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Generation of CRISPR/Cas9-mediated bicistronic knock-in <i>ins1-cre</i> driver mice

  • Hasegawa Yoshikazu
    Laborarory Animal Resource Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
  • Yoshiki Atsushi
    Experimental Animal Division, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan
  • Ibrahim Abdelaziz E.
    Laborarory Animal Resource Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan Department of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, Suez Canal University, Ismailia, Egypt
  • Iseki Hiroyoshi
    Laborarory Animal Resource Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan International Institute for Integrative Sleep Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
  • Mizuno Seiya
    Laborarory Animal Resource Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
  • Tanimoto Yoko
    Laborarory Animal Resource Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
  • Ikeda Yoshihisa
    Laborarory Animal Resource Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan Charles River Laboratories Japan, Inc., 955 Kamibayashi, Ishioka, Ibaraki 315-0138, Japan
  • Oishi Hisashi
    Laborarory Animal Resource Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
  • Takahashi Satoru
    Laborarory Animal Resource Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan International Institute for Integrative Sleep Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan

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Other Title
  • Generation of CRISPR/Cas9-mediated bicistronic knock-in ins1-cre driver mice

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Abstract

In the present study, we generated novel cre driver mice for gene manipulation in pancreatic β cells. Using the CRISPR/Cas9 system, stop codon sequences of Ins1 were targeted for insertion of cre, including 2A sequences. A founder of C57BL/6J-Ins1em1 (cre) Utr strain was produced from an oocyte injected with pX330 containing the sequences encoding gRNA and Cas9 and a DNA donor plasmid carrying 2A-cre. (R26GRR x C57BL/6J-Ins1em1 (cre) Utr) F1 mice were histologically characterized for cre-loxP recombination in the embryonic and adult stages; cre-loxP recombination was observed in all pancreatic islets examined in which almost all insulin-positive cells showed tdsRed fluorescence, suggesting β cell-specific recombination. Furthermore, there were no significant differences in results of glucose tolerance test among genotypes (homo/hetero/wild). Taken together, these observations indicated that C57BL/6J-Ins1em1 (cre) Utr is useful for studies of glucose metabolism and the strategy of bicistronic cre knock-in using the CRISPR/Cas9 system could be useful for production of cre driver mice.

Journal

  • Experimental Animals

    Experimental Animals 65 (3), 319-327, 2016

    Japanese Association for Laboratory Animal Science

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