Novel tag-and-exchange (RMCE) strategies generate master cell clones with predictable and stable transgene expression properties.
Site-specific recombinases have revolutionized the systematic generation of transgenic cell lines and embryonic stem cells/animals and will ultimately also reveal their potential in the genetic modification of induced pluripotent stem cells. Introduced in 1994, our Flp recombinase-mediated cassette exchange strategy permits the exchange of a target cassette for a cassette with the gene of interest, introduced as a part of an exchange vector. The process is "clean" in the sense that it does not co-introduce prokaryotic vector parts; neither does it leave behind a selection marker. Stringent selection principles provide master cell lines permitting subsequent recombinase-mediated cassette exchange cycles in the absence of a drug selection and with a considerable efficiency (approximately 10%). Exemplified by Chinese hamster ovary cells, the strategy proves to be successful even for cell lines with an unstable genotype.
Qiao
Junhua
J
Department of Molecular Biotechnology/Epigenetic Regulation, Helmholtz Centre for Infection Research, Braunschweig, Germany.
Oumard
André
A
Wegloehner
Wolfgang
W
Bode
Juergen
J
D000818:Animals; D016466:CHO Cells; D002999:Clone Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004254:DNA Nucleotidyltransferases / Q000235:genetics*; D005434:Flow Cytometry; D018390:Gene Targeting; D049452:Green Fluorescent Proteins / Q000235:genetics; D011995:Recombination, Genetic; D019076:Transgenes
D049452:Green Fluorescent Proteins; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase