Format

Send to

Choose Destination
J Gene Med. 2004 Jan;6(1):32-42.

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

Author information

1
INSERM U373, Institut Necker, Paris, France.

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 beta-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 beta-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 beta-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.

PMID:
14716675
DOI:
10.1002/jgm.442
[Indexed for MEDLINE]

Supplemental Content

Loading ...
Support Center