Tissue Selective Expression of Conditionally-Regulated ROCK by Gene Targeting to a Defined Locus

Genesis. 2009 Jul;47(7):440-6. doi: 10.1002/dvg.20519.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Cells, Cultured
  • DNA Primers
  • Embryonic Stem Cells / enzymology
  • Exons
  • Gene Expression Regulation, Enzymologic*
  • Gene Targeting*
  • Genetic Vectors
  • Humans
  • Hypoxanthine Phosphoribosyltransferase / genetics
  • Immunohistochemistry
  • Keratin-14 / genetics
  • Mice
  • Polymerase Chain Reaction
  • Promoter Regions, Genetic
  • Recombination, Genetic
  • Tamoxifen / analogs & derivatives
  • Tamoxifen / pharmacology
  • rho-Associated Kinases / genetics*

Substances

  • DNA Primers
  • Keratin-14
  • Tamoxifen
  • afimoxifene
  • Hypoxanthine Phosphoribosyltransferase
  • rho-Associated Kinases