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Tissue selective expression of conditionally‐regulated ROCK by gene targeting to a defined locus
Author(s) -
Samuel Michael S.,
Munro June,
Bryson Sheila,
Forrow Stephen,
Stevenson David,
Olson Michael F.
Publication year - 2009
Publication title -
genesis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.093
H-Index - 110
eISSN - 1526-968X
pISSN - 1526-954X
DOI - 10.1002/dvg.20519
Subject(s) - biology , exon , microbiology and biotechnology , gene , genetics
ROCK kinases regulate actin‐myosin structures downstream of Rho GTPases. We generated mice expressing 4‐hydroxytamoxifen (4HT)‐regulated human ROCK II ( ROCKII:mER ™) under the transcriptional control of the cytokeratin14 ( K14 ) promoter. The K14‐ROCKII:mER ™ minigene was recombineered into a novel cloning vector containing the promoter and first exon of the human HPRT gene, and second and third exons of the mouse Hprt gene. Homologous recombination into the Hprt locus, which is deleted for the promoter and first two exons in HM1 embryonic stem cells, reconstitutes a functional Hprt gene, allowing for growth in HAT (hypoxanthine‐aminopterin‐thymidine) medium. K14 ‐promoter‐driven ROCKII:mER™ expression was restricted to a superficial cell layer in embryoid bodies, with increased ROCK substrate phosphorylation induced by 4HT. ROCKII:mER™‐expressing primary murine keratinocytes responded to 4HT with increased substrate phosphorylation and cytoskeleton rearrangements, indicating that ROCKII:mER™ activity is regulated by 4HT in the target tissue. K14‐ROCKII:mER ™ mice will be valuable for examining the role of ROCK in skin development and cancer. genesis 47:440–446, 2009. © 2009 Wiley‐Liss, Inc.