Gene transfer and genetic modification of embryonic stem cells by Cre- and Cre-PR-expressing MESV-based retroviral vectors.
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.
Psarras
Stelios
S
INSERM U373, Institut Necker, Paris, France.
Karagianni
Niki
N
Kellendonk
Christoph
C
Tronche
François
F
Cosset
François-Loic
FL
Stocking
Carol
C
Schirrmacher
Volker
V
Boehmer Hv
Harald von
Hv
Khazaie
Khashayarsha
K
D000818:Animals; D005347:Fibroblasts; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D007150:Immunohistochemistry; D019426:Integrases / Q000096:biosynthesis* / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D011980:Receptors, Progesterone / Q000235:genetics*; D012190:Retroviridae; D039901:Totipotent Stem Cells; D019076:Transgenes
D011980:Receptors, Progesterone; C045073:Cre recombinase; D019426:Integrases