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the TAP-Hygro or βgeok cassettes. For each engineering step, cells harboring theBAC were transformed with the Red/ET expression plasmid pR6K-αβγby stan-dard procedures. Single colonies were picked and grown in 5 ml Luria (L) brothovernight. Then, 0.7 ml was transferred into 70 ml of L-broth (without glucose)and grown at 37 °C.At OD600 = 0.1–0.15, 0.7 ml of 10% L-arabinose (wt/vol) wasadded to induce Red protein expression. At OD600 0.25–0.4, cells were cen-trifuged (10 min, 3,800g, –5 °C, Sorvall SS34 rotor; plastic tubes) and resus-pended in 30 ml ice-cold 10% glycerol (vol/vol) (repeated three times). After thefinal centrifugation, decanting, and wiping the tube with a tissue, cells wereresuspended in a minimum residual volume (<500 µl final resuspended vol-ume) and 50 µl aliquots were immediately electroporated with 0.3–1 µg of lin-ear DNA fragment (PCR product or a fragment excised from a plasmid).Colonies were identified on selection plates containing appropriate combina-tions of the following antibiotics (tetracycline 25 µg/ml for pR6K-αβγ, chloram-phenicol 12.5 µg/ml, ampicillin 50 µg/ml, gentamicin 3 µg/ml, kanamycin20 µg/ml, hygromycin 50 µg/ml).

BAC DNA was prepared using the Large Construct Maxiprep Kit (Qiagen,Düsseldorf, Germany). Five liters yielded 270 µg. After PI-SceI digestion of thetargeting construct (10 µg, 20 U enzyme, 200 µl final volume, 37 °C, overnightincubation), the DNA was extracted with phenol-chloroform, precipitated withethanol, and resuspended in PBS.

ES cells. Mouse E14Tg2a (ref. 24) ES cells were cultured without feeders inmedium supplemented with recombinant leukemia inhibitory factor (LIF) asdescribed24. Electroporation was carried out with a BioRad Gene Pulser usingstandard conditions (3 µF, 800 V), adding 90 µg DNA in 90 µl PBS (without cal-cium and magnesium ions) to 7.4 × 10 7 cells, resuspended in 710 µl PBS. Cellswere seeded at a density of 2 ×106 cells/10 cm plate.At 24 h after electroporation,drug selection was started at the following concentrations: G418 (200 µg/ml);hygromycin (160 µg/ml).

ES cell injections were done using standard procedures25.Protein nuclear extracts from ES cells, embryoid bodies, and mouse organs

were done with the CellLytic NuClear extraction kit from Sigma(Deisenhofen, Germany). Western blotting was done following standard pro-cedures. The protein A part of the TAP tag11 was visualized with a peroxidase-coupled anti-peroxidase rabbit antibody (Sigma) only (no first antibody), andthe ECL system (Amersham).

AcknowledgmentsWe wish to thank Konstantinos Anastassiadis, William Brown, Frank van derHoeven, Robin Lovell-Badge, and Daniela Nebenius-Oosthuizen for discussionsand help. This work was partly funded by a grant from the VolkswagenFoundation, Program on Conditional Mutagenesis. The work was initiated at theEuropean Molecular Biology Laboratory (EMBL), Heidelberg.

Competing interests statementThe authors declare competing financial interests: see the Nature Biotechnologywebsite (http://www.nature.com/naturebiotechnology) for details.

Received 2 October 2002; accepted 15 January 2003

1. Muyrers, J.P., Zhang, Y. & Stewart, A.F. Techniques: recombinogenic engineering—new options for cloning and manipulating DNA. Trends Biochem. Sci. 26, 325–331(2001).

2. Copeland, N.G., Jenkins, N.A. & Court, D.L. Recombineering: a powerful new tool formouse functional genomics. Nat. Rev. Genet. 2, 769–779 (2001).

3. Heintz, N. BAC to the future: the use of bac transgenic mice for neuroscienceresearch. Nat. Rev. Neurosci. 2, 861–870 (2001).

4. Zhang, Y., Buchholz, F., Muyrers, J.P. & Stewart, A.F. A new logic for DNA engineeringusing recombination in Escherichia coli. Nat. Genet. 20, 123–128 (1998).

5. Muyrers, J.P., Zhang, Y., Testa, G. & Stewart, A.F. Rapid modification of bacterial arti-ficial chromosomes by ET-recombination. Nucleic Acids Res. 27, 1555–1557 (1999).

6. Zhang, Y., Muyrers, J.P., Testa, G.& Stewart, A.F.DNA cloning by homologous recom-bination in Escherichia coli. Nat. Biotechnol. 18, 1314–1317 (2000).

7. Testa, G. & Stewart, A.F. Creating a translocation. Engineering interchromosomaltranslocations in the mouse. EMBO Rep. 1, 120–121 (2000).

8. Rowley, J.D. The critical role of chromosome translocations in human leukemias.Annu. Rev. Genet. 32, 495–519 (1998).

9. Nilson, I. et al. Exon/intron structure of the human ALL-1 (MLL) gene involved intranslocations to chromosomal region 11q23 and acute leukaemias. Br. J. Haematol.93, 966–972 (1996).

10. Angrand, P.O., Daigle, N., van der Hoeven, F., Scholer, H.R. & Stewart, A.F. Simplifiedgeneration of targeting constructs using ET recombination. Nucleic Acids Res. 27,e16 (1999).

11. Rigaut, G. et al. A generic protein purification method for protein complex characteri-zation and proteome exploration. Nat. Biotechnol. 17, 1030–1032 (1999).

12. Buchholz, F., Angrand, P.O. & Stewart, A.F. Improved properties of FLP recombinaseevolved by cycling mutagenesis. Nat. Biotechnol. 16, 657–662 (1998).

13. Zambrowicz, B.P. et al. Disruption of overlapping transcripts in the ROSA βgeo26gene trap strain leads to widespread expression of β-galactosidase in mouseembryos and hematopoietic cells. Proc. Natl. Acad. Sci. USA 94, 3789–3794 (1997).

14. Moens, C.B., Auerbach, A.B., Conlon, R.A., Joyner, A.L. & Rossant, J. A targetedmutation reveals a role for N-myc in branching morphogenesis in the embryonicmouse lung. Genes Dev. 6, 691–704 (1992).

15. Muyrers, J.P. et al. Point mutation of bacterial artificial chromosomes by ET recombi-nation. EMBO Rep. 1, 239–243 (2000).

16. Ellis, H.M., Yu, D., DiTizio, T. & Court, D.L. High-efficiency mutagenesis, repair, andengineering of chromosomal DNA using single-stranded oligonucleotides.Proc. Natl.Acad. Sci. USA 98, 6742–6746 (2001).

17. Baer, A. & Bode, J. Coping with kinetic and thermodynamic barriers: RMCE, an effi-cient strategy for the targeted integration of transgenes. Curr. Opin. Biotechnol. 12,473–480 (2001).

18. Meyers, E.N., Lewandoski, M. & Martin, G.R. An Fgf8 mutant allelic series generatedby Cre- and Flp-mediated recombination. Nat. Genet. 18, 136–141 (1998).

19. Nagy, A. et al. Dissecting the role of N-myc in development using a single targetingvector to generate a series of alleles. Curr. Biol. 8, 661-664 (1998).

20. Andreas, S., Schwenk, F., Kuter-Luks, B., Faust, N. & Kuhn, R. Enhanced efficiencythrough nuclear localization signal fusion on phage PhiC31-integrase: activity com-parison with Cre and FLPe recombinase in mammalian cells. Nucleic Acids Res. 30,2299-2306 (2002).

21. Buchholz, F. & Stewart, A.F. Alteration of Cre recombinase site specificity by sub-strate-linked protein evolution. Nat. Biotechnol. 19, 1047–1052 (2001).

22. Ayton, P. et al. Truncation of the Mll gene in exon 5 by gene targeting leads to earlypreimplantation lethality of homozygous embryos. Genesis 30, 201–212 (2001).

23. Benes, V., Kilger, C., Voss, H., Paabo, S. & Ansorge, W. Direct primer walking on P1plasmid DNA. Biotechniques 23, 98–100 (1997).

24. Nichols, J., Evans, E.P. & Smith, A.G. Establishment of germ-line-competent embry-onic stem (ES) cells using differentiation-inhibiting activity. Development 110,1341–1348 (1990).

25. Joyner, A.L. (ed.). Gene Targeting. A Practical Approach (Oxford University Press,New York, 2000).

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Site-specific gene targetingin mouse embryonic stemcells with intact bacterialartificial chromosomesYi Yang and Brian Seed*

Published online 10 March 2003; doi:10.1038/nbt803

Homologous recombination in Escherichia coli simplifies the gen-eration of gene targeting constructs for transduction into mouseembryonic stem (ES) cells1–7. Taking advantage of the extensivehomology provided by intact bacterial artificial chromosomes(BACs), we have developed an efficient method for preparing tar-geted gene disruptions in ES cells. Correctly integrated cloneswere identified by a simple screening procedure based on chro-mosomal fluorescence in situ hybridization (FISH). To date, fivemutant lines have been generated and bred to homozygosity bythis approach.

Site-specific gene disruption in mice has been an important toolfor the analysis of gene function in vivo8,9. However, the process isresource intensive and the targeting frequencies typically are low.As currently practiced, creation of a targeting vector requires theformation in bacteria of a replica of the desired disrupted gene, fol-lowed by introduction of the replica into ES cells and identificationof the properly targeted DNA. Orthotopic integration can be

Department of Molecular Biology, Massachusetts General Hospital, andDepartment of Genetics, Harvard Medical School, Boston, MA 02114.*Corresponding author (seed@molbio.mgh.harvard.edu).

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detected by blot hybridization after digestion of the ES DNA withrestriction enzymes that cleave outside the transfected sequences,or by PCR using primers that are positioned in the flanking regionand the disrupted gene. With either method, the flankingsequences of the replica cannot be too long. Although previousstudies have shown some correlation between the length of thegenomic sequences included in the targeting construct and the tar-geting efficiency in ES cells10,11, the effect of very long flankingsequences has not been systematically studied. Data from haplo-type mapping of human populations and studies of meiotic andmitotic recombination frequencies in lower eukaryotes support theidea that favored sites for initiation of DNA exchange can be sepa-rated by up to several tens of kilobases from one another in euchro-matin. If these findings are relevant to mitotic recombination in EScells, it may be necessary to use very long flanking sequences toobtain high-frequency homologous recombination. Creating suchlong flanking arms by conventional cloning is cumbersome andfrequently impractical. To facilitate the creation of the replica inE. coli, several groups have used bacterial in vivo recombination1–7.

The recombination-promoting plasmid in our study contains thebacteriophage λ red genes α and β under the regulation of thearaBAD promoter4,12. Inclusion of the γ gene increased the totalyield of colonies following electroporation, but the frequency ofcorrect recombination events was low (data not shown). Consistentwith this, it has been reported elsewhere that neither gam nor com-promise of recBC are required for λ red–mediated recombination oflinear DNA fragments with the E. coli chromosome13.

Representative results from this strategy are shown in Figure 1.We introduced the zeor/neor dual selection cassette into a BACclone containing the mouse Fancg gene (also known as Xrcc9),replacing exons 1–10 (Fig. 1A). Using primers G34/Pzeo andPsv/G40 (see Experimental protocol), we screened chlorampheni-col- and zeocin-resistant colonies by PCR to identify correct tar-geting events at the 5′ and 3′ ends, respectively (Fig. 1B). The PCRproduct amplified from exons 5 and 6 was seen only in the wild-type BAC (Fig. 1B).

To determine whether any unintended rearrangements occurredduring modification, we compared EcoRV and SpeI digestion pat-terns of the mutant BACs with those of the wild-type BAC andfound no gross alterations (Fig. 1C). Blot hybridization yielded frag-ment patterns predicted from the mouse genomic sequence (Fig.1A,D). The fidelity of this procedure was further confirmed by shot-gun sequencing of the mutant BAC (data not shown).

To identify correct targeting events, we devised a simpleapproach based on PCR and FISH (Fig. 2A). The BAC is linearizedso that flanking vector arms remain on either side of the insert.G418-resistant colonies are first screened for the presence of thesearms by PCR and any colonies containing either of the vector seg-ments are discarded. The remaining colonies are then screened byFISH. If the mutant BAC replaces one of the wild-type gene loci,there should be no net gain in gene copy number as detected by FISHusing the BAC as probe. In contrast, random integration shouldresult in the appearance of an additional hybridization signal.

We used this strategy to disrupt the Fancg locus (Fig. 2B,C). Weelectroporated the modified BAC into ES cells and carried outPCR on the genomic DNA extracted from G418-resistant clones.Clone 68 showed both the control PCR fragment and the BAC vec-tor fragment, whereas clones C38 and C52 showed only the con-trol fragment (Fig. 2B). We carried out TSA-FISH on these clonesalong with wild-type ES cells using the entire BAC as probe. Wedetected two signals in clones C38 and C52 as well as in untarget-ed ES cells and an extra signal in clone C68 (Fig. 2C), suggestingthat C38 and C52 reflect correct targeting events and C68 is theresult of random integration. Although TSA-FISH has beenreported to be reliable for the detection of regions <10 kb14, wecarried out genomic DNA hybridization to exclude the possibilitythat G418 resistance could be ascribed to the random integrationof a zeo/neo fragment that was too small to be detected by TSA-FISH (Fig. 2D). The expected patterns were found with all threeprobes, indicating that the zeor/neor marker had integrated into theFancg locus and the 20 kb surrounding genomic region had notundergone any disruption other than the desired targeting event

Figure 1. Generation of Fancg (Xrcc9) knockout construct. (A) Structures of wild-type (WT) and mutant gene (MT). Gray blocks represent homologoussequences, open blocks exons, black vertical arrows SpeI sites, and open vertical arrows EcoRV sites. Short horizontal arrows indicate primer locations.FRT, Flp recombination target. (B) Results of PCR using primers indicated in (A) show correct integration at the 5′ and the 3′ sites and deletion of exons 5and 6. M, molecular size markers. (C) Restriction digestion of DNAs from wild-type (WT) and two mutant BACs (1 and 2) by SpeI and EcoRV. (D) DNAblotting of gel in (C) with 5′ probe indicated in (A).

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(Fig. 2D). As expected, DNA hybridization alone could not distin-guish orthotopic (C38 and C52) from ectopic (C68) integrationevents (Fig. 2D). We estimate that the region encompassed by theblotting data represents 1/5–1/7 of the intact BAC. Although largeDNAs are known to be susceptible to breakage by hydrodynamicshearing, the protocol used here minimizes the manipulation ofthe BAC DNA in vitro, and to date no evidence of adventitiousrearrangement of the targeting site has been seen. Consistent withthis, it has been reported that cells modified by gene targeting withone DNA fragment rarely incorporate a second DNA fragmentsimultaneously15.

The results of several experiments are summarized in Table 1.Usually 1 × 103–2 × 103 G418-resistant colonies could be obtainedfrom electroporation of 107 cells. For the five genes studied, we cal-culate an average effective targeting efficiency of 15% and an esti-mated absolute targeting efficiency of ∼ 10–5 without selectionenrichment. Historical gene targeting efficiencies vary from 10–3 toundetectable as a function of the locus targeted, vector design, andselection methods16. Literature reports suggest the average efficien-cy is closer to 10–6–10–7, which is the mean targeting efficiency ofthe well-studied Hprt locus17. Our mean ratio of homologous to

nonhomologous recombination events is 1.5 × 10–1, whereas that oftraditional methods falls between 10–2 and 10–5 (ref. 15). The effi-ciency we observe is close to the homologous recombination ratefound in nuclear extracts from mouse embryonic fibroblast cells18.

To prove that the ES clones identified by FISH represented correcttargeting events, we injected several ES clones into blastocysts,obtained germline chimeras, and bred the resulting progeny tohomozygosity. Viable homozygous null mice were identified withdisruptions in Fancg, Kbras1, Dok3, and Pag, as verified by genomicPCR genotyping and RT-PCR (Fig. 3A,B). Breeding of heterozygousMap3k7ip2 mutant mice did not result in viable homozygous off-spring, but homozygous embryos were detected at embryonic day9.5 (Fig. 3A,B). To date, all ES clones that have shown germlinetransmission have yielded homozygous mice. To evaluate locusintegrity around the targeted site, we amplified genomic markerswithin 100 kb flanking the targeting site in DNA from both the wild-type and mutant mice. All the markers tested were preserved in themutant mice (Fig. 3C), suggesting that an intact BAC can replace theendogenous gene locus by homologous recombination withoutadventitious deletion by undesired recombination between repeatedsequence elements in the BAC arms.

Figure 2. Targeting of ES cells by intact BACs. (A) Screening homologous recombination by PCR and FISH. Broken gray blocks represent largehomologous regions and black blocks BAC vector sequences. WT, wild-type locus; MT, mutant locus; FRT, Flp recombination target. (B) PCR analysis ofgenomic DNA extracted from Fancg-targeted ES clones C38, C52, and C68 and untargeted ES cells. BAC DNA was used as positive control. PCRfragments from the 5′ and the 3′ of BAC vector and the internal controls are indicated on the left. (C) FISH analysis of cell lines in (B). (D) Blot hybridizationof genomic DNA extracted from indicated clones and untargeted ES cells using probes indicated in (A) and a probe specific for zeor/neor cassette.

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Table 1. Summary of gene targeting efficiency

PCR screening (96 colonies) FISH screening

Gene No 5′ 3′ 5′ and 3′ No BAC Colonies 2 spots 3 spots Effective targetingsignala BAC BAC BAC signalb screened efficiencyc

Fancg 36 4 6 28 22 (37%) 4 3 (75%) 1 28%Kbras1 1 16 12 15 52 (55%) 27 6 (22%) 21 12%Pag 6 10 5 32 43 (48%) 16 7 (44%) 9 21%Dok3 6 12 9 55 14 (16%) 11 5 (45%) 6 7%Map3k7ip2 16 8 16 27 29 (36%) 10 2 (20%) 8 7%

aA high PCR failure rate (internal control negative) was seen with Fancg before the introduction of improved protocols.bPercentage calculation denominator excludes PCR failures.cThe percentage with no vector signal multiplied by the percentage with 2 FISH spots.

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Experimental protocolMice. All mouse strains were raised and maintained with protocols approvedby the institutional animal care and usage committee of MassachusettsGeneral Hospital.

Sequences of oligonucleotides. Pzeo: 5′-GGC CAG GGT GTT GTC CGGCAC C-3′; Psv: 5′-AAG GTT GGG CTT CGG AAT CG-3′; Pa: 5′-ACA GATGCG TAA GGA GAA AAT AC-3′; Pb: 5′-CGC CCT ATA GTG AGT CGT ATTAC-3′; Pc: 5′-ATA GTG TCA CCT AAA TAG CTT GG-3′; Pd: 5′-GGC ACGACA GGT TTC CCG ACT GG-3′. Control primers: 5′-GAG GAC ATC TTTCCC TCA GGC-3′; 5′-CAG AGG CTC TGA GTA AGA CC-3′. Primers fortargeting Fancg have been described19.

ES cell screening. BACs were modified as previously described2–6. DNA waspurified, digested with NotI, extracted with phenol-chloroform, and resus-pended in 0.1× TE buffer at 1 µg/µl. We electroporated 107 cells with 30 µg ofBAC DNA at 0.25 kV, 960 µF with a Bio-Rad Gene Pulser (Richmond, CA) andselected for transformants with 400 µg/ml G418 (Invitrogen, Carlsbad, CA).PCR reactions contained 0.1 µg of genomic DNA, 0.2 mM dNTPs, 0.5 µM ofprimers, and 1 unit of Taq polymerase in 20 µl of 1× reaction buffer (Roche,Indianapolis, IN) and were performed at 94 °C, 3 min, 1 cycle; 94 °C, 45 s, 55 °C,45 s and 72 °C, 45 s, 35 cycles; 72 °C, 5 min, 1 cycle.

TSA-FISH. BACs were labeled using the Prime-It Fluor FluorescenceLabeling Kit (Stratagene, La Jolla, CA). Manufacturers’ protocols were fol-lowed and 35 µg COT-1 DNA (Invitrogen) was used to suppress nonspecif-

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Gene targeting by homologous recombination allows precisegenetic manipulation. However, the complexity and expense of theprocess often discourage investigators from applying it routinely.Several reports have described systems for constructing targetingvectors by modifying BACs or PACs through recombination in E. coli1–7. Although the use of intact BACs as targeting constructs isan attractive strategy, genomic PCR or DNA blotting are only feasi-ble when a small part of the BAC is taken as the homologous regionin the targeting construct. We have simplified the identification oforthotopic integration by using FISH with the entire BAC as probe.To reduce the number of FISH experiments required, we used PCRto detect the vector sequences attached to the ends of the BAC as aprimary screening method.

The system can be used to modify BACs, PACs, or P1 clones with-out need for special strains or libraries3,5. Although more data mustbe collected to accurately estimate the increase in efficiency withintact BACs, anecdotal evidence suggests the procedure providessubstantially higher targeting frequencies. For instance, we did notobtain any correctly targeted clones from 192 colonies using con-ventional procedures to target Fancg. The method also provides asimpler approach to more sophisticated genomic manipulations,such as conditional knockouts, ‘knock-ins’, and large-scale chromo-somal engineering.

Figure 3. Generation of knockout mice. (A) Genotyping of mouse tail genomic DNA by PCR with primers amplifying the wild-type (WT) locus and themutant (MT) locus. (B) RT-PCR analysis of mRNA extracted from mouse splenocytes with primers amplifying wild-type gene transcripts and primersamplifying coding regions of either B2m (β2-microglobulin) mRNA (300 bp) or Casp8 (caspase-8) mRNA (500 bp) as internal controls. (C) Analysis ofgenomic markers within 100 kb of the targeting site. Distance of the markers from the targeting site and the sizes of the PCR fragments are indicated.Some markers (M9 for the Fancg locus and M3 for Kbras1 locus) are absent in corresponding BACs, suggesting that these markers are outside thegenomic inserts of the BACs.

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ic signals. The probe was dissolved in Hybrisol VI (Ventana, Tucson, AZ),denatured at 75 °C for 10 min, and incubated at 42 °C for 1 h. Slides seededwith 105 cells per sample were fixed in 4% (wt/vol) paraformaldehyde inPBS (pH 7.4) for 10 min at room temperature. They were then dehydrated 2 min each in 80%, 85%, 95%, and 100% ethanol; air-dried; denatured in70% (vol/vol) deionized formamide (American Bioanalytical, Natick, MA),2× SSC (pH 7.0) at 72 °C for 10 min; and dehydrated through ice-cold 70%,80%, 95%, and 100% ethanol. We applied 4 µl of 10 ng/µl probe per samplearea. Hybridization was done overnight in a humid chamber at 42 °C. Slideswere washed for 7 min in 2× SSC (pH 7.4) at 72 °C three times and once in0.2× SSC (pH 7.4) at 72 °C for 7 min. Signals were amplified using TSAFluorescence Systems (NEN Life Science Products, Boston, MA). Slideswere counterstained with DAPI (Sigma, St. Louis, MO) in 4× SSC, mountedin Vectashield (Vector Laboratories, Burlingame, CA), and observed undera Zeiss Axioplan 2 fluorescence microscope.

AcknowledgmentsWe thank Naifang Lu and Jeannie T. Lee for help in developing the FISH protocol, Naifang Lu for blastocyst injections, and Vidya Kunjathoor, YanhongMa, and Amy Stirman for assistance. This work was supported by grants fromthe US National Institutes of Health (AI27849, AI46731, and HL66678 to B.S.)and a postdoctoral fellowship from the Cancer Research Institute (to Y.Y.).

Competing financial interestsThe authors declare that they have no competing financial interests.

Received 27 August 2002; accepted 16 January 2003

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