Proc. Nat!. Acad. Sci. USAVol. 88, pp. 9498-9502, November 1991Genetics

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. WILSONVerna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030

Communicated by Salih J. Wakil, July 24, 1991 (receivedfor review May 8, 1991)

ABSTRACT Two fundamentally different pathways forhomologous recombination have been identified in mammaliancells. For most chromosomal recombination events, two copiesof a homologous sequence recombine to yield two copies in theproducts; such events are said to be conservative because thenumber of copies is preserved. By contrast, virtually allextrachromosomal recombination events are nonconservative;two copies recombine to give a product containg a singleintact copy (the other copy is destroyed in the mechanis).Since gene targeting involves an introduced (extrachromo-somal) plasmid and a chromosomal target, it was not clearwhich pathway would apply. We used a marked vector todetermine whether targeted integrants were products of re-combination events that involved two copies (the conservativepathway) or three copies (the nonconservative pathway) of thehomologous sequence. Among 51 gene targeting events, weidentified 17 homologous integrants and analyzed their struc-tures. All match the predictions for a conservative pathway.We conclude that the principal pathway for gene targeting inmammalian cells is conservative.

Gene targeting is a powerful tool for manipulating mammaliangenomes in precise ways (1-3). Learning what parametersaffect the frequency oftargeted recombination in mammaliancells 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 ofmetabolism. Previous studies ofchromosomal and extrachro-mosomal homologous recombination in mammalian cell linessuggest that two different pathways of recombination areused in mammalian cells (10-14).The predominant pathway for extrachromosomal recombi-

nation is nonconservative and can be described by the single-strand annealing (SSA) model of homologous recombination(12). There exist other nonconservative models; SSA is usedhere for the purposes of illustration. In this model, two copiesof a homologous sequence recombine to make a single copy.The model proposes a stripping away of strands from a break,pairing ofexposed homologies, and repair ofresidual gaps andsingle-stranded extensions. Since one copy ofthe homologoussequence is destroyed in the process, the recombination eventis said to be nonconservative.The primary pathway for homologous chromosomal re-

combination in mammalian cells is thought to be conservative(10, 11). Chromosomal recombination in yeast, which is alsoconservative, is stimulated by double-stranded breaks in away that can be described by the double-strand-break repair(DSBR) model for homologous recombination (15-17). Otherconservative models do not require double-stranded breaks,but DSBR serves as an appropriate example here becausemammalian targeted recombination is stimulated by double-stranded breaks in the vector (4, 18). The model predicts thattwo copies of a homologous sequence pair on either side of

a double-stranded break, forming adjacent Holliday junc-tions. These junctions can be resolved to give linked orunlinked products. In both cases, the process starts with twocopies ofthe homologous sequence and produces two copies.Since the number of copies is maintained in the recombina-tion event, the overall process is said to be conservative.Thus, there are two possibilities for the nature of targeted

recombination in mammalian cells: like extrachromosomalrecombination, it might be nonconservative, or, like chro-mosomal recombination, it might be conservative. We askedwhether the products of targeted integration matched theexpectations ofa conservative pathway or a nonconservativepathway. The chromosomal target for this study was theChinese hamster ovary (CHO) adenine phosphoribosyltrans-ferase (APRT) gene. The ease of selection for and against thisgene facilitates generation of the large number of targetedclones needed for pathway determination. In this paper,targeted integration into the APRT gene is shown to occur ina way that is consistent with a conservative pathway.

MATERIALS AND METHODSPlasmid Construction. Plasmid pAG6 (Fig. 1) was derived

from plasmid pAG1 (19), which is a modified pSV2gptplasmid containing the structural hamster APRT gene in theunique BamHI site. The plasmid backbone of pAG6 wasaltered by filling in the HindIII site near the simian virus 40promoter; the GPT gene is still expressible. The APRT genewas altered by inserting a 37-base-pair (bp) polylinker into theunique Xho I site in the third exon. This modificationdestroyed the Xho I site and created an insertional, frameshiftmutation ofAPRT. The polylinker contains a HindIII site andit was at this site that the plasmid was linearized for theexperiments.

Cells and Transfection Protocol. The hemizygous APRT-CHO cell line ATS49tg has been described (6). The APRTdeficiency results from a 3-bp deletion in the Mbo II site inexon V ofthe sole copy ofthe APRT gene (20). The linearizedplasmid was introduced into the cells by electroporation.Electroporation conditions were as follows: 800 V, 25 .F, 1x 107 cells in Hepes-buffered glucose (21), and 20 ,ug ofDNAper cuvette with a 4-mm gapped electrode (Bio-Rad GenePulser). Cells were plated after electroporation at 5 X 105cells per 100-mm dish in Dulbecco's modified Eagle's me-dium, 10%o fetal bovine serum, 2% nonessential amino acids,penicillin, and streptomycin and selected 24 hr later in HATmedium (100 ,uM hypoxanthine/0.4 ,uM aminopterin/16 ,tMthymidine; Sigma) for random integration or in ALASA me-dium (50 uM azaserine/25 uM alanosine/100 ,uM adenine;Sigma, National Cancer Institute, and Sigma, respectively) fortargeted events (22). After 13 days in selection, HAT-resistant(HATr) colonies were stained and counted; ALASAr colonieswere subcloned. Each ALASAr clone was tested in HATmedium for a preliminary assignment to recombinant class.

Abbreviations: SSA, single-strand annealing; DSBR, double-strand-break repair; APRT, adenine phosphoribosyltransferase; GPT, gua-nine phosphoribosyltransferase.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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GProc. Natl. Acad. Sci. USA 88 (1991) 9499

LH

J pAG6

xLI I' i>-~~APRT-GPT-

Target Conversion

j ~~~~~~~APRT+GPT- in Fig. 1 (6). Target convertants arise by correction of themutation at the endogenous APRT locus. Vector convertantsarise by correction of the vector mutation followed byrandom integration elsewhere in the genome. Integration ofthe vector yields two copies of the APRT gene at theendogenous locus. Only the integration clones can distin-guish between the two recombination pathways.

Predictions of Structures for Targeted Integrants. The ex-periment was designed to distinguish between a conservative(e.g., DSBR) and a nonconservative (e.g., SSA) recombina-tion pathway. The DSBR model predicts strand invasion ofthe chromosome by the homologous ends of the vector,leading to pairing between vector and chromosomal se-quences except at the region of the polylinker. Before repairsynthesis can begin, that nonhomology must be removed.Thus, the polylinker would be lost and replaced by thechromosomal Xho I site. The structure of the resultingintegrant will contain two genes, each possessing an Xho Isite and no polylinker (Fig. 2A). If the heteroduplex overlaps

Vector Conversion A DSBR

xAPRT+GPT+

x

Integration APRT+GPT+

_~~[ ' - - a zs {Z:X X/H

FIG. 1. Substrates and products expected from recombinationbetween pAG6 and the chromosomal APRT. Heavy lines, vectorsequence; 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 theXho I site (indicated by X). The vector was linearized at the Hindillinsertional mutation in APRT (indicated by H). Targeted colonies canbe of three classes: target convertants, vector convertants, andintegrants (described in the text).

*1-

HaB

H

H+E

X+B

16.9

5.8 7.1 1.3, 2.7i I*I

1 2.2* 3.9 1 4.5 2.4

DNA Isolation and Analysis. DNA was isolated fromALASA'clones. For Southern blot analysis, 10 ,ug of cellular DNA wasdigested with restriction enzymes (Boehringer Mannheim)and run on 0.6% or 0.8% agarose gels. DNA was transferredout of gels onto nylon membranes (Zetaprobe, Bio-Rad) bycapillary blotting. The filters were probed with the 3.9-kilobase (kb) BamHI fragment from plasmid pAGi (i.e., thewild-type APRT gene), which was isolated from agarose andlabeled with [a- 2P]dCTP by the random-priming method(Boehringer Mannheim).PCR was performed under standard conditions with prim-

ers optimized for magnesium concentration. The same APRTinternal primer was used for all amplifications (5'-GAGAACCCCAGAGAATTCGGTAGC-3'). To amplify theleft-hand gene copy, the opposing primer was made to theplasmid sequence (5'-CTGCATTCTAGTTGTGGTT-TGTCC-3'). To amplify the right-hand gene copy, the op-posing primer was made to the chromosomal sequence (5'-TfCATCCCCCTGGTGTTl GAGAGC-3'). Both primer setsamplify a 2.6-kb fragment. PCR products were phenol/chloroform extracted and ethanol precipitated prior to diges-tion. The digestion products were resolved on 2% NuSieveGTG agarose gels.

RESULTSClasses of Recombination Events. Gene targeting at APRT

produces three classes of homologous recombinants, shown

B SSA

^ ~~~,.

cF- I I ~H E H H

B

H+E

X+B

14.7 i 2.2

, 5.8 7.1 *1.3 5 2.2- . . -.0

2.2 , 3.9 6.9

FIG. 2. Reactants and integration products of a DSBR-like path-way (A) or of a SSA-like pathway (B). Vectors are drawn above thechromosomal target and x (A) or arrows (B) indicate the sites forrecombination. Below the products are the expected fragment sizesfor blotting analysis. H, HindIII; E, EcoRI; X, Xho I; B, Bgl II. Sizesare in kb. Solid bar is the parental APRT mutation. It may or may notbe present (+/-) in the downstream copy of the gene.

H X H

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9500 Genetics: Pennington and Wilson

the parental mutation, it may be corrected during the eventto generate two wild-type copies of the gene.

A nonconservative pathway would yield a different inte-gration product (Fig. 2B). Since one copy of the gene isdestroyed in this pathway, to get two copies in the product,the reaction must start with three. Given the high efficiencyof end-joining in mammalian cells (23-26), this is easilyimagined as one copy on the chromosome and two copiespresent as a dimer of the input vector. Following the SSAmodel proposed by Lin et al. (12), a break occurs in thechromosomal sequence, an exonuclease exposes singlestrands, and the dimer of the input vector anneals with thechromosome at complementary sequences. Repair synthesiscompletes the process. The product consists of two genes,one containing the wild-type Xho I site and the secondcontaining the polylinker. Depending on the position of theheteroduplex, the parental mutation may be corrected toresult in two wild-type copies of the gene. [An alternativeversion of this pathway (27) could generate the same productbeginning with a single-stranded chromosomal gap, which,for example, could be generated during replication.]The fundamental difference between the integration prod-

ucts of the two pathways is the presence or absence ofpolylinker sequences. For a colony to be selected as APRT',one copy of the gene must contain an Xho I site but the othercopy is not subject to selection and its structure distinguishesbetween the conservative and nonconservative pathways.An Xho I site indicates a conservative pathway and a HindIIIsite indicates a nonconservative pathway (Fig. 2).

Identification of Integrants. The linearized vector wasintroduced into ATS-49tg cells by electroporation. After 24hr, the majority of cells were subjected to ALASA selectionto identify targeted (APRT+) recombinants; a minority wereselected in HAT medium to identify random (GPT+) recom-binants. Random integrants arose at an average frequency of1.6 x 1O-4; targeted recombinants arose at an averagefrequency of 2.1 x 10-7 (Table 1). A total of 51 targetedcolonies were isolated from five experiments. Their struc-tures were determined by characterization of phenotype, bySouthern blotting analysis, and by PCR analysis.Seventeen of the 51 APRT+ colonies were GPT- (HAT

sensitive), suggesting that they were target convertants (seeFig. 1). Southern blotting and PCR analysis showed one ofthese to be a vector convertant. Targeted integrants weredistinguished from vector convertants in the remaining 34GPT+ (HAT) clones by Southern blotting. Genomic DNAsfrom these clones were digested with an appropriate restric-tion enzyme and the resulting blots were probed with theAPRT gene to determine whether the parental locus wasintact. For a HindIII digestion (Fig. 3A), the integrants willlose 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 con-vertants will retain the parental 8.5-kb band and have a new

band of an unpredictable size. Fig. 2A shows four integrants(lanes 2-5) and four vector convertants (lanes 6-9). Seven-teen targeted integrants and 18 vector convertants wereobtained (Table 1). The classification of the vector convert-ants was confirmed by PCR analysis; their structures will bepresented in detail elsewhere.

Analysis of the Targeted Integrants. The absence of the2.2-kb band in the HindIII digestion for all the integrants (Fig.3A) suggested a conservative pathway. To assign the inte-grants more precisely to the appropriate pathway, we usedtwo pairs of restriction enzyme digestions. If an integrantarose by the conservative pathway, the downstream geneshould have an Xho I site, giving a diagnostic 2.7-kb band ina HindlI/EcoRI digestion and diagnostic 4.5- and 2.4-kbbands in an Xho I/Bgl II digestion (Fig. 2A). The 2.7- and2.4-kb bands are identical to bands from the parental locus(Fig. 3B; lanes 1 and 6). By contrast, if an integrant arose bythe nonconservative pathway, the downstream gene shouldhave a HindIII site, giving diagnostic bands of 2.2 and 0.5 kbin a Hindll/EcoRI digestion and a diagnostic 6.9-kb band inan Xho I/Bgl II digestion (Fig. 2B). Like the four integrantsshown in Fig. 3B (lanes 2-5 and 7-10), all 17 integrants gavethe diagnostic bands expected from the conservative path-way.To identify wild-type and mutant copies ofthe APRT gene,

all 17 integrants were subjected to PCR analysis (Fig. 4). Theleft- and right-hand copies of the APRT gene were separatelyamplified using one primer within APRT and an opposingprimer either in the plasmid (for the left-hand copy) or in thechromosome (for the right-hand copy). The 2.6-kb PCRproducts were digested with Mbo II to distinguish betweenwild-type and mutant (i.e., parental) sequences. A wild-typegene gives diagnostic fragments of975 and 499 bp (Fig. 4, lanewt). A mutant gene gives a diagnostic fragment of 1474 bp(lane P). Eleven integrants had a wild-type APRT gene on theleft and a mutant copy on the right (lanes 3); five integrantshad two wild-type copies (lanes 1); one integrant had a mutantgene on the left and a wild-type gene on the right (lanes 2).

DISCUSSIONWe set out to determine whether targeted recombination inmammalian cells is conservative or nonconservative. As weillustrated by using the SSA model, a nonconservative path-way requires participation of three copies to give a targetedintegrant containing two copies (Fig. 2B). By contrast, aconservative 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 whethertargeted integrants arise by recombination of the chromo-somal sequence with a monomer of the vector (conservative)or a dimer of the vector (nonconservative). Involvement of amonomeric versus a dimeric vector was determined bylinearizing vector DNA at the site of an insertion mutation in

Table 1. Frequencies of random and targeted recombination and distribution of targeted events by classEvent

Random Targeted Target VectorExp. frequency frequency Integrants convertants convertants

1 8.7 x 10-5 1.5 x 10-7 1 2 42 9.6 x 10-5 2.9 x 10-7 5 4 53 2.7 x 10-4 2.2 x 10-7 3 5 24 2.0 x 10-4 2.0 x 10-7 5 3 35 1.3 x 10-4 2.1 x 10-7 3 2 4

Total 1.6 x 10-4 2.1 x 1O-7 17 (33%) 16 (31%) 18 (35%)Frequencies are calculated from the actual number of cells subjected to selection; 5 x 107 cells were treated per

experiment. The targeted colonies were divided into three classes based on phenotypes and blotting analysis. Convertantswere ultimately differentiated by the presence of the parental mutation at the endogenous locus, as determined by PCRanalysis.

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Proc. Natl. Acad. Sci. USA 88 (1991) 9501

Hindlil + EcoRI XhoI + BglII

2 3 4 5

7 acBe

58 a

6 7 8 9 10

6.9 -NC

0 4Ww 4.5 - C

_0 fm - - 39

C- 27- *aw.

NC- 22-w 2.4

142 40- 22

3 40

FIG. 3. (A) Identification ofHAT' clones by type of product.HindIII digestions of DNA fromtargeted GPT+ clones. Lanes: 1,parent cell line (partially ob-scured); 2-5, integrants; 6-9, vec-tor convertants. (B) Assignment ofintegrants to a recombinationpathway. Parental (lane 1) and in-tegrant (lanes 2-5) DNAs weredigested with HindIll and EcoRI.Lanes 6-10 are the same DNAsdigested with Xho I and Bgl II. C,diagnostic bands for the conserva-tive product; NC, diagnosticbands for the nonconversativeproduct. Sizes are in kb.

the APRT gene and analyzing targeted integrants for se-quences characteristic of a dimeric junction. Among 51targeted recombinants, 17 targeted integrants were identi-fied; none arises from a dimeric vector. We conclude, there-fore, that the primary pathway for targeted recombination inmammalian cells is conservative.

It could be argued that we did not observe the products ofthe nonconservative pathway because dimers of the inputvector did not form in CHO cells under the conditions of ourexperiments. To address this concern, we examined thestructures of some of the random integrants that arose as

by-products of our targeted recombination experiments.More than half of the analyzed clones contained integrationsof dimeric or concatemeric vector (unpublished observa-tions). These results confirm that CHO cells join DNA endswith high efficiency, which seems to be a general feature ofDNA metabolism in mammalian cells (23-26). Thus, it islikely that dimers of the vector were available in transfectedcells in our experiments.The essential feature of a nonconservative pathway, as it

applies to targeted integration, is that three copies of ahomologous sequence recombine to give two copies in theintegrant. Given the capacity ofmammalian cells tojoinDNAends, the most likely way to involve three copies would seemto be a dimer of the vector interacting with a single chromo-somal target (Fig. 2B). However, it is formally possible for asingle vector to recombine with two copies of the chromo-somal target on sister chromatids as shown in Fig. 5. Theintegrant produced by this nonconservative pathway wouldhave two copies of the APRT gene, each with an Xho I site,which matches the integrant structure we observed. In thispathway, however, the right-hand copy of the APRT genemust contain the parental Mbo II deletion and the left-handcopy must be the wild-type locus (Fig. 5). Among our 17integrants, we identified 6 that do not match this expectation:5 contained two wild-type copies of the APRT gene and 1contained the parental mutation in the left-hand gene. In

addition, in an analysis of 14 integrants that arose by targetedrecombination at the same locus using a vector linearized atthe unmodified Xho I site or at a nearby Pst I site, 7 wereshown to contain 2 wild-type copies ofthe APRT gene (G. M.Adair, personal communication). Thus, 13 of 31 integrants at

Set 1 Set 2

_.,I_

I9 I

bll975 499 250 9031

1 474 250 903

Set I Set 2wt 1 2 3 1 2 3 P

1474_975-903-

499-

250-

FIG. 4. Predominant integrant structure is shown. Wavy lines,chromosomal sequence; boxes, APRT homologous regions; straightline, plasmid sequence; solid bar, mutation. Relative positions ofPCR primers are shown and the Mbo II digestion fragments of theamplified fragment are shown below. The left-hand gene, amplifiedby 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 diagnosticMbo 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 theright locus; 3, clone with wild-type gene at the left locus and mutantgene at the right locus.

6.914.7_

8.5~~~~~~

20

A 2 3 4 5 6 7 8 9

.: .z.

B

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9502 Genetics: Pennington and Wilson

x x

FIG. 5. An alternative nonconservative pathway for gene target-ing. Substrates for the recombination are one vector (heavy lines)and two chromosomal APRT sequences on sister chromatids (thinlines). The product is indistinguishable from the conservative pre-diction except that this (nonconservative) product must contain theparental mutation (solid bar).

the APRT locus do not match the expectations of the non-conservative pathway shown in Fig. 5. In contrast, theseintegrants are allowed by a conservative pathway (such asDSBR) where they can arise by formation of a heteroduplexthat covers the deletion followed by mismatch repair orreplication (28).Our results support the conclusions of a previous study,

which showed that a double-stranded gap in the vector couldbe repaired during targeted integration onto the chromosome(29). Gap repair is an expectation of the DSBR model ofrecombination but by itself cannot differentiate between theDSBR model (i.e., a conservative pathway) and the noncon-servative pathway shown in Fig. 5. In addition, we wereconcerned that the ectopic site ofthe target gene (simian virus40 large tumor antigen) and the possible growth advantageconferred by expression of the large tumor antigen gene(which would gain an enhancer by a conservative pathwaybut not by a nonconservative pathway) might bias the anal-ysis against integrants derived from the nonconservativepathway. In the experiments described here the recombina-tion target is the endogenous APRT gene in CHO cells and theanalysis of the targeted integrants focuses on the selectivelyneutral copy of the gene.Our analysis of targeted integrants indicates that the path-

way for gene targeting in mammalian cells is similar to that inyeast, which is described by the DSBR model (15). Thatmodel predicts that the nonhomologous ends used in thisexperiment would be corrected by using the resident APRTgene as the template for repair synthesis. Resolution of therecombination intermediate could give the crossover prod-uct, resulting in integration, or the noncrossover product,which releases the vector from the chromosome. In yeast thenoncrossover products can be detected by correction ofchromosomal mutations or by correction ofthe vector, whichexists as a stable or unstable extrachromosomal plasmid (30).The target and vector convertants we detect are analogous tothe two kinds of noncrossover products in yeast, except thatwe detect vector convertants as ectopic integrants rather thanas extrachromosomal elements. This difference may reflectthe propensity for random integration in mammalian cells andits paucity in yeast (24, 30).

Preliminary analysis ofvector convertants in this study anda previous one (6) suggests an aspect of conservative recoim-bination and the DSBR model that may be especially relevant

in mammalian cells-that is, the opportunity to copy chro-mosomal sequences onto the ends of the invading vector. Inmammalian cells where a linear product can be incorporatedrandomly into the genome, copying of chromosomal se-quences onto one or both ends of a vector could allow afragment ofa gene to complete itself. The APRT gene may bean ideal targeting system for detecting such an event becauseof its small size. Copying, if it occurs, may not be extensiveenough to yield a selectable product for larger genes.

We thank Drs. R. S. Nairn and G. M. Adairfor helpful discussionsand critical reading of the manuscript, Dr. G. M. Adair for providingthe ATS-49tg cells, and Kathleen Marburger for technical guidance.The Wilson laboratory and the Baylor Molecular Biology Groupprovided useful discussions. We are grateful to Dr. Dana Carroll forsuggesting the alternative nonconservative recombination modelshown in Fig. 5. This work was supported by National Institutes ofHealth Grants GM 38219 and DK 42678, American Cancer SocietyGrant CD-420, and Cystic Fibrosis Foundation Grant Z112 to J.H.W.

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