NDLI: Derivation of rat embryonic stem cells and generation of protease-activated receptor-2 knockout rats

Content Provider	Springer Nature Link
Author	Yamamoto, Satoshi Nakata, Mitsugu Sasada, Reiko Ooshima, Yuki Ya, Takashi Shizawa, Tadahiro Tsukimi, Yasuhiro Takeyama, Michiyasu Matsumoto, Yoshio Hashimoto, Tadatoshi
Copyright Year	2011
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.
Starting Page	743
Ending Page	755
Page Count	13
File Format	PDF
ISSN	09628819
Journal	Transgenic Research
Volume Number	21
Issue Number	4
e-ISSN	15739368
Language	English
Publisher	Springer Netherlands
Publisher Date	2011-10-15
Publisher Place	Dordrecht
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Rat Embryonic stem cells Gene targeting Knockout PAR-2 Plant Genetics & Genomics Plant Sciences Animal Genetics and Genomics Molecular Medicine Human Genetics
Content Type	Text
Resource Type	Article
Subject	Genetics Animal Science and Zoology Biotechnology Agronomy and Crop Science

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1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
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3	National Digital Library of India Office, Indian Institute of Technology Kharagpur	The administrative and infrastructural headquarters of the project	Dr. B. Sutradhar bsutra@ndl.gov.in
4	Project PI / Joint PI	Principal Investigator and Joint Principal Investigators of the project	Dr. B. Sutradhar bsutra@ndl.gov.in Prof. Saswat Chakrabarti will be added soon
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