Enhanced purification of cell-permeant Cre and germline transmission after transduction into mouse embryonic stem cells.
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.
Stem Cell Engineering Group, Institute of Reconstructive Neurobiology, Life & Brain Center and Hertie Foundation, University of Bonn, Bonn, Germany.
Michael
M
Peitz
Richard
R
Jäger
Christoph
C
Patsch
Andrea
A
Jäger
Angela
A
Egert
Hubert
H
Schorle
Frank
F
Edenhofer
D000818:Animals; D015139:Blotting, Southern; D019556:COS Cells; D049109:Cell Proliferation; D002522:Chlorocebus aethiops; D053595:Embryonic Stem Cells / Q000378:metabolism*; D018014:Gene Transfer Techniques; D005838:Genotype; D015151:Immunoblotting; D017404:In Situ Hybridization, Fluorescence; D019426:Integrases / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism; D011995:Recombination, Genetic; D014161:Transduction, Genetic
D011994:Recombinant Proteins; C045073:Cre recombinase; D019426:Integrases