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

✍ Scribed by Stelios Psarras; Niki Karagianni; Christoph Kellendonk; François Tronche; François-Loic Cosset; Carol Stocking; Volker Schirrmacher; Harald von Boehmer; Khashayarsha Khazaie

• Publisher: John Wiley and Sons
• Year: 2004
• Tongue: English
• Weight: 354 KB
• Volume: 6
• Category: Article
• ISSN: 1099-498X
• DOI: 10.1002/jgm.442

✦ Synopsis

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. Copyright © 2003 John Wiley & Sons, Ltd.