Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: a tool for efficient genetic engineering of mammalian genomes.
Conditional mutagenesis is a powerful tool to analyze gene functions in mammalian cells. The site-specific recombinase Cre can be used to recombine loxP-modified alleles under temporal and spatial control. However, the efficient delivery of biologically active Cre recombinase to living cells represents a limiting factor. In this study we compared the potential of a hydrophobic peptide modified from Kaposi fibroblast growth factor with a basic peptide derived from HIV-TAT to promote cellular uptake of recombinant Cre. We present the production and characterization of a Cre protein that enters mammalian cells and subsequently performs recombination with high efficiency in a time- and concentration-dependent manner. Histidine-tagged Cre recombinase transduced inefficiently unless fused to a nuclear localization signal (NLS). Fusion of NLS-Cre to the fibroblast growth factor transduction peptide did not improve the transducibility, whereas fusion with the TAT peptide significantly enhanced cellular uptake and subsequent recombination. More than 95% recombination efficiency in fibroblast cells, as well as murine embryonic stem cells, was achieved after His-TAT-NLS-Cre transduction. Efficient recombination could also be obtained in primary splenocytes ex vivo. We expect that application of His-TAT-NLS-Cre, which can be produced readily in large quantities from a bacterial source, will expand our abilities to manipulate mammalian genomes.
Peitz
Michael
M
Institute for Genetics, University of Cologne, Weyertal 121, 50931 Cologne, Germany.
Pfannkuche
Kurt
K
Rajewsky
Klaus
K
Edenhofer
Frank
F
D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology; D004625:Embryo, Nonmammalian; D005346:Fibroblast Growth Factors / Q000494:pharmacology*; D015696:Gene Products, tat / Q000494:pharmacology*; D005818:Genetic Engineering / Q000379:methods*; D016678:Genome; D057927:Hydrophobic and Hydrophilic Interactions; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013154:Spleen / Q000378:metabolism; D013234:Stem Cells / Q000378:metabolism; D014161:Transduction, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D015696:Gene Products, tat; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D005346:Fibroblast Growth Factors; C045073:Cre recombinase; D019426:Integrases