title: Inducible cassette exchange: a rapid and efficient system enabling conditional gene expression in embryonic stem and primary cells.
abstract: Genetic modification is critically enabling for studies addressing specification and maintenance of cell fate; however, methods for engineering modifications are inefficient. We demonstrate a rapid and efficient recombination system in which an inducible, floxed cre allele replaces itself with an incoming transgene. We target this inducible cassette exchange (ICE) allele to the (HPRT) locus and demonstrate recombination in murine embryonic stem cells (ESCs) and primary cells from derivative ICE mice. Using lentivectors, we demonstrate recombination at a randomly integrated ICE locus in human ESCs. To illustrate the utility of this system, we insert the myogenic regulator, Myf5, into the ICE locus in each platform. This enables efficient directed differentiation of mouse and human ESCs into skeletal muscle and conditional myogenic transdetermination of primary cells cultured in vitro. This versatile tool is thus well suited to gain-of-function studies probing gene function in the specification and reprogramming of cell fate.
authors: Lillehei Heart Institute and Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota, USA.
authors: Michelina
authors: M
authors: Iacovino
authors: Darko
authors: D
authors: Bosnakovski
authors: Holger
authors: H
authors: Fey
authors: Danielle
authors: D
authors: Rux
authors: Gagan
authors: G
authors: Bajwa
authors: Elisabeth
authors: E
authors: Mahen
authors: Ana
authors: A
authors: Mitanoska
authors: Zhaohui
authors: Z
authors: Xu
authors: Michael
authors: M
authors: Kyba
mesh_terms: D000483:Alleles; D000818:Animals; D002454:Cell Differentiation; D004318:Doxycycline / Q000494:pharmacology; D018274:Electroporation; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000528:radiation effects; D015870:Gene Expression; D056426:Genetic Loci; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D024510:Muscle Development; D016254:Mutagenesis, Insertional / Q000379:methods*; D017570:MyoD Protein / Q000235:genetics / Q000378:metabolism; D051787:Myogenic Regulatory Factor 5 / Q000235:genetics / Q000378:metabolism; D018008:Myogenin / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods*; D019076:Transgenes
chemical_list: C497417:MYF5 protein, human; C497418:Myf5 protein, mouse; D017570:MyoD Protein; C057363:MyoD1 myogenic differentiation protein; C503801:Myog protein, mouse; D051787:Myogenic Regulatory Factor 5; D018008:Myogenin; D004318:Doxycycline