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

Yoshikazu Hasegawa; Yoshikazu Hoshino; Abdelaziz E. Ibrahim; Kanako Kato; Yoko Daitoku; Yoko Tanimoto; Yoshihisa Ikeda; Hisashi Oishi; Satoru Takahashi; Atsushi Yoshiki; Ken-ichi Yagami; Hiroyoshi Iseki; Seiya Mizuno; Fumihiro Sugiyama

doi:10.1538/expanim.16-0016

Originals

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

Yoshikazu Hasegawa, Yoshikazu Hoshino, Abdelaziz E. Ibrahim, Kanako Kato, Yoko Daitoku, Yoko Tanimoto, Yoshihisa Ikeda, Hisashi Oishi, Satoru Takahashi, Atsushi Yoshiki, Ken-ichi Yagami, Hiroyoshi Iseki, Seiya Mizuno, Fumihiro Sugiyama

Author information

Yoshikazu Hasegawa
Laborarory Animal Resource Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
Yoshikazu Hoshino
Laborarory Animal Resource Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
Hoshino Laboratory Animals, Inc., 1405 Kouda, Bando, Ibaraki 306-0606, Japan
Abdelaziz E. Ibrahim
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
Kanako Kato
Laborarory Animal Resource Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
Yoko Daitoku
Laborarory Animal Resource Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
Yoko Tanimoto
Laborarory Animal Resource Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
Yoshihisa Ikeda
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
Hisashi Oishi
Laborarory Animal Resource Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
Satoru Takahashi
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
Atsushi Yoshiki
Experimental Animal Division, RIKEN BioResource Center, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan
Ken-ichi Yagami
Laborarory Animal Resource Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
Hiroyoshi Iseki
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
Seiya Mizuno
Laborarory Animal Resource Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
Fumihiro Sugiyama
Laborarory Animal Resource Center, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan

Keywords: bicistronic, cre-loxP, CRISPR/Cas9, pancreatic β cells, mouse

JOURNAL OPEN ACCESS
Supplementary material

2016 Volume 65 Issue 3 Pages 319-327

DOI https://doi.org/10.1538/expanim.16-0016

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Details

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-Ins1^{em1 (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-Ins1^{em1 (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-Ins1^{em1 (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.

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