was obtained from Stratagene. PlasmidspE26-7 and pRPMG were gifts of J. Corden. pE26-7 containsa 13-kb genomic fragment of mouse RPII215 with a singlepoint mutation at position 6819 that confers a-amanitinresistance (22). pRPMG ("RNA polymerase minigene") con-tains a 19-kb insert composed of the entire mouse RPII215genomic sequence (23) minus the first intron; the minigenealso carries the a-amanitin resistance (ama9 point mutation.Plasmid pE26-7 was digested with EcoRI and EcoRV, and the5.8-kb fragment containing the point mutation was subclonedinto the EcoRI site of pUC19. The Sph I and HindIII sites inthe pUC polylinker were subsequently destroyed, generatingplasmid pamaHS. The 0.6-kb Sph I-HindIII fragment con-taining the mutation was subcloned into M13mpl9. Site-directed mutagenesis was carried out using the "pulse"modification (24) to the procedure described by Zoller andSmith (25) and employing a uracil-substituted DNA template(26). The Sph I-HindIII insert was isolated from the mutantphage produced and used to replace the wild-type sequencein pamaHS, thereby creating pamaHSB.

Culturing, Electroporation, and Selection of ES Cells. ES-D3 cells (27) were cultured on a feeder layer of mitomycinC-inactivated primary mouse embryo fibroblasts (27) in Dul-becco's modified Eagle's medium/15% fetal calf serum/0.1mM 2-mercaptoethanol/1 mM sodium pyruvate. Prior toelectroporation, ES-D3 cells were resuspended in phosphate-buffered saline at a concentration of 6.25 x 106 cells per ml,and vector DNA was added. The cells were aliquoted intoelectroporation cuvettes (0.8 ml each) and subjected to asingle 250-V pulse at 500 ,uF in a Bio-Rad Gene Pulser. Theywere subsequently plated onto gelatinized tissue culture

Abbreviations: ES, embryonic stem; RFLP, restriction fragmentlength polymorphism; amar, a-amanitin resistance; PCR, polymer-ase chain reaction.tTo whom reprint requests should be addressed.

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dishes in Buffalo rat liver cell-conditioned (BRL) medium(28) at a density of 2.5 x 106 cells per 90-mm dish. Smallaliquots were removed and plated separately to determine thepercentage of cells surviving electroporation and plating.BRL medium was changed 24 hr after electroporation, andselection was begun after an additional 24 hr; thereafter, dailymedium changes were made for 10 days. Selection mediumfor cells electroporated with pRPMG or with pamaHSBinitially contained 1 jtg of a-amanitin per ml (Calbiochem);the concentration was increased to 2 ttg/ml after 2 days ofselection. Cells electroporated with pMClNeo-PolyA wereselected in 100 ,ug of G418 per ml (GIBCO). Resistantcolonies were picked with finely drawn-out Pasteur pipettesand cultured on feeder layers.

Southern Blot Analysis. Genomic DNA (5 ,ug per reaction)was digested with appropriate restriction enzymes, separatedon a 0.7% agarose gel, transferred to nitrocellulose, andhybridized to a random-primed probe (Pharmacia oligolabel-ing kit) by standard methods (29). Blots were washed at a finalconcentration of 0.1 x SSC (0.015 M NaCI/0.0015 M sodiumcitrate)/0.1% SDS at 650C.DNA Amplification and Sequencing. Two-hundred nano-

grams of genomic DNA was amplified by the polymerasechain reaction (PCR) (30) using Taq DNA polymerase (Ther-mus aquaticus DNA polymerase) (Perkin-Elmer/Cetus).The recommended reaction buffer was adjusted to an optimalconcentration of 1.2 mM MgCl2. Primers for PCR were RP1(5'-GCTGTGTAGCCCAGGTTGAG-3') and RP3 (5'-GGG-TGGAGAAACGATGGTGC-3'). Reactions were performedon an Ericomp thermal cycler set for an initial incubation at94°C (31/2 min) followed by 30 cycles of 94°C (1 min), 57°C (1min), 72°C (2 min), and a final incubation at 72°C (7 min). Theamplified product was digested with Sph I and HindIII andseparated on a 0.7% agarose gel, and the 600-bp fragment waspurified using Geneclean (Bio 101). This fragment was ligatedinto pUC18, and recombinant plasmids were sequenced usingSequenase (USBC) and the RP2 primer (5'-AGTGGGTGT-GAGACCAGCCA-3').

RESULTSTargeting Scheme. The pamaHSB targeting vector used for

the site-specific mutagenesis of the RPII215 chromosomallocus of ES cells is illustrated in Fig. 1. The vector containsa 5.8-kb EcoRI fragment, extending from intron 7 to exon 21of a doubly mutant RPII215 allele. The first mutation is asingle base-pair change (A to G) at position 6819 in exon 15,which confers dominant resistance to the mushroom toxina-amanitin (22). Since the 5.8-kb genomic fragment lacks thepromoter and 5' and 3' coding regions ofRPII215, integrationof pamaHSB into random sites in the host cell chromosomedoes not confer a-amanitin resistance. Thus, only recombi-nation events that introduce the amar point mutation into thenative RPII215 locus would be selected by growing cellselectroporated with pamaHSB in medium containing a-amanitin.

Using site-directed mutagenesis in M13, we introduced asecond point mutation into the targeting vector 20 bp down-stream from the first. The replacement of C with T at thisposition destroyed a BamHI site, while the amino acidencoded (isoleucine) remained the same. The silent BamHImutation was created to provide an independent means ofscreening amar colonies for targeted clones. Since the dis-tance between them was very short, we expected the twopoint mutations to cosegregate in all targeting events.

Generation of Targeted amar ES Cell Clones. PlasmidpamaHSB was digested with EcoRI prior to electroporationinto ES-D3 cells. ES-D3 cells were also electroporated in theabsence ofDNA to determine the spontaneous mutation rateto a-amanitin resistance, and with pMClNeo-PolyA (9) or an

IXb E

I IBH

IH Xb

amaHSB

An

VVT CAG AAT GTA GAG GGTC hTA CAT CTC C

SGCCCG

BarnH

AAG CGG ATC CCATTC GCC TAG GGT

AMA CAG GAT GTA GAG GGC AAG CGG ATT CCAGTC CTA CAT CTC CCG TTC GCC TAA GGT

Asp

FIG. 1. Targeting vector for the site-specific mutagenesis of themouse RPI1215 gene. The top panel depicts a portion of the RPII215genomic sequence. Exons are shown in black, with exon numberabove; introns are in white. Xb, Xba I; E, EcoRI; B, BamHI; H,HindIII. The solid black bar depicts the 5.8-kb genomic fragment ofthe amar mutant allele of RPJI215 that was used as the insert in thepamaHSB targeting vector. The sequence of the wild-type RPII215gene beginning at nucleotide 6816 in exon 15 and the same region ofthe pamaHSB vector are shown in the bottom panel. The pointmutation that confers a-amanitin resistance and the silent pointmutation that creates a BamHI restriction fragment length polymor-phism (RFLP) are indicated by arrows.

amar RPII215 minigene (pRPMG) to assess the frequency ofrandom integration and expression of introduced DNA. Afterelectroporation, the cells were plated in BRL medium andallowed to recover for 48 hr. Selection with a-amanitin orwith G418 was then begun and continued for 10 days.Resistant clones were picked, expanded, and analyzed.Table 1 gives the results of three independent gene target-

ing experiments. Pooling the data from experiments 2 and 3,a single spontaneous amar clone arose from 3 x 106 ES-D3cells that survived electroporation in the absence ofDNA. Inparallel, 107 ES-D3 cells surviving electroporation with thepamaHSB targeting vector yielded a total of 180 amar clones.The elevated frequency at which the latter clones weregenerated, in contrast to the spontaneous mutation rate,strongly suggests that they were derived from gene targeting.To determine the frequency of random integration events

and the efficiency of electroporation, we used a plasmid thatcould be selected with the same drug as the targeted clones.Thus, the positive control plasmid pRPMG, which containsa functional amar RPII215 minigene, was electroporated andselected with a-amanitin in parallel to the pamaHSB targetingvector (see Table 1). In both experiments 2 and 3, targetedamar clones were generated at a frequency of 1 in 30 cells thatexpressed stably integrated pRPMG DNA (correcting for cellnumbers and for the molarity of the introduced DNA). Theoverall efficiency of electroporation in experiment 3 (seeTable 1, electroporation of pRPMG and pMClNeo-PolyA)was high relative to the other two experiments, and theabsolute number of targeted amar clones generated in thisexperiment was 5-fold greater than in experiment 2.

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Table 1. Gene targeting by electroporation of pamaHSB to generate amar ES cells

DNA electroporated ES cells ES cells Drug-resistant BamHI Frequency*

Exp. Plasmid nM ,ug electroporated, no. surviving, no. colonies, no. RFLP pRPMG pMClNeo-PolyA

1 pMClNeo-PolyA 10.5 20 5.0 x 106 3.9 x 105 71 G418pamaHSB 9.4 400 5.0 x 10 3.9 x 106 t 1/3 t

2 No DNA 5.0 x 106 5.2 x 105 OamapRPMG 3.3 35 5.0 x 106 5.2 x 105 30 amapamaHSB 9.4 400 5.0 X 107 5.2 x 106 30 ama 7/22 1/29

3 No DNA 2.5 x 107 2.5 x 106 1 ama 0/1pRPMG 3.8 40 5.0 x 106 5.0 x 105 180 amapMClNeo-PolyA 21 40 5.0 x 106 5.0 x 105 289 G418pamaHSB 9.4 400 5.0 x 107 5.0 x 106 150 ama 12/29 1/30 1/9

*Frequency of amar colonies from pamaHSB electroporation as compared to number of random integrations of pRPMG or pMClNeo-PolyA(corrected to equivalent DNA molarity and surviving cell numbers).

tSelection in experiment 1 was with a-amanitin (Boehringer Mannheim), which was past its expiration date, at a concentration of 1 ,ug/ml. Thisproduced many false positive clones. It was necessary to pool the clones on individual plates and reselect using fresh a-amanitin (Calbiochem)at 2 ,g/ml. An accurate targeting frequency cannot be calculated, but the three twice-selected clones that were analyzed did arise from differentoriginal plates. Subsequent experiments used fresh a-amanitin (Calbiochem) at 2 ug/ml.

Characterization of amar ES Cell Clones by Southern BlotHybridizations. DNA from the amar clones generated byelectroporation of ES-D3 cells with pamaHSB was subjectedto Southern blot analysis to screen for the BamHI RFLP. Theprobe used was a 1.8-kb Bgl II-HindIII restriction fragmentof RPII215 that spans the BamHI site in exon 15 (see Fig. 2).As shown in Fig. 2, digestion of wild-type ES-D3 DNA withBamHI followed by hybridization to this probe yielded two

-amaHSB

H XE H XII

E

I i IB B B

5.2 5.8- probe

B X E H

1 1-

0IW0

-ps

_.-

5.8-

5.2--s

FIG. 2. Southern blot analysis of DNA from the parent ES-D3cell line and a typical amar targeted ES cell clone (9-3). A map of thenative RPJJ215 chromosomal locus is given, with the solid black barrepresenting the 1.8-kb Bgl II-HindIII fragment used as a probe, andthe open bar representing the 5.8-kb insert of the pamaHSB targetingvector. Lanes of the autoradiogram are arranged in pairs, ES-D3 onthe left and clone 9-3 on the right. Fragment sizes are given in kb. B,BamHI; X, Xba I; E, EcoRI; H, HindIII.

bands of 5.8 and 5.2 kb. In contrast, 30-40% of the amarclones generated in each of three independent experimentspossessed the introduced BamHI RFLP (see Table 1). South-ern blot analysis of one such clone (9-3), generated inexperiment 1, is shown in Fig. 2. Digestion ofDNA from thisclone with BamHI gave rise to a new band of 11 kb, whereasthe intensities of the 5.8- and 5.2-kb bands were halved aspredicted. When subjected to additional restriction digestswith enzymes that cleave outside the region homologous tothe introduced DNA, the restriction patterns of wild-typeES-D3 and clone 9-3 were identical across a region spanning27.5 kb and encompassing essentially the whole RPII215locus (Fig. 2). Identical results for 20 of 54 clones analyzedstrongly suggest that the simultaneous acquisition of a-amanitin resistance and the BamHI polymorphism by theseclones was the result of a gene targeting event. None of the54 amar clones contained either plasmid or RPII215 se-quences integrated at random sites elsewhere in the genome(data not shown).

Amplification and Sequence Analysis of Target Loci of ama'ES Cell Clones. Surprisingly, 60-70% of the amar clonesgenerated in each experiment did not possess the BamHIRFLP. However, these clones arose at a much higher ratethan the spontaneous mutation frequency (see Table 1,experiments 2 and 3), suggesting that they too resulted fromgene targeting events. To determine whether these clonespossessed the specific amar point mutation (A toG at position6819 in exon 15) and to confirm that the clones possessing theBamHI polymorphism also had the specific amar mutation,we amplified the targeted region using PCR (30), subclonedthe amplified fragment, and sequenced the DNA from indi-vidual subclones derived from six amar colonies. Two amarcolonies that possessed the BamHI polymorphism by South-ern analysis were found to have the two expected pointmutations in one allele (see Fig. 3 for sequence of antisensestrand). Four amar colonies that had not demonstrated theBamHI RFLP were confirmed to have intact BamHI sites inboth alleles but had acquired the amar point mutation (T to Cin the antisense strand) at position 6819 in one allele (see Fig.3). No other mutations were detected within a 140-bp regioncentered around position 6819 in any of the targeted amarclones analyzed. The single spontaneous amar mutant thatarose after electroporation of ES-D3 cells in the absence ofDNA also possessed an intact BamHI site but, in contrast,was wild-type at position 6819 (data not shown). This latterobservation supports the conclusion that the clones possess-ing the specific point mutation at position 6819 resulted fromgene targeting events.

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Amnar BamrpaG T =

pamnaHSB a- A

G A T C

_...l s

,,W. f

aw* -. . F

_&a"

G A T C., .

IAsp

AmZ lam

G A T C

-*#. -oft

O" db

1.1

::B:_. * Bam--f Hi

RP11215(WT) a T GG

Arras Bam+

Heteroduplex formationI

FIG. 3. DNA sequence analysis of amar targeted ES cell clones.(Upper) Region ofthe RPIJ215 gene that was amplified from the amarclones by PCR and subsequently sequenced. RP1, RP2, and RP3were the 20-bp primers used for PCR or for sequencing. B, theBamHI site in exon 15; S, Sph I; H, HindIII. (Lower) Sequenceladders generated from the antisense strand of a particular allele(position 6816 at top). Right: wild-type allele of amar clone SA.Center: targeted allele of amar clone 5A, which has a single pointmutation as indicated by the arrow. Left: targeted allele ofclone 18B,which has two specific point mutations as indicated by the arrows.Clones SA and 18B were generated in experiment 2.

DISCUSSIONThe experiments presented here demonstrate the feasibilityof introducing specific point mutations into an endogenousgene in ES cells. We have introduced two point mutationsinto the RPII215 chromosomal locus, at a frequency of 1targeted cell per 30 that expressed stably integrated DNAafter electroporation. This relative efficiency is higher than isgenerally achieved by electroporation and direct positiveselection and is comparable to that achieved by a positive-negative selection strategy (11, 15). One explanation for thehigh relative efficiency that we observed may be that, byusing a vector containing only two single base-pair substitu-tions within 5.8 kb of homology, we avoided large internalregions of nonhomology that may hinder recombination bygene conversion (31). A similar relative frequency was ob-served for the disruption of the Hox).1 gene by microinjec-tion of a vector containing only 20 bp of nonhomology (13).

Implicit in our targeting scheme was the assumption thatthe short distance (20 bp) between the two point mutations inthe introduced DNA would result in their cosegregation in themajority oftargeted recombination events. In fact, 30-40% ofthe selected amar events contained both mutations and aremost easily explained by gene conversion or double cross-over. In contrast, the remaining 60-70%o of the amar colonieshad the specific amar point mutation but lacked the BamHIpolymorphism. It is very unlikely that these clones arosefrom frequent crossovers or resolution of gene conversionwithin the 20-nucleotide region. Rather, we suggest that eachpoint mutation was independently corrected by a DNAmismatch repair pathway. The specific mismatches in ourexperiment would be two GT mispairs if the sense strand ofthe introduced DNA invaded and formed a heteroduplex withthe endogenous gene (see Fig. 4) or two AC mispairs if theantisense strand were involved. Previous experiments haveshown that =95% of GT mismatches and 75% of AC mis-matches in mammalian cells undergo correction (21, 32).Over 90% ofGT mismatches are corrected to GC (21, 32, 33),whereas there is no directional bias for AC repair (21). Thedirectionality of GT repair would account for the high pro-portion of targeted clones with only one point mutation, byfavoring the amar phenotype in the case of the first mismatchbut restoring the BamHI site in the second case (see Fig. 4).The targeted clones containing both point mutations couldhave arisen from independent mismatch repair of the AC

=G T=T G

Independent mismatch repair

-G -c-

-C -G-

Amar Bam+

-C-A

Amar Bam-

II

Amas(selected against)

III, IV

FIG. 4. Proposed mechanism for the targeted introduction ofpoint mutations into RPII215: Heteroduplex formation and indepen-dent DNA mismatch repair. The top panel depicts the endogenousRPII21S locus (solid lines) and the homologous region of the pama-HSB targeting vector (open lines). WT, wild type; s, sense strand; a,antisense strand. The specific base pairs shown confer a-amanitin-resistance or sensitivity and the presence or absence of a BamHIrestriction site as indicated. The center panel depicts the heterodu-plex formed ifthe sense strand ofthe pamaHSB vector is the invadingstrand. The possible products resulting from independent DNAmismatch repair and subsequent resolution of the heteroduplex areshown below (roman numerals). The predominance ofGT mismatchrepair to GC is illustrated by the thick arrow. In three targetingexperiments, 60-70% of the amar clones analyzed were from class I.

The remaining 30-40o were from class II. These could have arisenfrom AC mismatch repair following invasion of the antisense strandof pamaHSB or, alternatively, from double crossover or geneconversion. Classes III and IV, in which the first mismatch isrepaired to AT, would not survive selection with a-amanitin.

mismatches of a heteroduplex formed by the invasion of thepamaHSB antisense strand. Efficient DNA mismatch repairenzymes might also account for the high relative targetingfrequency.

Segregation of point mutations was also observed in ex-periments in which heteroduplex DNAs containing severalmismatches were injected into mammalian cells (34) and inextrachromosomal recombination experiments performed bythe same group in which homologous DNAs were coinjectedinto mammalian cells (35). The authors postulated that inde-pendent mismatch repair was responsible for the segregationin the former case and gene conversion was responsible in thelatter. Given their results, our observations on RPII215 aremost likely not locus-specific. If DNA mismatch repair of aheteroduplex is involved in gene targeting, then some pointmutations may be easier to introduce than others, dependingon the efficiency and directionality of repair of the specificmismatch. Further experiments will be necessary to confirmthis hypothesis, but it should be considered in devising futurestrategies for the site-specific mutagenesis of mammaliangenes.

It has been suggested that repair of a heteroduplex inter-mediate was involved in the introduction of de novo muta-tions in the target locus in two other gene targeting studies

RPI S B-

RP2

H

RP3

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(20, 36). However, in each case, the target locus or the vectorDNA contained a large deletion as well as single base-pairmismatches in the region of homology. The absence of denovo mutations in the targeted RPII215 genes described heresuggests that heteroduplex-induced mutagenesis (36) mayrequire a large internal region of nonhomology near singlebase-pair mismatches.

Previous studies have shown that resistance to a-amanitininhibits the in vitro differentiation of certain cells (37). Theamar ES cell clones we have generated will be useful forfurther investigation of this phenomenon, both in vitro and inES cell-derived chimeric and transgenic mice. In addition,the strategy for site-specific mutagenesis described herecould easily be extended to those mammalian genes for whichpoint mutations can be directly selected. The direct correla-tion between the absolute number of targeted clones and theefficiency of electroporation suggests that point mutationscould also be introduced into nonselectable genes. For ex-ample, in our most efficient experiment, one amar clone wasgenerated per 3.3 x 104 ES cells surviving electroporation.Although detection and isolation ofthese rare targeted eventswould be laborious, it should be possible to screen pools ofelectroporated ES cells by PCR of the target locus andhybridization to mutation-specific oligonucleotides. Furtheroptimization of gene transfer efficiency, perhaps by micro-injecting DNA, would increase the absolute number of tar-geting events and thereby ease the screening process.The introduction of specific point mutations into the coding

or regulatory regions of mammalian genes will allow fine-structure analysis of the functions of protein domains and ofcis-acting DNA elements. When combined with the ability oftotipotent ES cells to contribute to the mouse germ line, thisapproach will make it possible to generate mice carryingspecific point mutations, hence providing a powerful meansto address a wide variety of biological questions.

We thank J. Corden for the generous gift ofthe pE26-7 and pRPMGplasmids. We are grateful to A. Joyner, A. Gossler, A. Auerbach,and C. Moens for advice on culturing ES cells. We also thank J.Rossant and P. Dubreuil for critical reading of the manuscript. Thiswork was supported by grants from the Medical Research Counciland the National Cancer Institute of Canada. J.E. was supported bya studentship from the Natural Sciences and Engineering ResearchCouncil of Canada.

1. Lin, F.-L., Sperle, K. & Sternberg, N. (1985) Proc. Natl. Acad.Sci. USA 82, 1391-1395.

2. Smithies, O., Gregg, R. G., Boggs, S. S., Koralewski, M. A. &Kucherlapati, R. S. (1985) Nature (London) 317, 230-234.

3. Thomas, K. R., Folger, K. R. & Capecchi, M. R. (1986) Cell44, 419-428.

4. Thompson, S., Clarke, A. R., Pow, A. M., Hooper, M. L. &Melton, D. W. (1989) Cell 56, 313-321.

5. Schwartzberg, P. L., Goff, S. P. & Robertson, E. J. (1989)Science 246, 799-803.

6. Zijlstra, M., Li, E., Sajjadi, F., Subramani, S. & Jaenisch, R.(1989) Nature (London) 342, 435-438.

7. Koller, B. H., Hagemann, L. J., Doetschman, T., Hagaman,J. R., Huang, S., Williams, P. J., First, N. L., Maeda, N. &Smithies, 0. (1989) Proc. Natl. Acad. Sci. USA 86,8927-8931.

8. Capecchi, M. R. (1989) Trends Genet. 5, 70-76.9. Thomas, K. R. & Capecchi, M. R. (1987) Cell 51, 503-512.

10. Doetschman, T., Maeda, N. & Smithies, 0. (1988) Proc. Natl.Acad. Sci. USA 85, 8583-8587.

11. Mansour, S. L., Thomas, K. R. & Capecchi, M. R. (1988)Nature (London) 336, 348-352.

12. Sedivy, J. M. & Sharp, P. A. (1989) Proc. Natl. Acad. Sci.USA 86, 227-231.

13. Zimmer, A. & Gruss, P. (1989) Nature (London) 338, 150-153.14. Joyner, A. L., Skarnes, W. C. & Rossant, J. (1989) Nature

(London) 338, 153-156.15. Johnson, R. S., Sheng, M., Greenberg, M. E., Kolodner,

R. D., Papaioannou, V. E. & Spiegelman, B. M. (1989) Sci-ence 245, 1234-1236.

16. Koller, B. H. & Smithies, 0. (1989) Proc. Natl. Acad. Sci. USA86, 8932-8935.

17. Doetschman, T., Gregg, R. G., Maeda, N., Hooper, M. L.,Melton, D. W., Thompson, S. & Smithies, 0. (1987) Nature(London) 330, 576-578.

18. Baker, M. D., Pennell, N., Bosnoyan, L. & Shulman, M. J.(1988) Proc. Natl. Acad. Sci. USA 85, 6432-6436.

19. Jasin, M. & Berg, P. (1988) Genes Dev. 2, 1353-1363.20. Brinster, R. L., Braun, R. E., Lo, D., Avarbock, M. R., Oram,

F. & Palmiter, R. D. (1989) Proc. Natl. Acad. Sci. USA 86,7087-7091.

21. Brown, T. C. & Jiricny, J. (1988) Cell 54, 705-711.22. Bartolomei, M. S. & Corden, J. L. (1987) Mol. Cell. Biol. 7,

586-594.23. Ahearn, J. M., Jr., Bartolomei, M. S., West, M. L., Cisek,

L. J. & Corden, J. L. (1987) J. Biol. Chem. 262, 10695-10705.24. Nisbet, I. T. & Beilharz, M. W. (1985) Gene Anal. Tech. 2,

23-29.25. Zoller, M. J. & Smith, M. (1982) Nucleic Acids Res. 10,

6487-6500.26. Kunkel, T. A., Roberts, J. D. & Zakour, R. A. (1987) Methods

Enzymol. 154, 367-382.27. Doetschman, T. C., Eistetter, H., Katz, M., Schmidt, W. &

Kemler, R. (1985) J. Embryol. Exp. Morphol. 87, 27-45.28. Smith, A. G. & Hooper, M. L. (1987) Dev. Biol. 121, 1-9.29. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular

Cloning:A Laboratory Manual (Cold Spring Harbor Lab., ColdSpring Harbor, NY).

30. Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn,G. T., Erlich, H. A. & Arnheim, N. (1985) Science 230, 1350-1354.

31. Ellis, J. & Bernstein, A. (1989) Mol. Cell. Biol. 9, 1621-1627.32. Brown, T. C. & Jiricny, J. (1987) Cell 50, 945-950.33. Wiebauer, K. & Jiricny, J. (1989) Nature (London) 339, 234-

236.34. Folger, K. R., Thomas, K. & Capecchi, M. R. (1985) Mol. Cell.

Biol. 5, 70-74.35. Folger, K. R., Thomas, K. & Capecchi, M. R. (1985) Mol. Cell.

Biol. 5, 59-69.36. Thomas, K. R. & Capecchi, M. R. (1986) Nature (London) 324,

34-38.37. Crerar, M. M., Leather, R., David, E. & Pearson, M. L. (1983)

Mol. Cell. Biol. 3, 946-955.

4684 Genetics: Steeg et al.

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