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目的应用CRISPR/Cas9技术构建miRNA-29b1基因敲除小鼠。方法针对miRNA-29b1基因设计一段sgRNA,sgRNA和Cas9体外转录后显微注射至C57BL/6小鼠受精卵细胞。小鼠出生后取其基因组DNA进行测序以鉴定基因型,同时取小鼠心、肝、脾、肺、肾等脏器研磨后提取总RNA,通过real-time PCR分析miRNA-29b1在这些脏器中的表达。结果设计了20 bp的miRNA-29b1sgRNA并与Cas9一起进行了体外转录,显微注射小鼠受精卵细胞后获得miRNA-29b1基因突变小鼠。测序结果表明突变小鼠有两种基因型,一种为10 bp的缺失突变;另一种为22 bp的缺失突变,同时伴有3 bp的插入突变。与野生型小鼠相比,基因突变小鼠心、肝、脾、肺、肾等组织中miRNA-29b1表达量下降明显。结论应用CRISPR/Cas9技术成功构建miRNA-29b1基因敲除小鼠。
Objective To construct miRNA-29b1 knockout mice using CRISPR / Cas9 technique. Methods A series of sgRNA, sgRNA and Cas9 were designed for miRNA-29b1 gene transcription in vitro and then injected into C57BL / 6 mice fertilized eggs. After the birth of the mice, the genomic DNA was sequenced to identify the genotype. At the same time, the total RNA was extracted from the heart, liver, spleen, lung, kidney and other organs after being ground. The miRNA-29b1 was analyzed by real-time PCR in these organs In the expression. Results A 20 bp miRNA-29b1sgRNA was designed and transcribed in vitro with Cas9. The miRNA-29b1 mutant mice were obtained after microinjection of mouse fertilized egg cells. Sequencing results showed that the mutant mice had two genotypes, one with 10 bp deletion mutation and the other with 22 bp deletion mutation accompanied by a 3 bp insertion mutation. Compared with wild type mice, miRNA-29b1 expression in heart, liver, spleen, lung, kidney and other tissues of gene mutation mice decreased significantly. Conclusion The miRNA-29b1 knockout mice were successfully constructed using CRISPR / Cas9 technology.