期刊
GENESIS
卷 45, 期 8, 页码 508-517出版社
WILEY
DOI: 10.1002/dvg.20321
关键词
embryonic stem cells; inducible gene expression; site-specific recombination; protein transduction; germline competency; gene targeting
Continuous expression of Cre recombinase has the potential to yield toxic side effects in various cell types, thereby limiting applications of the Cre/IoxP system for conditional mutagenesis. In this study, we investigate the potential of Cre protein transduction to overcome this limitation. COS-7, CV1-5B, and mouse embryonic stem (ES) cells treated with cell-permeant Cre (HTNCre) maintain a normal growth behavior employing Cre concentrations sufficient to induce recombination in more than 90% of the cells, whereas continuous application of high doses resulted in markedly reduced proliferation. HTNCre-treated ES cells maintain a normal karyotype and are still able to contribute to the germline. Moreover, we present an enhanced HTNCre purification protocol that allows the preparation of a concentrated glycerol stock solution, thereby enabling a considerable simplification of the Cre protein transduction procedure. The protocol described here allows rapid and highly efficient conditional mutagenesis of cultured cells.
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