RRC ID 12712
Author Yamamoto S, Nakata M, Sasada R, Ooshima Y, Yano T, Shinozawa T, Tsukimi Y, Takeyama M, Matsumoto Y, Hashimoto T.
Title Derivation of rat embryonic stem cells and generation of protease-activated receptor-2 knockout rats.
Journal Transgenic Res
Abstract One of the remarkable achievements in knockout (KO) rat production reported during the period 2008-2010 is the derivation of authentic embryonic stem (ES) cells from rat blastocysts using a novel culture medium containing glycogen synthase kinase 3 and mitogen-activated protein kinase kinase inhibitors (2i medium). Here, we report gene-targeting technology via homologous recombination in rat ES cells, demonstrating its use through production of a protease-activated receptor-2 gene (Par-2) KO rat. We began by generating germline-competent ES cells from Dark Agouti rats using 2i medium. These ES cells, which differentiate into cardiomyocytes in vitro, can produce chimeras with high ES cell contribution when injected into blastocysts. We then introduced a targeting vector with a neomycin-resistant gene driven by the CAG promoter to disrupt Par-2. After a 7-day drug selection, 489 neomycin-resistant colonies were obtained. Following screening by polymerase chain reaction (PCR) genotyping and quantitative PCR analysis, we confirmed three homologous recombinant clones, resulting in chimeras that transmitted the Par-2 targeted allele to offspring. Par-2 KO rats showed a loss of Par-2 messenger RNA expression in their stomach cells and a lack of PAR-2 mediated smooth muscle relaxation in the aorta as indicated by pharmacological testing. Compared with mice, rats offer many advantages in biomedical research, including a larger body size; consequently, they are widely used in scientific investigation. Thus, the establishment of a gene-targeting technology using rat ES cells will be a valuable tool in human disease model production and drug discovery.
Volume 21(4)
Pages 743-55
Published 2012-8-1
DOI 10.1007/s11248-011-9564-0
PMID 22002084
MeSH Animals Animals, Genetically Modified* Blastocyst / cytology Cell Culture Techniques Embryonic Stem Cells* Gene Knockout Techniques* Gene Targeting Homologous Recombination Myocytes, Cardiac / cytology Rats Receptor, PAR-2 / genetics*
IF 1.856
Times Cited 23
DNA material Rat BAC RNB1 RNB1-222L22