Gene transfer and genetic modification of embryonic stem cells by Cre- and Cre-PR-expressing MESV-based retroviral vectors

Stelios Psarras, Niki Karagianni, Christoph Kellendonk, François Tronche, François Loic Cosset, Carol Stocking, Volker Schirrmacher, Harald von Boehmer, Khashayarsha Khazaie

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Background. Genetic modification of embryonic stem (ES) cells represents a powerful tool for transgenic and developmental experiments. We report that retroviral constructs based on murine embryonal stem cell virus (MESV) can efficiently deliver and express Cre recombinase or a post-translationally inducible Cre-Progesterone receptor (Cre.PR) fusion in mouse fibroblasts and ES cells. Methods. To study the vectors a sensitive reporter cell line, 3TZ, was derived from the murine 3T6 fibroblast line that expresses β-galactosidase only upon Cre-mediated recombination. This was used together with the ROSA26-R ES cell Cre-reporter system or unmodified mouse ES cells as targets of infection. Efficiency of gene transfer was evaluated immunohistochemically by the use of an anti-Cre polyclonal antibody, and by monitoring the expression of β-galactosidase. Results. Infection of the 3TZ cells with high titer 718C or 719CP virus revealed efficient gene transduction of constitutive or hormone-inducible recombinase activity, respectively. The vectors efficiently transduced murine ES cells with Cre, Cre-PR (fusion of Cre and progesterone receptor) or β-galactosidase. Cre-mediated recombination in more than 60% of ROSA26-R ES cells was achieved when infected by a VSV-G-pseudotyped MESV retrovirus at MOI of 50. Conclusions. The MESV-based retroviral systems, when combined with hormone inducible Cre, represent efficient tools for the transfer of Cre activity in ES cells.

Original languageEnglish (US)
Pages (from-to)32-42
Number of pages11
JournalJournal of Gene Medicine
Volume6
Issue number1
DOIs
StatePublished - Jan 2004
Externally publishedYes

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Cell Fusion
Progesterone Receptors
Embryonic Stem Cells
Galactosidases
Stem Cells
Viruses
Genes
Genetic Recombination
Fibroblasts
Hormones
Recombinases
Retroviridae
Infection
Cell Line
Antibodies
Mouse Embryonic Stem Cells

Keywords

  • Adenoviral vectors
  • Cre recombinase
  • Cre.PR fusion
  • ES cells
  • Gene transfer
  • MESV retroviral

ASJC Scopus subject areas

  • Genetics

Cite this

Gene transfer and genetic modification of embryonic stem cells by Cre- and Cre-PR-expressing MESV-based retroviral vectors. / Psarras, Stelios; Karagianni, Niki; Kellendonk, Christoph; Tronche, François; Cosset, François Loic; Stocking, Carol; Schirrmacher, Volker; von Boehmer, Harald; Khazaie, Khashayarsha.

In: Journal of Gene Medicine, Vol. 6, No. 1, 01.2004, p. 32-42.

Research output: Contribution to journalArticle

Psarras, S, Karagianni, N, Kellendonk, C, Tronche, F, Cosset, FL, Stocking, C, Schirrmacher, V, von Boehmer, H & Khazaie, K 2004, 'Gene transfer and genetic modification of embryonic stem cells by Cre- and Cre-PR-expressing MESV-based retroviral vectors', Journal of Gene Medicine, vol. 6, no. 1, pp. 32-42. https://doi.org/10.1002/jgm.442
Psarras, Stelios ; Karagianni, Niki ; Kellendonk, Christoph ; Tronche, François ; Cosset, François Loic ; Stocking, Carol ; Schirrmacher, Volker ; von Boehmer, Harald ; Khazaie, Khashayarsha. / Gene transfer and genetic modification of embryonic stem cells by Cre- and Cre-PR-expressing MESV-based retroviral vectors. In: Journal of Gene Medicine. 2004 ; Vol. 6, No. 1. pp. 32-42.
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AU - Tronche, François

AU - Cosset, François Loic

AU - Stocking, Carol

AU - Schirrmacher, Volker

AU - von Boehmer, Harald

AU - Khazaie, Khashayarsha

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N2 - Background. Genetic modification of embryonic stem (ES) cells represents a powerful tool for transgenic and developmental experiments. We report that retroviral constructs based on murine embryonal stem cell virus (MESV) can efficiently deliver and express Cre recombinase or a post-translationally inducible Cre-Progesterone receptor (Cre.PR) fusion in mouse fibroblasts and ES cells. Methods. To study the vectors a sensitive reporter cell line, 3TZ, was derived from the murine 3T6 fibroblast line that expresses β-galactosidase only upon Cre-mediated recombination. This was used together with the ROSA26-R ES cell Cre-reporter system or unmodified mouse ES cells as targets of infection. Efficiency of gene transfer was evaluated immunohistochemically by the use of an anti-Cre polyclonal antibody, and by monitoring the expression of β-galactosidase. Results. Infection of the 3TZ cells with high titer 718C or 719CP virus revealed efficient gene transduction of constitutive or hormone-inducible recombinase activity, respectively. The vectors efficiently transduced murine ES cells with Cre, Cre-PR (fusion of Cre and progesterone receptor) or β-galactosidase. Cre-mediated recombination in more than 60% of ROSA26-R ES cells was achieved when infected by a VSV-G-pseudotyped MESV retrovirus at MOI of 50. Conclusions. The MESV-based retroviral systems, when combined with hormone inducible Cre, represent efficient tools for the transfer of Cre activity in ES cells.

AB - Background. Genetic modification of embryonic stem (ES) cells represents a powerful tool for transgenic and developmental experiments. We report that retroviral constructs based on murine embryonal stem cell virus (MESV) can efficiently deliver and express Cre recombinase or a post-translationally inducible Cre-Progesterone receptor (Cre.PR) fusion in mouse fibroblasts and ES cells. Methods. To study the vectors a sensitive reporter cell line, 3TZ, was derived from the murine 3T6 fibroblast line that expresses β-galactosidase only upon Cre-mediated recombination. This was used together with the ROSA26-R ES cell Cre-reporter system or unmodified mouse ES cells as targets of infection. Efficiency of gene transfer was evaluated immunohistochemically by the use of an anti-Cre polyclonal antibody, and by monitoring the expression of β-galactosidase. Results. Infection of the 3TZ cells with high titer 718C or 719CP virus revealed efficient gene transduction of constitutive or hormone-inducible recombinase activity, respectively. The vectors efficiently transduced murine ES cells with Cre, Cre-PR (fusion of Cre and progesterone receptor) or β-galactosidase. Cre-mediated recombination in more than 60% of ROSA26-R ES cells was achieved when infected by a VSV-G-pseudotyped MESV retrovirus at MOI of 50. Conclusions. The MESV-based retroviral systems, when combined with hormone inducible Cre, represent efficient tools for the transfer of Cre activity in ES cells.

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