Unmodified Cre recombinase crosses the membrane.
Site-specific recombination in genetically modified cells can be achieved by the activity of Cre recombinase from bacteriophage P1. Commonly an expression vector encoding Cre is introduced into cells; however, this can lead to undesired side-effects. Therefore, we tested whether cell-permeable Cre fusion proteins can be directly used for lox-specific recombination in a cell line tailored to shift from red to green fluorescence after loxP-specific recombination. Comparison of purified recombinant Cre proteins with and without a heterologous 'protein transduction domain' surprisingly showed that the unmodified Cre recombinase already possesses an intrinsic ability to cross the membrane border. Addition of purified recombinant Cre enyzme to primary bone marrow cells isolated from transgenic C/EBPalpha(fl/fl) mice also led to excision of the 'floxed' C/EBPalpha gene, thus demonstrating its potential for in vivo applications. We conclude that Cre enyzme itself or its intrinsic membrane-permeating moiety are attractive tools for direct manipulation of mammalian cells.
Will
Elke
E
Department of Cell and Virus Genetics, Heinrich-Pette-Institute for Experimental Virology and Immunology, Martinistrasse 52, D-20251 Hamburg, Germany.
Klump
Hannes
H
Heffner
Nicole
N
Schwieger
Maike
M
Schiedlmeier
Bernhard
B
Ostertag
Wolfram
W
Baum
Christopher
C
Stocking
Carol
C
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases