Τίτλος:
Gene transfer and genetic modification of embryonic stem cells by Cre- and Cre-PR-expressing MESV-based retroviral vectors
Γλώσσες Τεκμηρίου:
Αγγλικά
Περίληψη:
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 © 2004 John Wiley & Sons, Ltd.
Συγγραφείς:
Psarras, S.
Karagianni, N.
Kellendonk, C.
Tronche, F.
Cosset, F.-L.
Stocking, C.
Schirrmacher, V.
von Boehmer, H.
Khazaie, K.
Περιοδικό:
The Journal of Gene Medicine
Λέξεις-κλειδιά:
adenovirus vector; beta galactosidase; cre recombinase; DNA; polyclonal antibody; progesterone receptor; retrovirus vector; cre recombinase; integrase; progesterone receptor, animal cell; article; cell line; controlled study; DNA modification; embryo; embryo cell; enzyme activity; fibroblast; gene induction; gene transfer; genetic recombination; genetic transduction; human; human cell; immunohistochemistry; mouse; nonhuman; priority journal; protein expression; protein processing; reporter gene; stem cell; target cell; transgenics; Vesicular stomatitis virus; viral gene delivery system; animal; biosynthesis; gene therapy; gene transfer; gene vector; genetics; methodology; Retrovirus; totipotent stem cell; transgene; transgenic mouse, Murinae; unidentified retrovirus, Animals; Fibroblasts; Gene Therapy; Gene Transfer Techniques; Genetic Vectors; Immunohistochemistry; Integrases; Mice; Mice, Transgenic; Receptors, Progesterone; Retroviridae; Totipotent Stem Cells; Transgenes
