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Generation of CRISPR/Cas9-mediated bicistronic knock-inins1-cre driver mice

机译:CRISPR / Cas9介导的双顺反子敲入的产生ins1-cre驱动程序小鼠

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摘要

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.
机译:在本研究中,我们生成了用于胰腺β细胞基因操纵的新型cre驱动程序小鼠。使用CRISPR / Cas9系统,将Ins1的终止密码子序列靶向cre的插入,包括2A序列。 C57BL / 6J-Ins1em1(cre)Utr 菌株的创建者是由注射了含有编码gRNA和Cas9序列的pX330的卵母细胞和带有2A-cre的DNA供体质粒产生的。 (R26GRR x C57BL / 6J-Ins1 em1(cre)Utr )F1小鼠在胚胎和成年期的组织学特征是cre-loxP重组。在所检查的所有胰岛中均观察到cre-loxP重组,其中几乎所有胰岛素阳性细胞均显示tdsRed荧光,表明β细胞特异性重组。此外,基因型(纯合/杂合/野生)之间的葡萄糖耐量测试结果没有显着差异。综上所述,这些观察结果表明C57BL / 6J-Ins1 em1(cre)Utr 可用于葡萄糖代谢的研究,而使用CRISPR / Cas9系统的双顺反子cre敲入策略可用于生产cre驱动小鼠。

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