Stem cell engineering using transducible Cre recombinase.
Embryonic stem (ES) cells have become a major focus of scientific interest both as a potential donor source for regenerative medicine and as a model system for tissue development and pathobiology. Tight and efficient methods for genetic engineering are required to exploit ES cells as disease models and to generate specific somatic phenotypes by lineage selection or instruction. In 1990s, the application of site-specific recombinases (SSRs) such as Cre has revolutionized mammalian genetics by providing a reliable and efficient means to delete, insert, invert, or exchange chromosomal DNA in a conditional manner. Despite these significant advances, the available technology still suffers from limitations, including unwanted side effects elicited by the random integration of Cre expression vectors and leak activity of inducible or presumptive cell type-specific Cre expression systems. These challenges can be met by combining the Cre/loxP recombination system with direct intracellular delivery of Cre by protein transduction, thus enabling rapid and highly efficient conditional mutagenesis in ES cells and ES cell-derived somatic progeny. Modified recombinant variants of Cre protein induce recombination in virtually 100% of human ES (hES) and mouse ES (mES) cells. Here, we present methods for generating purified transducible Cre protein from Escherichia coli and its transduction into ES cells and their neural progeny.
Nolden
Lars
L
Institute of Reconstructive Neurobiology, University of Bonn-Life & Brain Center and Hertie Foundation, Germany.
Edenhofer
Frank
F
Peitz
Michael
M
Brüstle
Oliver
O
D000818:Animals; D018014:Gene Transfer Techniques; D006801:Humans; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000502:physiology; D013234:Stem Cells / Q000166:cytology / Q000502:physiology*; D023822:Tissue Engineering / Q000379:methods*; D014161:Transduction, Genetic
C045073:Cre recombinase; D019426:Integrases