• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of narLink to Publisher's site
Nucleic Acids Res. Oct 1, 1996; 24(19): 3707–3713.
PMCID: PMC146162

A polyoma-based episomal vector efficiently expresses exogenous genes in mouse embryonic stem cells.

Abstract

We describe the ability of novel episomally maintained vectors to efficiently promote gene expression in embryonic stem (ES) cells as well as in established mouse cell lines. Extrachromosomal maintenance of our vectors is based on the presence of polyoma virus DNA sequences, including the origin of replication harboring a mutant enhancer (PyF101), and a modified version of the polyoma early region (LT20) encoding the large T antigen only. Reporter gene expression from such extrachromosomally replicating vectors was approximately 10-fold higher than expression from replication-incompetent control plasmids. After transfection of different ES cell lines, the polyoma virus-derived plasmid variant pMGD20neo (7.2 kb) was maintained episomally in 16% of the G418-resistant clones. No chromosomal integration of pMGD20neo vector DNA was detected in ES cells that contained episomal vector DNA even after long term passage. The vector's replication ability was not altered after insertion of up to 10 kb hprt gene fragments. Besides undifferentiated ES cells, the polyoma-based vectors were also maintained extrachromosomally in differentiating ES cells and embryoid bodies as well as in established mouse cell lines.

Full Text

The Full Text of this article is available as a PDF (130K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Bronson SK, Smithies O. Altering mice by homologous recombination using embryonic stem cells. J Biol Chem. 1994 Nov 4;269(44):27155–27158. [PubMed]
  • Doetschman TC, Eistetter H, Katz M, Schmidt W, Kemler R. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J Embryol Exp Morphol. 1985 Jun;87:27–45. [PubMed]
  • Doetschman T, Shull M, Kier A, Coffin JD. Embryonic stem cell model systems for vascular morphogenesis and cardiac disorders. Hypertension. 1993 Oct;22(4):618–629. [PubMed]
  • Keller GM. In vitro differentiation of embryonic stem cells. Curr Opin Cell Biol. 1995 Dec;7(6):862–869. [PubMed]
  • Gassmann M, Fandrey J, Bichet S, Wartenberg M, Marti HH, Bauer C, Wenger RH, Acker H. Oxygen supply and oxygen-dependent gene expression in differentiating embryonic stem cells. Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):2867–2872. [PMC free article] [PubMed]
  • Gassmann M, Donoho G, Berg P. Maintenance of an extrachromosomal plasmid vector in mouse embryonic stem cells. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1292–1296. [PMC free article] [PubMed]
  • Treisman R, Novak U, Favaloro J, Kamen R. Transformation of rat cells by an altered polyoma virus genome expressing only the middle-T protein. Nature. 1981 Aug 13;292(5824):595–600. [PubMed]
  • Fujimura FK, Deininger PL, Friedmann T, Linney E. Mutation near the polyoma DNA replication origin permits productive infection of F9 embryonal carcinoma cells. Cell. 1981 Mar;23(3):809–814. [PubMed]
  • Soeda E, Arrand JR, Smolar N, Walsh JE, Griffin BE. Coding potential and regulatory signals of the polyoma virus genome. Nature. 1980 Jan 31;283(5746):445–453. [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]
  • Soriano P, Montgomery C, Geske R, Bradley A. Targeted disruption of the c-src proto-oncogene leads to osteopetrosis in mice. Cell. 1991 Feb 22;64(4):693–702. [PubMed]
  • Yenofsky RL, Fine M, Pellow JW. A mutant neomycin phosphotransferase II gene reduces the resistance of transformants to antibiotic selection pressure. Proc Natl Acad Sci U S A. 1990 May;87(9):3435–3439. [PMC free article] [PubMed]
  • Zhu ZY, Veldman GM, Cowie A, Carr A, Schaffhausen B, Kamen R. Construction and functional characterization of polyomavirus genomes that separately encode the three early proteins. J Virol. 1984 Jul;51(1):170–180. [PMC free article] [PubMed]
  • Selden RF, Howie KB, Rowe ME, Goodman HM, Moore DD. Human growth hormone as a reporter gene in regulation studies employing transient gene expression. Mol Cell Biol. 1986 Sep;6(9):3173–3179. [PMC free article] [PubMed]
  • Hasty P, Rivera-Pérez J, Bradley A. The length of homology required for gene targeting in embryonic stem cells. Mol Cell Biol. 1991 Nov;11(11):5586–5591. [PMC free article] [PubMed]
  • Mansour SL, Thomas KR, Deng CX, Capecchi MR. Introduction of a lacZ reporter gene into the mouse int-2 locus by homologous recombination. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7688–7692. [PMC free article] [PubMed]
  • Robertson E, Bradley A, Kuehn M, Evans M. Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector. Nature. 1986 Oct 2;323(6087):445–448. [PubMed]
  • Muller WJ, Naujokas MA, Hassell JA. Isolation of large T antigen-producing mouse cell lines capable of supporting replication of polyomavirus-plasmid recombinants. Mol Cell Biol. 1984 Nov;4(11):2406–2412. [PMC free article] [PubMed]
  • Adra CN, Boer PH, McBurney MW. Cloning and expression of the mouse pgk-1 gene and the nucleotide sequence of its promoter. Gene. 1987;60(1):65–74. [PubMed]
  • Lacks S, Greenberg B. Complementary specificity of restriction endonucleases of Diplococcus pneumoniae with respect to DNA methylation. J Mol Biol. 1977 Jul;114(1):153–168. [PubMed]
  • Doetschman T, Gregg RG, Maeda N, Hooper ML, Melton DW, Thompson S, Smithies O. Targetted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature. 1987 Dec 10;330(6148):576–578. [PubMed]
  • Ramírez-Solis R, Bradley A. Advances in the use of embryonic stem cell technology. Curr Opin Biotechnol. 1994 Oct;5(5):528–533. [PubMed]
  • Melton DW. Gene targeting in the mouse. Bioessays. 1994 Sep;16(9):633–638. [PubMed]
  • Hasty P, Rivera-Pérez J, Chang C, Bradley A. Target frequency and integration pattern for insertion and replacement vectors in embryonic stem cells. Mol Cell Biol. 1991 Sep;11(9):4509–4517. [PMC free article] [PubMed]
  • Berstine EG, Hooper ML, Grandchamp S, Ephrussi B. Alkaline phosphatase activity in mouse teratoma. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3899–3903. [PMC free article] [PubMed]
  • Conkie D, Affara N, Harrison PR, Paul J, Jones K. In situ localization of globin messenger RNA formation. II. After treatment of Friend virus-transformed mouse cells with dimethyl sulfoxide. J Cell Biol. 1974 Nov;63(2 Pt 1):414–419. [PMC free article] [PubMed]
  • Klebe RJ, Chen T, Ruddle FH. Controlled production of proliferating somatic cell hybrids. J Cell Biol. 1970 Apr;45(1):74–82. [PMC free article] [PubMed]
  • O'Hare K. Replication of polyoma plasmid recombinants in mouse cells. J Mol Biol. 1981 Sep 5;151(1):203–210. [PubMed]
  • Mulligan RC, Berg P. Expression of a bacterial gene in mammalian cells. Science. 1980 Sep 19;209(4463):1422–1427. [PubMed]
  • Mével-Ninio M, Lutfalla G, Bertolotti R. A polyoma-derived plasmid vector maintained episomally in both E. coli and mouse hepatoma cells. Exp Cell Res. 1986 Sep;166(1):63–76. [PubMed]
  • Kusano T, Uehara H, Saito H, Segawa K, Oishi M. Multicopy plasmid with a structure related to the polyoma virus genome. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1789–1793. [PMC free article] [PubMed]
  • Ariizumi K, Ariga H. New class of polyomavirus mutant that can persist as free copies in F9 embryonal carcinoma cells. Mol Cell Biol. 1986 Nov;6(11):3920–3927. [PMC free article] [PubMed]
  • Nishimori K, Kohda T, Fujiwara J, Oishi M. Establishment of composite DNA derived from L factor as a plasmid in mouse embryonal carcinoma (F9) cells. Mol Cell Biol. 1988 May;8(5):2097–2104. [PMC free article] [PubMed]
  • Nilsson SV, Magnusson G. T-antigen expression by polyoma mutants with modified RNA splicing. EMBO J. 1983;2(12):2095–2101. [PMC free article] [PubMed]
  • Hasty P, Rivera-Pérez J, Bradley A. The role and fate of DNA ends for homologous recombination in embryonic stem cells. Mol Cell Biol. 1992 Jun;12(6):2464–2474. [PMC free article] [PubMed]
  • Bautch VL. Effects of polyoma virus oncogenes in transgenic mice. Mol Biol Med. 1989 Aug;6(4):309–317. [PubMed]
  • Helseth A, Siegal GP, Haug E, Bautch VL. Transgenic mice that develop pituitary tumors. A model for Cushing's disease. Am J Pathol. 1992 May;140(5):1071–1080. [PMC free article] [PubMed]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • PubMed
    PubMed
    PubMed citations for these articles

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...