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Proc Natl Acad Sci U S A. 1996 August 20; 93(17): 9067–9072. | PMCID: PMC38596 |
Single-copy transgenic mice with chosen-site integration. S K Bronson, E G Plaehn, K D Kluckman, J R Hagaman, N Maeda, and O Smithies Department of Pathology, University of North Carolina, Chapel Hill 27599-7525, USA. Abstract We describe a general way of introducing transgenes into the mouse germ line for comparing different sequences without the complications of variation in copy number and insertion site. The method uses homologous recombination in embryonic stem (ES) cells to generate mice having a single copy of a transgene integrated into a chosen location in the genome. To test the method, a single copy murine bcl-2 cDNA driven by either a chicken beta-actin promoter or a human beta-actin promoter has been inserted immediately 5' to the X-linked hypoxanthine phosphoribosyltransferase locus by a directly selectable homologous recombination event. The level of expression of the targeted bcl-2 transgene in ES cells is identical in independently isolated homologous recombinants having the same promoter yet varies between the different promoters. In contrast, the expression of bcl-2 transgenes having the same (chicken beta-actin) promoter varies drastically when they are independently integrated at random insertion sites. Both promoters direct broad expression of the single-copy transgene in mice derived from the respective targeted ES cells. In vitro and in vivo, the human beta-actin promoter consistently directed a higher level of transgene expression than the chicken beta-actin promoter. Full text Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.9M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References. Images in this article Click on the image to see a larger version. These references are in PubMed. This may not be the complete list of references from this article. - Palmiter RD, Brinster RL. Germ-line transformation of mice. Annu Rev Genet. 1986;20:465–499. [PubMed]
- Meisler MH. Insertional mutation of 'classical' and novel genes in transgenic mice. Trends Genet. 1992 Oct;8(10):341–344. [PubMed]
- Costantini F, Radice G, Lee JL, Chada KK, Perry W, Son HJ. Insertional mutations in transgenic mice. Prog Nucleic Acid Res Mol Biol. 1989;36:159–169. [PubMed]
- Caskey CT, Kruh GD. The HPRT locus. Cell. 1979 Jan;16(1):1–9. [PubMed]
- Reid LH, Shesely EG, Kim HS, Smithies O. Cotransformation and gene targeting in mouse embryonic stem cells. Mol Cell Biol. 1991 May;11(5):2769–2777. [PubMed]
- Quitschke WW, Lin ZY, DePonti-Zilli L, Paterson BM. The beta actin promoter. High levels of transcription depend upon a CCAAT binding factor. J Biol Chem. 1989 Jun 5;264(16):9539–9546. [PubMed]
- Nuñez G, London L, Hockenbery D, Alexander M, McKearn JP, Korsmeyer SJ. Deregulated Bcl-2 gene expression selectively prolongs survival of growth factor-deprived hemopoietic cell lines. J Immunol. 1990 May 1;144(9):3602–3610. [PubMed]
- Tybulewicz VL, Crawford CE, Jackson PK, Bronson RT, Mulligan RC. Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene. Cell. 1991 Jun 28;65(7):1153–1163. [PubMed]
- Gunning P, Leavitt J, Muscat G, Ng SY, Kedes L. A human beta-actin expression vector system directs high-level accumulation of antisense transcripts. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4831–4835. [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. 326(6110):292–295. [PubMed]
- Rossant J, Vijh KM, Grossi CE, Cooper MD. Clonal origin of haematopoietic colonies in the postnatal mouse liver. Nature. 1986 Feb 6;319(6053):507–511. [PubMed]
- Lock LF, Takagi N, Martin GR. Methylation of the Hprt gene on the inactive X occurs after chromosome inactivation. Cell. 1987 Jan 16;48(1):39–46. [PubMed]
- Gartler SM, Dyer KA, Goldman MA. Mammalian X chromosome inactivation. Mol Genet Med. 1992;2:121–160. [PubMed]
- Willard HF, Brown CJ, Carrel L, Hendrich B, Miller AP. Epigenetic and chromosomal control of gene expression: molecular and genetic analysis of X chromosome inactivation. Cold Spring Harb Symp Quant Biol. 1993;58:315–322. [PubMed]
- Shaw-White JR, Denko N, Albers L, Doetschman TC, Stringer JR. Expression of the lacZ gene targeted to the HPRT locus in embryonic stem cells and their derivatives. Transgenic Res. 1993 Jan;2(1):1–13. [PubMed]
- Crossley M, Orkin SH. Regulation of the beta-globin locus. Curr Opin Genet Dev. 1993 Apr;3(2):232–237. [PubMed]
- Januzzi JL, Azrolan N, O'Connell A, Aalto-Setälä K, Breslow JL. Characterization of the mouse apolipoprotein Apoa-1/Apoc-3 gene locus: genomic, mRNA, and protein sequences with comparisons to other species. Genomics. 1992 Dec;14(4):1081–1088. [PubMed]
- Stacey A, Schnieke A, McWhir J, Cooper J, Colman A, Melton DW. Use of double-replacement gene targeting to replace the murine alpha-lactalbumin gene with its human counterpart in embryonic stem cells and mice. Mol Cell Biol. 1994 Feb;14(2):1009–1016. [PubMed]
- Detloff PJ, Lewis J, John SW, Shehee WR, Langenbach R, Maeda N, Smithies O. Deletion and replacement of the mouse adult beta-globin genes by a "plug and socket" repeated targeting strategy. Mol Cell Biol. 1994 Oct;14(10):6936–6943. [PubMed]
- Wu H, Liu X, Jaenisch R. Double replacement: strategy for efficient introduction of subtle mutations into the murine Col1a-1 gene by homologous recombination in embryonic stem cells. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2819–2823. [PubMed]
- Askew GR, Doetschman T, Lingrel JB. Site-directed point mutations in embryonic stem cells: a gene-targeting tag-and-exchange strategy. Mol Cell Biol. 1993 Jul;13(7):4115–4124. [PubMed]
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