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Nucleic Acids Res. 1993 October 11; 21(20): 4783–4787.
PMCID: PMC331506

A method for the generation of YAC transgenic mice by pronuclear microinjection.


Yeast artificial chromosomes (YACs) represent the latest generation of vectors which have the great advantage of large insert size. The introduction of YACs into mammalian cells and organisms has become an important goal, since it offers the potential to study the control of large and complex transcription units and identify genes by complementation. Microinjection into the nucleus is the most direct and efficient way of delivering YAC DNA into cells, but requires the purification of the YAC from the remaining yeast chromosomes. Here we describe a detailed method for the isolation of pure, intact and highly concentrated YAC DNA. As a model system the murine tyrosinase gene was chosen and four YACs covering this locus were isolated. Introduction by homologous recombination in yeast of sequences permitting YAC amplification greatly facilitated the isolation of YAC DNA at high concentrations. YAC DNA stabilized in a salt and polyamine containing buffer did not compromise the survival of microinjected oocytes and was suitable for the generation of transgenic mice. Applications and benefits of this technique will be discussed.

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Selected References

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  • Burke DT, Carle GF, Olson MV. Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science. 1987 May 15;236(4803):806–812. [PubMed]
  • Pavan WJ, Hieter P, Reeves RH. Modification and transfer into an embryonal carcinoma cell line of a 360-kilobase human-derived yeast artificial chromosome. Mol Cell Biol. 1990 Aug;10(8):4163–4169. [PMC free article] [PubMed]
  • Huxley C, Hagino Y, Schlessinger D, Olson MV. The human HPRT gene on a yeast artificial chromosome is functional when transferred to mouse cells by cell fusion. Genomics. 1991 Apr;9(4):742–750. [PubMed]
  • Pachnis V, Pevny L, Rothstein R, Costantini F. Transfer of a yeast artificial chromosome carrying human DNA from Saccharomyces cerevisiae into mammalian cells. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5109–5113. [PubMed]
  • Jakobovits A, Moore AL, Green LL, Vergara GJ, Maynard-Currie CE, Austin HA, Klapholz S. Germ-line transmission and expression of a human-derived yeast artificial chromosome. Nature. 1993 Mar 18;362(6417):255–258. [PubMed]
  • Gnirke A, Barnes TS, Patterson D, Schild D, Featherstone T, Olson MV. Cloning and in vivo expression of the human GART gene using yeast artificial chromosomes. EMBO J. 1991 Jul;10(7):1629–1634. [PubMed]
  • Eliceiri B, Labella T, Hagino Y, Srivastava A, Schlessinger D, Pilia G, Palmieri G, D'Urso M. Stable integration and expression in mouse cells of yeast artificial chromosomes harboring human genes. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2179–2183. [PubMed]
  • Fernandez-Luna JL, Matthews RJ, Brownstein BH, Schreiber RD, Thomas ML. Characterization and expression of the human leukocyte-common antigen (CD45) gene contained in yeast artificial chromosomes. Genomics. 1991 Jul;10(3):756–764. [PubMed]
  • Strauss WM, Jaenisch R. Molecular complementation of a collagen mutation in mammalian cells using yeast artificial chromosomes. EMBO J. 1992 Feb;11(2):417–422. [PubMed]
  • Choi TK, Hollenbach PW, Pearson BE, Ueda RM, Weddell GN, Kurahara CG, Woodhouse CS, Kay RM, Loring JF. Transgenic mice containing a human heavy chain immunoglobulin gene fragment cloned in a yeast artificial chromosome. Nat Genet. 1993 Jun;4(2):117–123. [PubMed]
  • Strauss WM, Dausman J, Beard C, Johnson C, Lawrence JB, Jaenisch R. Germ line transmission of a yeast artificial chromosome spanning the murine alpha 1(I) collagen locus. Science. 1993 Mar 26;259(5103):1904–1907. [PubMed]
  • Gnirke A, Huxley C, Peterson K, Olson MV. Microinjection of intact 200- to 500-kb fragments of YAC DNA into mammalian cells. Genomics. 1993 Mar;15(3):659–667. [PubMed]
  • Schedl A, Beermann F, Thies E, Montoliu L, Kelsey G, Schütz G. Transgenic mice generated by pronuclear injection of a yeast artificial chromosome. Nucleic Acids Res. 1992 Jun 25;20(12):3073–3077. [PMC free article] [PubMed]
  • Schedl A, Montoliu L, Kelsey G, Schütz G. A yeast artificial chromosome covering the tyrosinase gene confers copy number-dependent expression in transgenic mice. Nature. 1993 Mar 18;362(6417):258–261. [PubMed]
  • Smith DR, Smyth AP, Moir DT. Amplification of large artificial chromosomes. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8242–8246. [PubMed]
  • Hill A, Bloom K. Genetic manipulation of centromere function. Mol Cell Biol. 1987 Jul;7(7):2397–2405. [PMC free article] [PubMed]
  • Larin Z, Monaco AP, Lehrach H. Yeast artificial chromosome libraries containing large inserts from mouse and human DNA. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4123–4127. [PubMed]
  • Kelsey G, Schedl A, Ruppert S, Niswander L, Magnuson T, Klebig ML, Rinchik EM, Schütz G. Physical mapping of the albino-deletion complex in the mouse to localize alf/hsdr-1, a locus required for neonatal survival. Genomics. 1992 Oct;14(2):275–287. [PubMed]
  • Anand R, Villasante A, Tyler-Smith C. Construction of yeast artificial chromosome libraries with large inserts using fractionation by pulsed-field gel electrophoresis. Nucleic Acids Res. 1989 May 11;17(9):3425–3433. [PMC free article] [PubMed]
  • Burgers PM, Percival KJ. Transformation of yeast spheroplasts without cell fusion. Anal Biochem. 1987 Jun;163(2):391–397. [PubMed]
  • Beermann F, Ruppert S, Hummler E, Bosch FX, Müller G, Rüther U, Schütz G. Rescue of the albino phenotype by introduction of a functional tyrosinase gene into mice. EMBO J. 1990 Sep;9(9):2819–2826. [PubMed]
  • Tanaka S, Yamamoto H, Takeuchi S, Takeuchi T. Melanization in albino mice transformed by introducing cloned mouse tyrosinase gene. Development. 1990 Feb;108(2):223–227. [PubMed]
  • Yokoyama T, Silversides DW, Waymire KG, Kwon BS, Takeuchi T, Overbeek PA. Conserved cysteine to serine mutation in tyrosinase is responsible for the classical albino mutation in laboratory mice. Nucleic Acids Res. 1990 Dec 25;18(24):7293–7298. [PMC free article] [PubMed]
  • Holdener-Kenny B, Sharan SK, Magnuson T. Mouse albino-deletions: from genetics to genes in development. Bioessays. 1992 Dec;14(12):831–839. [PubMed]
  • Smith DR, Smyth AP, Strauss WM, Moir DT. Incorporation of copy-number control elements into yeast artificial chromosomes by targeted homologous recombination. Mamm Genome. 1993;4(3):141–147. [PubMed]
  • Couto LB, Spangler EA, Rubin EM. A method for the preparative isolation and concentration of intact yeast artificial chromosomes. Nucleic Acids Res. 1989 Oct 11;17(19):8010–8010. [PMC free article] [PubMed]
  • Brinster RL, Chen HY, Trumbauer ME, Yagle MK, Palmiter RD. Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4438–4442. [PubMed]
  • Gosule LC, Schellman JA. DNA condensation with polyamines I. Spectroscopic studies. J Mol Biol. 1978 May 25;121(3):311–326. [PubMed]
  • Chattoraj DK, Gosule LC, Schellman A. DNA condensation with polyamines. II. Electron microscopic studies. J Mol Biol. 1978 May 25;121(3):327–337. [PubMed]

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