Enhanced purification of cell-permeant Cre and germline transmission after transduction into mouse embryonic stem cells |
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Authors: | Peitz Michael Jäger Richard Patsch Christoph Jäger Andrea Egert Angela Schorle Hubert Edenhofer Frank |
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Institution: | Stem Cell Engineering Group, Institute of Reconstructive Neurobiology, Life & Brain Center and Hertie Foundation, University of Bonn, Bonn, Germany. |
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Abstract: | Continuous expression of Cre recombinase has the potential to yield toxic side effects in various cell types, thereby limiting applications of the Cre/loxP 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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Keywords: | embryonic stem cells inducible gene expression site‐specific recombination protein transduction germline competency gene targeting |
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