Construction of miRNA-29b1 knockout mice based on CRISPR/Cas9 technology

VernacularTitle:利用 CRISPR／Cas9技术构建 miRNA－29 b1基因敲除小鼠
Author: Yong ZHAO ; Changhong SHI ; Ya ZHAO ; Zhiqian XIN ; Peijuan LIU ; Caiqin ZHANG ; Bing BAI ; Jieying BAI ; Hua WANG ; Hai ZHANG
Keywords: CRISPR/Cas9; miRNA-29b1; Gene knockout mice
From: Chinese Journal of Comparative Medicine 2016;26(12):1-4
CountryChina
Language:Chinese
Abstract: Objective To construct miRNA-29b1 gene knockout mice based on CRISPR/Cas9 technology. Methods To design and synthesize sgRNA according to the miRNA-29b1 sequence in Genbank .sgRNA and Cas9 were transcribed to RNA in vitro, these RNA were then microinjected into zygotes of C 57BL/6 mice.After mouse birth, the genome DNA was extracted and sequenced to identify its genotype; meanwhile , real-time PCR was used to assay the expression of miRNA-29b1 in the heart, liver, spleen, lung and kidney of mutated mice .Result A 20 bp sgRNA targeted on miRNA-29b1 was synthesized and transcribed to RNA with Cas 9.After microinjection, miRNA-29b1 gene-mutated mice were obtained.The sequencing results showed that there were two types of genotype for the mutated mice , one was 10 bp deletion, and another was 23 bp deletion accompanied with a 3 bp insertion.Compared with the wild-type mice, the expression of miRNA-29b1 in the heart, liver, spleen, lung and kidney was reduced significantly .Conclusions miRNA-29b1 gene knockout mice are constructed successfully by using CRISPR /Cas9 technology.