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Proc Natl Acad Sci U S A. Dec 1990; 87(24): 9918–9922.

Homologous recombination at c-fyn locus of mouse embryonic stem cells with use of diphtheria toxin A-fragment gene in negative selection.


In attempting to produce a mutant mouse with embryonic stem cells, the critical step is the efficient isolation of homologous recombinants; the frequency of the homologous recombination is usually low and the potency of the cells to differentiate into germ cells is unstable in culture. Here, we report an efficacious method for such isolation in which the diphtheria toxin A-fragment gene is used to negatively select nonhomologous recombinants. In contrast to the use of the herpes simplex virus thymidine kinase gene, the selection can be made singly by the neomycin analog G418 without using a drug such as ganciclovir, a nucleoside analog. At the c-fyn locus, the diphtheria-toxin negative selection enriched the recombinants about 10-fold, and half of the cells integrating with the neomycin phosphotransferase gene were homologous recombinants.

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  • Zijlstra M, Bix M, Simister NE, Loring JM, Raulet DH, Jaenisch R. Beta 2-microglobulin deficient mice lack CD4-8+ cytolytic T cells. Nature. 1990 Apr 19;344(6268):742–746. [PubMed]
  • Thomas KR, Capecchi MR. Targeted disruption of the murine int-1 proto-oncogene resulting in severe abnormalities in midbrain and cerebellar development. Nature. 1990 Aug 30;346(6287):847–850. [PubMed]
  • Joyner AL, Skarnes WC, Rossant J. Production of a mutation in mouse En-2 gene by homologous recombination in embryonic stem cells. Nature. 1989 Mar 9;338(6211):153–156. [PubMed]
  • DeChiara TM, Efstratiadis A, Robertson EJ. A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting. Nature. 1990 May 3;345(6270):78–80. [PubMed]
  • Schwartzberg PL, Goff SP, Robertson EJ. Germ-line transmission of a c-abl mutation produced by targeted gene disruption in ES cells. Science. 1989 Nov 10;246(4931):799–803. [PubMed]
  • Zimmer A, Gruss P. Production of chimaeric mice containing embryonic stem (ES) cells carrying a homoeobox Hox 1.1 allele mutated by homologous recombination. Nature. 1989 Mar 9;338(6211):150–153. [PubMed]
  • Johnson RS, Sheng M, Greenberg ME, Kolodner RD, Papaioannou VE, Spiegelman BM. Targeting of nonexpressed genes in embryonic stem cells via homologous recombination. Science. 1989 Sep 15;245(4923):1234–1236. [PubMed]
  • Williams RL, Hilton DJ, Pease S, Willson TA, Stewart CL, Gearing DP, Wagner EF, Metcalf D, Nicola NA, Gough NM. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature. 1988 Dec 15;336(6200):684–687. [PubMed]
  • Smith AG, Heath JK, Donaldson DD, Wong GG, Moreau J, Stahl M, Rogers D. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature. 1988 Dec 15;336(6200):688–690. [PubMed]
  • Jasin M, Berg P. Homologous integration in mammalian cells without target gene selection. Genes Dev. 1988 Nov;2(11):1353–1363. [PubMed]
  • Zijlstra M, Li E, Sajjadi F, Subramani S, Jaenisch R. Germ-line transmission of a disrupted beta 2-microglobulin gene produced by homologous recombination in embryonic stem cells. Nature. 1989 Nov 23;342(6248):435–438. [PubMed]
  • Mansour SL, Thomas KR, Capecchi MR. Disruption of the proto-oncogene int-2 in mouse embryo-derived stem cells: a general strategy for targeting mutations to non-selectable genes. Nature. 1988 Nov 24;336(6197):348–352. [PubMed]
  • Semba K, Nishizawa M, Miyajima N, Yoshida MC, Sukegawa J, Yamanashi Y, Sasaki M, Yamamoto T, Toyoshima K. yes-related protooncogene, syn, belongs to the protein-tyrosine kinase family. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5459–5463. [PMC free article] [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Thomas KR, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell. 1987 Nov 6;51(3):503–512. [PubMed]
  • Palmiter RD, Behringer RR, Quaife CJ, Maxwell F, Maxwell IH, Brinster RL. Cell lineage ablation in transgenic mice by cell-specific expression of a toxin gene. Cell. 1987 Jul 31;50(3):435–443. [PubMed]
  • Hooper M, Hardy K, Handyside A, Hunter S, Monk M. HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells. Nature. 1987 Mar 19;326(6110):292–295. [PubMed]
  • Suda Y, Hirai S, Suzuki M, Ikawa Y, Aizawa S. Active ras and myc oncogenes can be compatible, but Sv40 large T antigen is specifically suppressed with normal differentiation of mouse embryonic stem cells. Exp Cell Res. 1988 Sep;178(1):98–113. [PubMed]
  • Suda Y, Suzuki M, Ikawa Y, Aizawa S. Mouse embryonic stem cells exhibit indefinite proliferative potential. J Cell Physiol. 1987 Oct;133(1):197–201. [PubMed]
  • Katoh K, Takahashi Y, Hayashi S, Kondoh H. Improved mammalian vectors for high expression of G418 resistance. Cell Struct Funct. 1987 Dec;12(6):575–580. [PubMed]
  • Suda Y, Aizawa S, Hirai S, Inoue T, Furuta Y, Suzuki M, Hirohashi S, Ikawa Y. Driven by the same Ig enhancer and SV40 T promoter ras induced lung adenomatous tumors, myc induced pre-B cell lymphomas and SV40 large T gene a variety of tumors in transgenic mice. EMBO J. 1987 Dec 20;6(13):4055–4065. [PMC free article] [PubMed]
  • Pawson T. Non-catalytic domains of cytoplasmic protein-tyrosine kinases: regulatory elements in signal transduction. Oncogene. 1988 Nov;3(5):491–495. [PubMed]
  • Pappenheimer AM., Jr Diphtheria toxin. Annu Rev Biochem. 1977;46:69–94. [PubMed]
  • Yamaizumi M, Mekada E, Uchida T, Okada Y. One molecule of diphtheria toxin fragment A introduced into a cell can kill the cell. Cell. 1978 Sep;15(1):245–250. [PubMed]
  • Maxwell IH, Maxwell F, Glode LM. Regulated expression of a diphtheria toxin A-chain gene transfected into human cells: possible strategy for inducing cancer cell suicide. Cancer Res. 1986 Sep;46(9):4660–4664. [PubMed]
  • Charron J, Malynn BA, Robertson EJ, Goff SP, Alt FW. High-frequency disruption of the N-myc gene in embryonic stem and pre-B cell lines by homologous recombination. Mol Cell Biol. 1990 Apr;10(4):1799–1804. [PMC free article] [PubMed]
  • Holliday R. Untwisting B-Z DNA. Trends Genet. 1989 Nov;5(11):355–356. [PubMed]
  • Wahls WP, Wallace LJ, Moore PD. The Z-DNA motif d(TG)30 promotes reception of information during gene conversion events while stimulating homologous recombination in human cells in culture. Mol Cell Biol. 1990 Feb;10(2):785–793. [PMC free article] [PubMed]
  • Wahls WP, Moore PD. Homologous recombination enhancement conferred by the Z-DNA motif d(TG)30 is abrogated by simian virus 40 T antigen binding to adjacent DNA sequences. Mol Cell Biol. 1990 Feb;10(2):794–800. [PMC free article] [PubMed]
  • Brinster RL, Braun RE, Lo D, Avarbock MR, Oram F, Palmiter RD. Targeted correction of a major histocompatibility class II E alpha gene by DNA microinjected into mouse eggs. Proc Natl Acad Sci U S A. 1989 Sep;86(18):7087–7091. [PMC free article] [PubMed]
  • Veillette A, Bolen JB. src-related protein tyrosine kinases. Cancer Treat Res. 1989;47:121–142. [PubMed]
  • Veillette A, Bookman MA, Horak EM, Bolen JB. The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56lck. Cell. 1988 Oct 21;55(2):301–308. [PubMed]
  • Steele RE, Deng JC, Ghosn CR, Fero JB. Structure and expression of fyn genes in Xenopus laevis. Oncogene. 1990 Mar;5(3):369–376. [PubMed]
  • Kawakami T, Pennington CY, Robbins KC. Isolation and oncogenic potential of a novel human src-like gene. Mol Cell Biol. 1986 Dec;6(12):4195–4201. [PMC free article] [PubMed]

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