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

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

65 ( 3 )  , pp.319 - 327 , 2016-07 , Japanese Association for Laboratory Animal Science
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.

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