摘要
目的:构建miR-125a-5p基因敲除小鼠并进行基因鉴定,并探讨基因敲除小鼠体内miR-125a-5p对靶基因Foxp3的调控。方法:利用CRISPR/Cas9基因编辑技术,以C57BL/6J小鼠为背景,对miR-125a-5p基因位点进行基因敲除,培育纯合miR-125a-5p基因敲除小鼠。采用裂解法提取鼠尾组织基因组DNA,进行PCR反应检测亲代纯合miR-125a-5p基因敲除小鼠基因型,并选用雌、雄均为纯合子的基因敲除鼠作为亲代种鼠进行配种繁殖。通过全自动蛋白表达分析系统检测小鼠脾脏组织Foxp3的蛋白表达。结果:成功构建miR-125a-5p基因敲除小鼠,琼脂糖凝胶电泳显示PCR产物为240 bp或0 bp单条带的为纯合型基因敲除(KO)小鼠,鉴定获得668 bp或428 bp单条带的为野生型(WT)小鼠,与预期的目的基因片段分子量大小一致,成功鉴定了miR-125a-5p基因敲除小鼠的基因型。选用雌、雄均为纯合子的基因敲除鼠进行配种繁育,获得了一批miR-125a-5p敲除纯合子基因型小鼠。与WT小鼠相比,miR-125a-5p基因敲除小鼠的脾脏组织中Foxp3表达降低(P<0.05)。结论:利用CRISPR/Cas9基因编辑技术成功构建了纯合miR-125a-5p基因敲除小鼠,miR-125a-5p基因的缺失参与靶基因Foxp3的表达调控,为进一步研究miR-125a-5p在自身免疫性疾病中的发生机制提供前期基础。
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 editing 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 expression 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 breeding,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 technology.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.
作者
王辞岚
李劲频
傅金凤
陈柳伶
谭舒婷
刘竞丽
Wang Cilan;Li Jinpin;Fu Jinfeng;Chen Liuling;Tan Shuting;Liu Jingli(Department of Neurology,The First Affiliated Hospital of Guangxi Medical University,Nanning 530021,China)
出处
《广西医科大学学报》
CAS
2023年第5期838-842,共5页
Journal of Guangxi Medical University
基金
国家自然科学基金资助项目(No.81960241)。
