Construction of miR-125a-5p knockout C57BL/6J mice

Objective:To construct miR-125a-5p knockout mice and conduct gene identification, and to explore the regulation of miR-125a-5p on target gene Foxp3 in gene knockout mice.Methods:CRISPR/Cas9 gene edit-ing technology was used to knock out miR-125a-5p gene locus in C57BL/6J mice, and homozygous miR-125a-5p knockout mice were generated.Genomic DNA was extracted from mouse tail tissue by cleavage method, and the genotypes of parental homozygous miR-125a-5p knockout mice were detected by PCR reaction.Both male and female homozygous gene knockout mice were selected as parental breeding mice for breeding.The expres-sion of Foxp3 protein in mouse spleen was detected by automatic protein expression analysis system.Results:The miR-125a-5p knockout mice were successfully constructed.Agarose gel electrophoresis showed that the PCR products with a single band of 240 bp or 0 bp were homozygous gene knockout (KO) mice, and the PCR products with a single band of 668 bp or 428 bp were identified as wild-type (WT) mice.The above results were consistent with the expected molecular weight of the target gene fragment, and the genotype of miR-125a-5p knockout mice was successfully identified.Male and female homozygous knockout mice were selected for breed-ing, and a batch of miR-125a-5p knockout homozygous genotype mice were obtained.Compared with wild-type mice, the expression of Foxp3 decreased in the spleen of miR-125a-5p knockout mice (P< 0.05).Conclusion:The homozygous miR-125a-5p knockout mice are successfully constructed by CRISPR/Cas9 gene editing tech-nology.The deletion of miR-125a-5p gene is involved in the regulation of target gene Foxp3 expression, which provides a preliminary basis for further study of the mechanism of miR-125a-5p in autoimmune diseases.