Proc. Nat!. Acad. Sci. USA Vol. 88, pp. 9498-9502, November 1991 Genetics
Gene targeting in Chinese hamster ovary cells is conservative (homologous recombination/gene correction/mammalian recombination/double-strand-break repair/single-strand anling)
SANDRA L. PENNINGTON AND JOHN H. WILSON Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030
Communicated by Salih J. Wakil, July 24, 1991 (received for review May 8, 1991)
Two fundamentally different pathways for ABSTRACT homologous recombination have been identified in mammalian cells. For most chromosomal recombination events, two copies of a homologous sequence recombine to yield two copies in the products; such events are said to be conservative because the number of copies is preserved. By contrast, virtually all extrachromosomal recombination events are nonconservative; two copies recombine to give a product containg a single intact copy (the other copy is destroyed in the mechanis). Since gene targeting involves an introduced (extrachromosomal) plasmid and a chromosomal target, it was not clear which pathway would apply. We used a marked vector to determine whether targeted integrants were products of recombination events that involved two copies (the conservative pathway) or three copies (the nonconservative pathway) of the homologous sequence. Among 51 gene targeting events, we identified 17 homologous integrants and analyzed their structures. All match the predictions for a conservative pathway. We conclude that the principal pathway for gene targeting in mammalian cells is conservative.
a double-stranded break, forming adjacent Holliday junctions. These junctions can be resolved to give linked or unlinked products. In both cases, the process starts with two copies of the homologous sequence and produces two copies. Since the number of copies is maintained in the recombination event, the overall process is said to be conservative. Thus, there are two possibilities for the nature of targeted recombination in mammalian cells: like extrachromosomal recombination, it might be nonconservative, or, like chromosomal recombination, it might be conservative. We asked whether the products of targeted integration matched the expectations of a conservative pathway or a nonconservative pathway. The chromosomal target for this study was the Chinese hamster ovary (CHO) adenine phosphoribosyltransferase (APRT) gene. The ease of selection for and against this gene facilitates generation of the large number of targeted clones needed for pathway determination. In this paper, targeted integration into the APRT gene is shown to occur in a way that is consistent with a conservative pathway.
Gene targeting is a powerful tool for manipulating mammalian genomes in precise ways (1-3). Learning what parameters affect the frequency of targeted recombination in mammalian cells may allow us to harness this normally rare event (4-6) to study gene expression, to make animal models for disease (7-9), and, ultimately, to correct human inborn errors of metabolism. Previous studies of chromosomal and extrachromosomal homologous recombination in mammalian cell lines suggest that two different pathways of recombination are used in mammalian cells (10-14). The predominant pathway for extrachromosomal recombination is nonconservative and can be described by the singlestrand annealing (SSA) model of homologous recombination (12). There exist other nonconservative models; SSA is used here for the purposes of illustration. In this model, two copies of a homologous sequence recombine to make a single copy. The model proposes a stripping away of strands from a break, pairing of exposed homologies, and repair ofresidual gaps and single-stranded extensions. Since one copy of the homologous sequence is destroyed in the process, the recombination event is said to be nonconservative. The primary pathway for homologous chromosomal recombination in mammalian cells is thought to be conservative (10, 11). Chromosomal recombination in yeast, which is also conservative, is stimulated by double-stranded breaks in a way that can be described by the double-strand-break repair (DSBR) model for homologous recombination (15-17). Other conservative models do not require double-stranded breaks, but DSBR serves as an appropriate example here because mammalian targeted recombination is stimulated by doublestranded breaks in the vector (4, 18). The model predicts that two copies of a homologous sequence pair on either side of
Plasmid Construction. Plasmid pAG6 (Fig. 1) was derived from plasmid pAG1 (19), which is a modified pSV2gpt plasmid containing the structural hamster APRT gene in the unique BamHI site. The plasmid backbone of pAG6 was altered by filling in the HindIII site near the simian virus 40 promoter; the GPT gene is still expressible. The APRT gene was altered by inserting a 37-base-pair (bp) polylinker into the unique Xho I site in the third exon. This modification destroyed the Xho I site and created an insertional, frameshift mutation of APRT. The polylinker contains a HindIII site and it was at this site that the plasmid was linearized for the
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Abbreviations: SSA, single-strand annealing; DSBR, double-strandbreak repair; APRT, adenine phosphoribosyltransferase; GPT, guanine phosphoribosyltransferase.
MATERIALS AND METHODS
experiments. Cells and Transfection Protocol. The hemizygous APRTCHO cell line ATS49tg has been described (6). The APRT deficiency results from a 3-bp deletion in the Mbo II site in exon V ofthe sole copy of the APRT gene (20). The linearized plasmid was introduced into the cells by electroporation. Electroporation conditions were as follows: 800 V, 25 .F, 1 x 107 cells in Hepes-buffered glucose (21), and 20 ,ug of DNA per cuvette with a 4-mm gapped electrode (Bio-Rad Gene Pulser). Cells were plated after electroporation at 5 X 105 cells per 100-mm dish in Dulbecco's modified Eagle's medium, 10%o fetal bovine serum, 2% nonessential amino acids, penicillin, and streptomycin and selected 24 hr later in HAT medium (100 ,uM hypoxanthine/0.4 ,uM aminopterin/16 ,tM thymidine; Sigma) for random integration or in ALASA medium (50 uM azaserine/25 uM alanosine/100 ,uM adenine; Sigma, National Cancer Institute, and Sigma, respectively) for targeted events (22). After 13 days in selection, HAT-resistant (HATr) colonies were stained and counted; ALASAr colonies were subcloned. Each ALASAr clone was tested in HAT medium for a preliminary assignment to recombinant class.
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Natl. Acad. Sci. USA 88 (1991) GProc. Genetics: Pennington and Wilson
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in Fig. 1 (6). Target convertants arise by correction of the mutation at the endogenous APRT locus. Vector convertants arise by correction of the vector mutation followed by random integration elsewhere in the genome. Integration of the vector yields two copies of the APRT gene at the endogenous locus. Only the integration clones can distinguish between the two recombination pathways. Predictions of Structures for Targeted Integrants. The experiment was designed to distinguish between a conservative (e.g., DSBR) and a nonconservative (e.g., SSA) recombination pathway. The DSBR model predicts strand invasion of the chromosome by the homologous ends of the vector, leading to pairing between vector and chromosomal sequences except at the region of the polylinker. Before repair synthesis can begin, that nonhomology must be removed. Thus, the polylinker would be lost and replaced by the chromosomal Xho I site. The structure of the resulting integrant will contain two genes, each possessing an Xho I site and no polylinker (Fig. 2A). If the heteroduplex overlaps A
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FIG. 1. Substrates and products expected from recombination between pAG6 and the chromosomal APRT. Heavy lines, vector sequence; stippled box, simian virus 40 early promoter and enhancer; solid bar, Mbo II deletion that renders the cell line APRT-; GPT, guanine phosphoribosyltransferase. This mutation is 471 bp from the Xho I site (indicated by X). The vector was linearized at the Hindill insertional mutation in APRT (indicated by H). Targeted colonies can be of three classes: target convertants, vector convertants, and integrants (described in the text).
DNA Isolation and Analysis. DNA was isolated from ALASA' clones. For Southern blot analysis, 10 ,ug of cellular DNA was digested with restriction enzymes (Boehringer Mannheim) and run on 0.6% or 0.8% agarose gels. DNA was transferred out of gels onto nylon membranes (Zetaprobe, Bio-Rad) by capillary blotting. The filters were probed with the 3.9kilobase (kb) BamHI fragment from plasmid pAGi (i.e., the wild-type APRT gene), which was isolated from agarose and labeled with [a- 2P]dCTP by the random-priming method (Boehringer Mannheim). PCR was performed under standard conditions with primers optimized for magnesium concentration. The same APRT internal primer was used for all amplifications (5'GAGAACCCCAGAGAATTCGGTAGC-3'). To amplify the left-hand gene copy, the opposing primer was made to the plasmid sequence (5'-CTGCATTCTAGTTGTGGTTTGTCC-3'). To amplify the right-hand gene copy, the opposing primer was made to the chromosomal sequence (5'TfCATCCCCCTGGTGTTl GAGAGC-3'). Both primer sets amplify a 2.6-kb fragment. PCR products were phenol/ chloroform extracted and ethanol precipitated prior to digestion. The digestion products were resolved on 2% NuSieve GTG agarose gels.
RESULTS Classes of Recombination Events. Gene targeting at APRT produces three classes of homologous recombinants, shown
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FIG. 2. Reactants and integration products of a DSBR-like path(A) or of a SSA-like pathway (B). Vectors are drawn above the chromosomal target and x (A) or arrows (B) indicate the sites for recombination. Below the products are the expected fragment sizes for blotting analysis. H, HindIII; E, EcoRI; X, Xho I; B, Bgl II. Sizes are in kb. Solid bar is the parental APRT mutation. It may or may not be present (+/-) in the downstream copy of the gene. way
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the parental mutation, it may be corrected during the event to generate two wild-type copies of the gene. A nonconservative pathway would yield a different integration product (Fig. 2B). Since one copy of the gene is destroyed in this pathway, to get two copies in the product, the reaction must start with three. Given the high efficiency of end-joining in mammalian cells (23-26), this is easily imagined as one copy on the chromosome and two copies present as a dimer of the input vector. Following the SSA model proposed by Lin et al. (12), a break occurs in the chromosomal sequence, an exonuclease exposes single strands, and the dimer of the input vector anneals with the chromosome at complementary sequences. Repair synthesis completes the process. The product consists of two genes, one containing the wild-type Xho I site and the second containing the polylinker. Depending on the position of the heteroduplex, the parental mutation may be corrected to result in two wild-type copies of the gene. [An alternative version of this pathway (27) could generate the same product beginning with a single-stranded chromosomal gap, which, for example, could be generated during replication.] The fundamental difference between the integration products of the two pathways is the presence or absence of polylinker sequences. For a colony to be selected as APRT', one copy of the gene must contain an Xho I site but the other copy is not subject to selection and its structure distinguishes between the conservative and nonconservative pathways. An Xho I site indicates a conservative pathway and a HindIII site indicates a nonconservative pathway (Fig. 2). Identification of Integrants. The linearized vector was introduced into ATS-49tg cells by electroporation. After 24 hr, the majority of cells were subjected to ALASA selection to identify targeted (APRT+) recombinants; a minority were selected in HAT medium to identify random (GPT+) recombinants. Random integrants arose at an average frequency of 1.6 x 1O-4; targeted recombinants arose at an average frequency of 2.1 x 10-7 (Table 1). A total of 51 targeted colonies were isolated from five experiments. Their structures were determined by characterization of phenotype, by Southern blotting analysis, and by PCR analysis. Seventeen of the 51 APRT+ colonies were GPT- (HAT sensitive), suggesting that they were target convertants (see Fig. 1). Southern blotting and PCR analysis showed one of these to be a vector convertant. Targeted integrants were distinguished from vector convertants in the remaining 34 GPT+ (HAT) clones by Southern blotting. Genomic DNAs from these clones were digested with an appropriate restriction enzyme and the resulting blots were probed with the APRT gene to determine whether the parental locus was intact. For a HindIII digestion (Fig. 3A), the integrants will lose the 8.5-kb parental band and gain either a 16.9-kb band, indicative of a conservative event, or 14.7- and 2.2-kb bands, indicating a nonconservative event. In contrast, vector convertants will retain the parental 8.5-kb band and have a new
Proc. Nad. Acad. Sci. USA 88 (1991) band of an unpredictable size. Fig. 2A shows four integrants (lanes 2-5) and four vector convertants (lanes 6-9). Seventeen targeted integrants and 18 vector convertants were obtained (Table 1). The classification of the vector convertants was confirmed by PCR analysis; their structures will be presented in detail elsewhere. Analysis of the Targeted Integrants. The absence of the 2.2-kb band in the HindIII digestion for all the integrants (Fig. 3A) suggested a conservative pathway. To assign the integrants more precisely to the appropriate pathway, we used two pairs of restriction enzyme digestions. If an integrant arose by the conservative pathway, the downstream gene should have an Xho I site, giving a diagnostic 2.7-kb band in a HindlI/EcoRI digestion and diagnostic 4.5- and 2.4-kb bands in an Xho I/Bgl II digestion (Fig. 2A). The 2.7- and 2.4-kb bands are identical to bands from the parental locus (Fig. 3B; lanes 1 and 6). By contrast, if an integrant arose by the nonconservative pathway, the downstream gene should have a HindIII site, giving diagnostic bands of 2.2 and 0.5 kb in a Hindll/EcoRI digestion and a diagnostic 6.9-kb band in an Xho I/Bgl II digestion (Fig. 2B). Like the four integrants shown in Fig. 3B (lanes 2-5 and 7-10), all 17 integrants gave the diagnostic bands expected from the conservative pathway.
To identify wild-type and mutant copies of the APRT gene, all 17 integrants were subjected to PCR analysis (Fig. 4). The left- and right-hand copies of the APRT gene were separately amplified using one primer within APRT and an opposing primer either in the plasmid (for the left-hand copy) or in the chromosome (for the right-hand copy). The 2.6-kb PCR products were digested with Mbo II to distinguish between wild-type and mutant (i.e., parental) sequences. A wild-type gene gives diagnostic fragments of975 and 499 bp (Fig. 4, lane wt). A mutant gene gives a diagnostic fragment of 1474 bp (lane P). Eleven integrants had a wild-type APRT gene on the left and a mutant copy on the right (lanes 3); five integrants had two wild-type copies (lanes 1); one integrant had a mutant gene on the left and a wild-type gene on the right (lanes 2).
DISCUSSION We set out to determine whether targeted recombination in mammalian cells is conservative or nonconservative. As we illustrated by using the SSA model, a nonconservative pathway requires participation of three copies to give a targeted integrant containing two copies (Fig. 2B). By contrast, a conservative pathway, as illustrated by the DSBR model, requires only two copies to give a targeted integrant (Fig. 2A). To distinguish between these pathways, we asked whether targeted integrants arise by recombination of the chromosomal sequence with a monomer of the vector (conservative) or a dimer of the vector (nonconservative). Involvement of a monomeric versus a dimeric vector was determined by linearizing vector DNA at the site of an insertion mutation in
Table 1. Frequencies of random and targeted recombination and distribution of targeted events by class Event Vector Random Target Targeted convertants convertants Integrants frequency frequency Exp. 4 2 1.5 x 10-7 1 1 8.7 x 10-5 5 5 4 2.9 x 10-7 2 9.6 x 10-5 2 5 2.2 x 10-7 3 2.7 x 10-4 3 5 3 3 2.0 x 10-4 2.0 x 10-7 4 4 2 3 1.3 x 10-4 2.1 x 10-7 5 18 (35%) 17 (33%) 16 (31%) 2.1 x 1O-7 1.6 x 10-4 Total x 5 were treated cells of cells to from the actual number are calculated per 107 subjected selection; Frequencies experiment. The targeted colonies were divided into three classes based on phenotypes and blotting analysis. Convertants were ultimately differentiated by the presence of the parental mutation at the endogenous locus, as determined by PCR
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Proc. Natl. Acad. Sci. USA 88 (1991)
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FIG. 3. (A) Identification of HAT' clones by type of product. HindIII digestions of DNA from targeted GPT+ clones. Lanes: 1, parent cell line (partially obscured); 2-5, integrants; 6-9, vector convertants. (B) Assignment of integrants to a recombination pathway. Parental (lane 1) and integrant (lanes 2-5) DNAs were digested with HindIll and EcoRI. Lanes 6-10 are the same DNAs digested with Xho I and Bgl II. C, diagnostic bands for the conservative product; NC, diagnostic bands for the nonconversative product. Sizes are in kb.
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the APRT gene and analyzing targeted integrants for secharacteristic of a dimeric junction. Among 51 targeted recombinants, 17 targeted integrants were identified; none arises from a dimeric vector. We conclude, therefore, that the primary pathway for targeted recombination in mammalian cells is conservative. It could be argued that we did not observe the products of the nonconservative pathway because dimers of the input vector did not form in CHO cells under the conditions of our experiments. To address this concern, we examined the structures of some of the random integrants that arose as by-products of our targeted recombination experiments. More than half of the analyzed clones contained integrations of dimeric or concatemeric vector (unpublished observations). These results confirm that CHO cells join DNA ends with high efficiency, which seems to be a general feature of DNA metabolism in mammalian cells (23-26). Thus, it is likely that dimers of the vector were available in transfected cells in our experiments. The essential feature of a nonconservative pathway, as it applies to targeted integration, is that three copies of a homologous sequence recombine to give two copies in the integrant. Given the capacity ofmammalian cells tojoin DNA ends, the most likely way to involve three copies would seem to be a dimer of the vector interacting with a single chromosomal target (Fig. 2B). However, it is formally possible for a single vector to recombine with two copies of the chromosomal target on sister chromatids as shown in Fig. 5. The integrant produced by this nonconservative pathway would have two copies of the APRT gene, each with an Xho I site, which matches the integrant structure we observed. In this pathway, however, the right-hand copy of the APRT gene must contain the parental Mbo II deletion and the left-hand copy must be the wild-type locus (Fig. 5). Among our 17 integrants, we identified 6 that do not match this expectation: 5 contained two wild-type copies of the APRT gene and 1 contained the parental mutation in the left-hand gene. In quences
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addition, in an analysis of 14 integrants that arose by targeted recombination at the same locus using a vector linearized at the unmodified Xho I site or at a nearby Pst I site, 7 were shown to contain 2 wild-type copies of the APRT gene (G. M. Adair, personal communication). Thus, 13 of 31 integrants at Set
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FIG. 4. Predominant integrant structure is shown. Wavy lines, chromosomal sequence; boxes, APRT homologous regions; straight line, plasmid sequence; solid bar, mutation. Relative positions of PCR primers are shown and the Mbo II digestion fragments of the amplified fragment are shown below. The left-hand gene, amplified by PCR primer set 1, is shown as wild-type; its diagnostic Mbo II digestion fragments are 975 and 499 bp. The right-hand gene, amplified by PCR primer set 2, is shown as mutant; its diagnostic Mbo II digestion fragment is 1474 bp. Lanes: wt, wild-type gene; P, parental, mutant pattern; 1, clone with wild-type gene at both loci; 2, clone with mutant gene at the left locus and wild-type gene at the right locus; 3, clone with wild-type gene at the left locus and mutant gene at the right locus.
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in mammalian cells-that is, the opportunity to copy chromosomal sequences onto the ends of the invading vector. In mammalian cells where a linear product can be incorporated randomly into the genome, copying of chromosomal sequences onto one or both ends of a vector could allow a fragment of a gene to complete itself. The APRT gene may be an ideal targeting system for detecting such an event because of its small size. Copying, if it occurs, may not be extensive enough to yield a selectable product for larger genes.
x
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FIG. 5. An alternative nonconservative pathway for gene targeting. Substrates for the recombination are one vector (heavy lines) and two chromosomal APRT sequences on sister chromatids (thin lines). The product is indistinguishable from the conservative prediction except that this (nonconservative) product must contain the parental mutation (solid bar).
the APRT locus do not match the expectations of the nonconservative pathway shown in Fig. 5. In contrast, these integrants are allowed by a conservative pathway (such as DSBR) where they can arise by formation of a heteroduplex that covers the deletion followed by mismatch repair or replication (28). Our results support the conclusions of a previous study, which showed that a double-stranded gap in the vector could be repaired during targeted integration onto the chromosome (29). Gap repair is an expectation of the DSBR model of recombination but by itself cannot differentiate between the DSBR model (i.e., a conservative pathway) and the nonconservative pathway shown in Fig. 5. In addition, we were concerned that the ectopic site of the target gene (simian virus 40 large tumor antigen) and the possible growth advantage conferred by expression of the large tumor antigen gene (which would gain an enhancer by a conservative pathway but not by a nonconservative pathway) might bias the analysis against integrants derived from the nonconservative pathway. In the experiments described here the recombination target is the endogenous APRT gene in CHO cells and the analysis of the targeted integrants focuses on the selectively neutral copy of the gene. Our analysis of targeted integrants indicates that the pathway for gene targeting in mammalian cells is similar to that in yeast, which is described by the DSBR model (15). That model predicts that the nonhomologous ends used in this experiment would be corrected by using the resident APRT gene as the template for repair synthesis. Resolution of the recombination intermediate could give the crossover product, resulting in integration, or the noncrossover product, which releases the vector from the chromosome. In yeast the noncrossover products can be detected by correction of chromosomal mutations or by correction ofthe vector, which exists as a stable or unstable extrachromosomal plasmid (30). The target and vector convertants we detect are analogous to the two kinds of noncrossover products in yeast, except that we detect vector convertants as ectopic integrants rather than as extrachromosomal elements. This difference may reflect the propensity for random integration in mammalian cells and its paucity in yeast (24, 30). Preliminary analysis of vector convertants in this study and a previous one (6) suggests an aspect of conservative recoimbination and the DSBR model that may be especially relevant
We thank Drs. R. S. Nairn and G. M. Adairfor helpful discussions and critical reading of the manuscript, Dr. G. M. Adair for providing the ATS-49tg cells, and Kathleen Marburger for technical guidance. The Wilson laboratory and the Baylor Molecular Biology Group provided useful discussions. We are grateful to Dr. Dana Carroll for suggesting the alternative nonconservative recombination model shown in Fig. 5. This work was supported by National Institutes of Health Grants GM 38219 and DK 42678, American Cancer Society Grant CD-420, and Cystic Fibrosis Foundation Grant Z112 to J.H.W. 1. Smithies, O., Gregg, R. G., Boggs, S. S., Koralewski, M. A. & Kucherlapati, R. S. (1985) Nature (London) 317, 230-234. 2. Thomas, K. R. & Capecchi, M. R. (1987) Cell 51, 503-512. 3. Doetschman, T., Maeda, N. & Smithies, 0. (1988) Proc. Natl. Acad. Sci. USA 85, 8583-8587. 4. Thomas, K. R., Folger, K. R. & Capecchi, M. R. (1986) Cell 44, 419-428. 5. Mansour, S. L., Thomas, K. R. & Capecchi, M. R. (1988) Nature (London) 336, 348-352. 6. Adair, G. M., Nairn, R. S., Wilson, J. H., Seidman, M. M., Brotherman, K. A., MacKinnon, C. & Scheerer, J. B. (1989) Proc. Natl. Acad. Sci. USA 86, 4574-4578. 7. Zimmer, A. & Gruss, P. (1989) Nature (London) 33, 150-153. 8. Zijlstra, M., Li, E., Sajjadi, F., Subramani, S. & Jaenisch, R. (1989) Nature (London) 342, 435-438. 9. Thompson, S., Clarke, A. R., Pow, A. M., Hooper, M. L. & Melton, D. W. (1989) Cell 56, 313-321. 10. Bollag, R. J. & Liskay, R. M. (1988) Genetics 119, 161-169. 11. Subramani, S. & Rubnitz, J. (1985) Mol. Cell. Biol. 5, 659-666. 12. Lin, F.-L., Sperle, K. & Sternberg, N. (1984) Mol. Cell. Biol. 4, 1020-1034. 13. Seidman, M. M. (1987) Mol. Cell. Biol. 7, 3561-3565. 14. Lin, F.-L., Sperle, K. & Sternberg, N. (1990) Mol. Cell. Biol. 10, 103-112. 15. Szostak, J. W., Orr-Weaver, T. L., Rothstein, R. J. & Stahl, F. W. (1983) Cell 33, 25-35. 16. Sun, H., Treco, D. & Szostak, J. W. (1991) Cell 64, 1155-1161. 17. Nickoloff, J. A., Chen, E. Y. & Heffron, F. (1986) Proc. Natl. Acad. Sci. USA 83, 7831-7835. 18. Rommerskirch, W., Graeber, I., Grassmann, M. & Grassmann, A. (1988) Nucleic Acids Res. 16, 941-952. 19. Porter, T., Pennington, S. L., Adair, G. M., Nairn, R. S. & Wilson, J. H. (1990) Nucleic Acids Res. 18, 5173-5180. 20. Adair, G. M., Nairn, R. S., Wilson, J. H., Scheerer, J. B. & Brotherman, K. A. (1990) Somatic Cell Mol. Genet. 16, 437441. 21. Chu, G., Hayakawa, H. & Berg, P. (1987) Nucleic Acids Res. 15, 1311-1326. 22. Nairn, R. S., Humphrey, R. M. & Adair, G. M. (1988) Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 53, 249-260. 23. Roth, D. B. & Wilson, J. H. (1986) Mol. Cell. Biol. 6, 42954304. 24. Roth, D. & Wilson, J. (1988) in Genetic Recombination, eds. Kucherlapati, R. & Smith, R. (Am. Soc. Microbiol., Washington), pp. 621-653. 25. Roth, D. B., Chang, X.-B. & Wilson, J. H. (1989) Mol. Cell. Biol. 9, 3049-3057. 26. Zheng, H., Chang, X.-B. & Wilson, J. H. (1989) Plasmid 22, 99-105. 27. Wake, C. T., Vemnaleone, F. & Wilson, J. H. (1985) Mol. Cell. Biol. 5, 2080-2089. 28. Orr-Weaver, T. L., Nicolas, A. & Szostak, J. W. (1988) Mol. Cell. Biol. 8, 5292-5298. 29. Jasin, M. & Berg, P. (1988) Genes Dev. 2, 1353-1363. 30. Orr-Weaver, T. L. & Szostak, J. W. (1983) Proc. Natd. Acad. USA 80, 4417-4421.
Gene targeting in Chinese hamster ovary cells is conservative (homologous recombination/gene correction/mammalian recombination/double-strand-break repair/single-strand anling)
SANDRA L. PENNINGTON AND JOHN H. WILSON Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030
Communicated by Salih J. Wakil, July 24, 1991 (received for review May 8, 1991)
Two fundamentally different pathways for ABSTRACT homologous recombination have been identified in mammalian cells. For most chromosomal recombination events, two copies of a homologous sequence recombine to yield two copies in the products; such events are said to be conservative because the number of copies is preserved. By contrast, virtually all extrachromosomal recombination events are nonconservative; two copies recombine to give a product containg a single intact copy (the other copy is destroyed in the mechanis). Since gene targeting involves an introduced (extrachromosomal) plasmid and a chromosomal target, it was not clear which pathway would apply. We used a marked vector to determine whether targeted integrants were products of recombination events that involved two copies (the conservative pathway) or three copies (the nonconservative pathway) of the homologous sequence. Among 51 gene targeting events, we identified 17 homologous integrants and analyzed their structures. All match the predictions for a conservative pathway. We conclude that the principal pathway for gene targeting in mammalian cells is conservative.
a double-stranded break, forming adjacent Holliday junctions. These junctions can be resolved to give linked or unlinked products. In both cases, the process starts with two copies of the homologous sequence and produces two copies. Since the number of copies is maintained in the recombination event, the overall process is said to be conservative. Thus, there are two possibilities for the nature of targeted recombination in mammalian cells: like extrachromosomal recombination, it might be nonconservative, or, like chromosomal recombination, it might be conservative. We asked whether the products of targeted integration matched the expectations of a conservative pathway or a nonconservative pathway. The chromosomal target for this study was the Chinese hamster ovary (CHO) adenine phosphoribosyltransferase (APRT) gene. The ease of selection for and against this gene facilitates generation of the large number of targeted clones needed for pathway determination. In this paper, targeted integration into the APRT gene is shown to occur in a way that is consistent with a conservative pathway.
Gene targeting is a powerful tool for manipulating mammalian genomes in precise ways (1-3). Learning what parameters affect the frequency of targeted recombination in mammalian cells may allow us to harness this normally rare event (4-6) to study gene expression, to make animal models for disease (7-9), and, ultimately, to correct human inborn errors of metabolism. Previous studies of chromosomal and extrachromosomal homologous recombination in mammalian cell lines suggest that two different pathways of recombination are used in mammalian cells (10-14). The predominant pathway for extrachromosomal recombination is nonconservative and can be described by the singlestrand annealing (SSA) model of homologous recombination (12). There exist other nonconservative models; SSA is used here for the purposes of illustration. In this model, two copies of a homologous sequence recombine to make a single copy. The model proposes a stripping away of strands from a break, pairing of exposed homologies, and repair ofresidual gaps and single-stranded extensions. Since one copy of the homologous sequence is destroyed in the process, the recombination event is said to be nonconservative. The primary pathway for homologous chromosomal recombination in mammalian cells is thought to be conservative (10, 11). Chromosomal recombination in yeast, which is also conservative, is stimulated by double-stranded breaks in a way that can be described by the double-strand-break repair (DSBR) model for homologous recombination (15-17). Other conservative models do not require double-stranded breaks, but DSBR serves as an appropriate example here because mammalian targeted recombination is stimulated by doublestranded breaks in the vector (4, 18). The model predicts that two copies of a homologous sequence pair on either side of
Plasmid Construction. Plasmid pAG6 (Fig. 1) was derived from plasmid pAG1 (19), which is a modified pSV2gpt plasmid containing the structural hamster APRT gene in the unique BamHI site. The plasmid backbone of pAG6 was altered by filling in the HindIII site near the simian virus 40 promoter; the GPT gene is still expressible. The APRT gene was altered by inserting a 37-base-pair (bp) polylinker into the unique Xho I site in the third exon. This modification destroyed the Xho I site and created an insertional, frameshift mutation of APRT. The polylinker contains a HindIII site and it was at this site that the plasmid was linearized for the
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Abbreviations: SSA, single-strand annealing; DSBR, double-strandbreak repair; APRT, adenine phosphoribosyltransferase; GPT, guanine phosphoribosyltransferase.
MATERIALS AND METHODS
experiments. Cells and Transfection Protocol. The hemizygous APRTCHO cell line ATS49tg has been described (6). The APRT deficiency results from a 3-bp deletion in the Mbo II site in exon V ofthe sole copy of the APRT gene (20). The linearized plasmid was introduced into the cells by electroporation. Electroporation conditions were as follows: 800 V, 25 .F, 1 x 107 cells in Hepes-buffered glucose (21), and 20 ,ug of DNA per cuvette with a 4-mm gapped electrode (Bio-Rad Gene Pulser). Cells were plated after electroporation at 5 X 105 cells per 100-mm dish in Dulbecco's modified Eagle's medium, 10%o fetal bovine serum, 2% nonessential amino acids, penicillin, and streptomycin and selected 24 hr later in HAT medium (100 ,uM hypoxanthine/0.4 ,uM aminopterin/16 ,tM thymidine; Sigma) for random integration or in ALASA medium (50 uM azaserine/25 uM alanosine/100 ,uM adenine; Sigma, National Cancer Institute, and Sigma, respectively) for targeted events (22). After 13 days in selection, HAT-resistant (HATr) colonies were stained and counted; ALASAr colonies were subcloned. Each ALASAr clone was tested in HAT medium for a preliminary assignment to recombinant class.
9498
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Natl. Acad. Sci. USA 88 (1991) GProc. Genetics: Pennington and Wilson
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in Fig. 1 (6). Target convertants arise by correction of the mutation at the endogenous APRT locus. Vector convertants arise by correction of the vector mutation followed by random integration elsewhere in the genome. Integration of the vector yields two copies of the APRT gene at the endogenous locus. Only the integration clones can distinguish between the two recombination pathways. Predictions of Structures for Targeted Integrants. The experiment was designed to distinguish between a conservative (e.g., DSBR) and a nonconservative (e.g., SSA) recombination pathway. The DSBR model predicts strand invasion of the chromosome by the homologous ends of the vector, leading to pairing between vector and chromosomal sequences except at the region of the polylinker. Before repair synthesis can begin, that nonhomology must be removed. Thus, the polylinker would be lost and replaced by the chromosomal Xho I site. The structure of the resulting integrant will contain two genes, each possessing an Xho I site and no polylinker (Fig. 2A). If the heteroduplex overlaps A
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FIG. 1. Substrates and products expected from recombination between pAG6 and the chromosomal APRT. Heavy lines, vector sequence; stippled box, simian virus 40 early promoter and enhancer; solid bar, Mbo II deletion that renders the cell line APRT-; GPT, guanine phosphoribosyltransferase. This mutation is 471 bp from the Xho I site (indicated by X). The vector was linearized at the Hindill insertional mutation in APRT (indicated by H). Targeted colonies can be of three classes: target convertants, vector convertants, and integrants (described in the text).
DNA Isolation and Analysis. DNA was isolated from ALASA' clones. For Southern blot analysis, 10 ,ug of cellular DNA was digested with restriction enzymes (Boehringer Mannheim) and run on 0.6% or 0.8% agarose gels. DNA was transferred out of gels onto nylon membranes (Zetaprobe, Bio-Rad) by capillary blotting. The filters were probed with the 3.9kilobase (kb) BamHI fragment from plasmid pAGi (i.e., the wild-type APRT gene), which was isolated from agarose and labeled with [a- 2P]dCTP by the random-priming method (Boehringer Mannheim). PCR was performed under standard conditions with primers optimized for magnesium concentration. The same APRT internal primer was used for all amplifications (5'GAGAACCCCAGAGAATTCGGTAGC-3'). To amplify the left-hand gene copy, the opposing primer was made to the plasmid sequence (5'-CTGCATTCTAGTTGTGGTTTGTCC-3'). To amplify the right-hand gene copy, the opposing primer was made to the chromosomal sequence (5'TfCATCCCCCTGGTGTTl GAGAGC-3'). Both primer sets amplify a 2.6-kb fragment. PCR products were phenol/ chloroform extracted and ethanol precipitated prior to digestion. The digestion products were resolved on 2% NuSieve GTG agarose gels.
RESULTS Classes of Recombination Events. Gene targeting at APRT produces three classes of homologous recombinants, shown
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FIG. 2. Reactants and integration products of a DSBR-like path(A) or of a SSA-like pathway (B). Vectors are drawn above the chromosomal target and x (A) or arrows (B) indicate the sites for recombination. Below the products are the expected fragment sizes for blotting analysis. H, HindIII; E, EcoRI; X, Xho I; B, Bgl II. Sizes are in kb. Solid bar is the parental APRT mutation. It may or may not be present (+/-) in the downstream copy of the gene. way
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the parental mutation, it may be corrected during the event to generate two wild-type copies of the gene. A nonconservative pathway would yield a different integration product (Fig. 2B). Since one copy of the gene is destroyed in this pathway, to get two copies in the product, the reaction must start with three. Given the high efficiency of end-joining in mammalian cells (23-26), this is easily imagined as one copy on the chromosome and two copies present as a dimer of the input vector. Following the SSA model proposed by Lin et al. (12), a break occurs in the chromosomal sequence, an exonuclease exposes single strands, and the dimer of the input vector anneals with the chromosome at complementary sequences. Repair synthesis completes the process. The product consists of two genes, one containing the wild-type Xho I site and the second containing the polylinker. Depending on the position of the heteroduplex, the parental mutation may be corrected to result in two wild-type copies of the gene. [An alternative version of this pathway (27) could generate the same product beginning with a single-stranded chromosomal gap, which, for example, could be generated during replication.] The fundamental difference between the integration products of the two pathways is the presence or absence of polylinker sequences. For a colony to be selected as APRT', one copy of the gene must contain an Xho I site but the other copy is not subject to selection and its structure distinguishes between the conservative and nonconservative pathways. An Xho I site indicates a conservative pathway and a HindIII site indicates a nonconservative pathway (Fig. 2). Identification of Integrants. The linearized vector was introduced into ATS-49tg cells by electroporation. After 24 hr, the majority of cells were subjected to ALASA selection to identify targeted (APRT+) recombinants; a minority were selected in HAT medium to identify random (GPT+) recombinants. Random integrants arose at an average frequency of 1.6 x 1O-4; targeted recombinants arose at an average frequency of 2.1 x 10-7 (Table 1). A total of 51 targeted colonies were isolated from five experiments. Their structures were determined by characterization of phenotype, by Southern blotting analysis, and by PCR analysis. Seventeen of the 51 APRT+ colonies were GPT- (HAT sensitive), suggesting that they were target convertants (see Fig. 1). Southern blotting and PCR analysis showed one of these to be a vector convertant. Targeted integrants were distinguished from vector convertants in the remaining 34 GPT+ (HAT) clones by Southern blotting. Genomic DNAs from these clones were digested with an appropriate restriction enzyme and the resulting blots were probed with the APRT gene to determine whether the parental locus was intact. For a HindIII digestion (Fig. 3A), the integrants will lose the 8.5-kb parental band and gain either a 16.9-kb band, indicative of a conservative event, or 14.7- and 2.2-kb bands, indicating a nonconservative event. In contrast, vector convertants will retain the parental 8.5-kb band and have a new
Proc. Nad. Acad. Sci. USA 88 (1991) band of an unpredictable size. Fig. 2A shows four integrants (lanes 2-5) and four vector convertants (lanes 6-9). Seventeen targeted integrants and 18 vector convertants were obtained (Table 1). The classification of the vector convertants was confirmed by PCR analysis; their structures will be presented in detail elsewhere. Analysis of the Targeted Integrants. The absence of the 2.2-kb band in the HindIII digestion for all the integrants (Fig. 3A) suggested a conservative pathway. To assign the integrants more precisely to the appropriate pathway, we used two pairs of restriction enzyme digestions. If an integrant arose by the conservative pathway, the downstream gene should have an Xho I site, giving a diagnostic 2.7-kb band in a HindlI/EcoRI digestion and diagnostic 4.5- and 2.4-kb bands in an Xho I/Bgl II digestion (Fig. 2A). The 2.7- and 2.4-kb bands are identical to bands from the parental locus (Fig. 3B; lanes 1 and 6). By contrast, if an integrant arose by the nonconservative pathway, the downstream gene should have a HindIII site, giving diagnostic bands of 2.2 and 0.5 kb in a Hindll/EcoRI digestion and a diagnostic 6.9-kb band in an Xho I/Bgl II digestion (Fig. 2B). Like the four integrants shown in Fig. 3B (lanes 2-5 and 7-10), all 17 integrants gave the diagnostic bands expected from the conservative pathway.
To identify wild-type and mutant copies of the APRT gene, all 17 integrants were subjected to PCR analysis (Fig. 4). The left- and right-hand copies of the APRT gene were separately amplified using one primer within APRT and an opposing primer either in the plasmid (for the left-hand copy) or in the chromosome (for the right-hand copy). The 2.6-kb PCR products were digested with Mbo II to distinguish between wild-type and mutant (i.e., parental) sequences. A wild-type gene gives diagnostic fragments of975 and 499 bp (Fig. 4, lane wt). A mutant gene gives a diagnostic fragment of 1474 bp (lane P). Eleven integrants had a wild-type APRT gene on the left and a mutant copy on the right (lanes 3); five integrants had two wild-type copies (lanes 1); one integrant had a mutant gene on the left and a wild-type gene on the right (lanes 2).
DISCUSSION We set out to determine whether targeted recombination in mammalian cells is conservative or nonconservative. As we illustrated by using the SSA model, a nonconservative pathway requires participation of three copies to give a targeted integrant containing two copies (Fig. 2B). By contrast, a conservative pathway, as illustrated by the DSBR model, requires only two copies to give a targeted integrant (Fig. 2A). To distinguish between these pathways, we asked whether targeted integrants arise by recombination of the chromosomal sequence with a monomer of the vector (conservative) or a dimer of the vector (nonconservative). Involvement of a monomeric versus a dimeric vector was determined by linearizing vector DNA at the site of an insertion mutation in
Table 1. Frequencies of random and targeted recombination and distribution of targeted events by class Event Vector Random Target Targeted convertants convertants Integrants frequency frequency Exp. 4 2 1.5 x 10-7 1 1 8.7 x 10-5 5 5 4 2.9 x 10-7 2 9.6 x 10-5 2 5 2.2 x 10-7 3 2.7 x 10-4 3 5 3 3 2.0 x 10-4 2.0 x 10-7 4 4 2 3 1.3 x 10-4 2.1 x 10-7 5 18 (35%) 17 (33%) 16 (31%) 2.1 x 1O-7 1.6 x 10-4 Total x 5 were treated cells of cells to from the actual number are calculated per 107 subjected selection; Frequencies experiment. The targeted colonies were divided into three classes based on phenotypes and blotting analysis. Convertants were ultimately differentiated by the presence of the parental mutation at the endogenous locus, as determined by PCR
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Proc. Natl. Acad. Sci. USA 88 (1991)
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FIG. 3. (A) Identification of HAT' clones by type of product. HindIII digestions of DNA from targeted GPT+ clones. Lanes: 1, parent cell line (partially obscured); 2-5, integrants; 6-9, vector convertants. (B) Assignment of integrants to a recombination pathway. Parental (lane 1) and integrant (lanes 2-5) DNAs were digested with HindIll and EcoRI. Lanes 6-10 are the same DNAs digested with Xho I and Bgl II. C, diagnostic bands for the conservative product; NC, diagnostic bands for the nonconversative product. Sizes are in kb.
2.4
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40
the APRT gene and analyzing targeted integrants for secharacteristic of a dimeric junction. Among 51 targeted recombinants, 17 targeted integrants were identified; none arises from a dimeric vector. We conclude, therefore, that the primary pathway for targeted recombination in mammalian cells is conservative. It could be argued that we did not observe the products of the nonconservative pathway because dimers of the input vector did not form in CHO cells under the conditions of our experiments. To address this concern, we examined the structures of some of the random integrants that arose as by-products of our targeted recombination experiments. More than half of the analyzed clones contained integrations of dimeric or concatemeric vector (unpublished observations). These results confirm that CHO cells join DNA ends with high efficiency, which seems to be a general feature of DNA metabolism in mammalian cells (23-26). Thus, it is likely that dimers of the vector were available in transfected cells in our experiments. The essential feature of a nonconservative pathway, as it applies to targeted integration, is that three copies of a homologous sequence recombine to give two copies in the integrant. Given the capacity ofmammalian cells tojoin DNA ends, the most likely way to involve three copies would seem to be a dimer of the vector interacting with a single chromosomal target (Fig. 2B). However, it is formally possible for a single vector to recombine with two copies of the chromosomal target on sister chromatids as shown in Fig. 5. The integrant produced by this nonconservative pathway would have two copies of the APRT gene, each with an Xho I site, which matches the integrant structure we observed. In this pathway, however, the right-hand copy of the APRT gene must contain the parental Mbo II deletion and the left-hand copy must be the wild-type locus (Fig. 5). Among our 17 integrants, we identified 6 that do not match this expectation: 5 contained two wild-type copies of the APRT gene and 1 contained the parental mutation in the left-hand gene. In quences
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addition, in an analysis of 14 integrants that arose by targeted recombination at the same locus using a vector linearized at the unmodified Xho I site or at a nearby Pst I site, 7 were shown to contain 2 wild-type copies of the APRT gene (G. M. Adair, personal communication). Thus, 13 of 31 integrants at Set
Set 2
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FIG. 4. Predominant integrant structure is shown. Wavy lines, chromosomal sequence; boxes, APRT homologous regions; straight line, plasmid sequence; solid bar, mutation. Relative positions of PCR primers are shown and the Mbo II digestion fragments of the amplified fragment are shown below. The left-hand gene, amplified by PCR primer set 1, is shown as wild-type; its diagnostic Mbo II digestion fragments are 975 and 499 bp. The right-hand gene, amplified by PCR primer set 2, is shown as mutant; its diagnostic Mbo II digestion fragment is 1474 bp. Lanes: wt, wild-type gene; P, parental, mutant pattern; 1, clone with wild-type gene at both loci; 2, clone with mutant gene at the left locus and wild-type gene at the right locus; 3, clone with wild-type gene at the left locus and mutant gene at the right locus.
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in mammalian cells-that is, the opportunity to copy chromosomal sequences onto the ends of the invading vector. In mammalian cells where a linear product can be incorporated randomly into the genome, copying of chromosomal sequences onto one or both ends of a vector could allow a fragment of a gene to complete itself. The APRT gene may be an ideal targeting system for detecting such an event because of its small size. Copying, if it occurs, may not be extensive enough to yield a selectable product for larger genes.
x
x
FIG. 5. An alternative nonconservative pathway for gene targeting. Substrates for the recombination are one vector (heavy lines) and two chromosomal APRT sequences on sister chromatids (thin lines). The product is indistinguishable from the conservative prediction except that this (nonconservative) product must contain the parental mutation (solid bar).
the APRT locus do not match the expectations of the nonconservative pathway shown in Fig. 5. In contrast, these integrants are allowed by a conservative pathway (such as DSBR) where they can arise by formation of a heteroduplex that covers the deletion followed by mismatch repair or replication (28). Our results support the conclusions of a previous study, which showed that a double-stranded gap in the vector could be repaired during targeted integration onto the chromosome (29). Gap repair is an expectation of the DSBR model of recombination but by itself cannot differentiate between the DSBR model (i.e., a conservative pathway) and the nonconservative pathway shown in Fig. 5. In addition, we were concerned that the ectopic site of the target gene (simian virus 40 large tumor antigen) and the possible growth advantage conferred by expression of the large tumor antigen gene (which would gain an enhancer by a conservative pathway but not by a nonconservative pathway) might bias the analysis against integrants derived from the nonconservative pathway. In the experiments described here the recombination target is the endogenous APRT gene in CHO cells and the analysis of the targeted integrants focuses on the selectively neutral copy of the gene. Our analysis of targeted integrants indicates that the pathway for gene targeting in mammalian cells is similar to that in yeast, which is described by the DSBR model (15). That model predicts that the nonhomologous ends used in this experiment would be corrected by using the resident APRT gene as the template for repair synthesis. Resolution of the recombination intermediate could give the crossover product, resulting in integration, or the noncrossover product, which releases the vector from the chromosome. In yeast the noncrossover products can be detected by correction of chromosomal mutations or by correction ofthe vector, which exists as a stable or unstable extrachromosomal plasmid (30). The target and vector convertants we detect are analogous to the two kinds of noncrossover products in yeast, except that we detect vector convertants as ectopic integrants rather than as extrachromosomal elements. This difference may reflect the propensity for random integration in mammalian cells and its paucity in yeast (24, 30). Preliminary analysis of vector convertants in this study and a previous one (6) suggests an aspect of conservative recoimbination and the DSBR model that may be especially relevant
We thank Drs. R. S. Nairn and G. M. Adairfor helpful discussions and critical reading of the manuscript, Dr. G. M. Adair for providing the ATS-49tg cells, and Kathleen Marburger for technical guidance. The Wilson laboratory and the Baylor Molecular Biology Group provided useful discussions. We are grateful to Dr. Dana Carroll for suggesting the alternative nonconservative recombination model shown in Fig. 5. This work was supported by National Institutes of Health Grants GM 38219 and DK 42678, American Cancer Society Grant CD-420, and Cystic Fibrosis Foundation Grant Z112 to J.H.W. 1. Smithies, O., Gregg, R. G., Boggs, S. S., Koralewski, M. A. & Kucherlapati, R. S. (1985) Nature (London) 317, 230-234. 2. Thomas, K. R. & Capecchi, M. R. (1987) Cell 51, 503-512. 3. Doetschman, T., Maeda, N. & Smithies, 0. (1988) Proc. Natl. Acad. Sci. USA 85, 8583-8587. 4. Thomas, K. R., Folger, K. R. & Capecchi, M. R. (1986) Cell 44, 419-428. 5. Mansour, S. L., Thomas, K. R. & Capecchi, M. R. (1988) Nature (London) 336, 348-352. 6. Adair, G. M., Nairn, R. S., Wilson, J. H., Seidman, M. M., Brotherman, K. A., MacKinnon, C. & Scheerer, J. B. (1989) Proc. Natl. Acad. Sci. USA 86, 4574-4578. 7. Zimmer, A. & Gruss, P. (1989) Nature (London) 33, 150-153. 8. Zijlstra, M., Li, E., Sajjadi, F., Subramani, S. & Jaenisch, R. (1989) Nature (London) 342, 435-438. 9. Thompson, S., Clarke, A. R., Pow, A. M., Hooper, M. L. & Melton, D. W. (1989) Cell 56, 313-321. 10. Bollag, R. J. & Liskay, R. M. (1988) Genetics 119, 161-169. 11. Subramani, S. & Rubnitz, J. (1985) Mol. Cell. Biol. 5, 659-666. 12. Lin, F.-L., Sperle, K. & Sternberg, N. (1984) Mol. Cell. Biol. 4, 1020-1034. 13. Seidman, M. M. (1987) Mol. Cell. Biol. 7, 3561-3565. 14. Lin, F.-L., Sperle, K. & Sternberg, N. (1990) Mol. Cell. Biol. 10, 103-112. 15. Szostak, J. W., Orr-Weaver, T. L., Rothstein, R. J. & Stahl, F. W. (1983) Cell 33, 25-35. 16. Sun, H., Treco, D. & Szostak, J. W. (1991) Cell 64, 1155-1161. 17. Nickoloff, J. A., Chen, E. Y. & Heffron, F. (1986) Proc. Natl. Acad. Sci. USA 83, 7831-7835. 18. Rommerskirch, W., Graeber, I., Grassmann, M. & Grassmann, A. (1988) Nucleic Acids Res. 16, 941-952. 19. Porter, T., Pennington, S. L., Adair, G. M., Nairn, R. S. & Wilson, J. H. (1990) Nucleic Acids Res. 18, 5173-5180. 20. Adair, G. M., Nairn, R. S., Wilson, J. H., Scheerer, J. B. & Brotherman, K. A. (1990) Somatic Cell Mol. Genet. 16, 437441. 21. Chu, G., Hayakawa, H. & Berg, P. (1987) Nucleic Acids Res. 15, 1311-1326. 22. Nairn, R. S., Humphrey, R. M. & Adair, G. M. (1988) Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 53, 249-260. 23. Roth, D. B. & Wilson, J. H. (1986) Mol. Cell. Biol. 6, 42954304. 24. Roth, D. & Wilson, J. (1988) in Genetic Recombination, eds. Kucherlapati, R. & Smith, R. (Am. Soc. Microbiol., Washington), pp. 621-653. 25. Roth, D. B., Chang, X.-B. & Wilson, J. H. (1989) Mol. Cell. Biol. 9, 3049-3057. 26. Zheng, H., Chang, X.-B. & Wilson, J. H. (1989) Plasmid 22, 99-105. 27. Wake, C. T., Vemnaleone, F. & Wilson, J. H. (1985) Mol. Cell. Biol. 5, 2080-2089. 28. Orr-Weaver, T. L., Nicolas, A. & Szostak, J. W. (1988) Mol. Cell. Biol. 8, 5292-5298. 29. Jasin, M. & Berg, P. (1988) Genes Dev. 2, 1353-1363. 30. Orr-Weaver, T. L. & Szostak, J. W. (1983) Proc. Natd. Acad. USA 80, 4417-4421.
