摘要
目的采用CRISPR/Cas 9技术构建微小RNA-22-3p(miR-22)基因敲除(miR-22^(-/-))小鼠,繁育miR-22^(-/-)小鼠并鉴定其基因型。方法使用CRISPR/Cas 9技术,构建miR-22^(-/-)基因工程小鼠,基因鉴定F0代miR-22^(-/-)小鼠后,待其性成熟后与同窝野生型小鼠交配得F1代miR-22^(-/-)小鼠,通过实时荧光定量聚合酶联反应(qPCR)从RNA水平上分析miR-22敲除效率。蛋白质印迹法(Western blot)检测miR-22与靶基因的相互作用。结果利用CRISPR/Cas 9技术成功构建了miR-22^(-/-)小鼠,对繁育获得的小鼠进行了基因鉴定,鉴定出了miR-22^(+/+)、miR-22+/-、miR-22^(-/-)三种稳定的基因型。qPCR检测结果证实miR-22^(-/-)小鼠与同窝野生型小鼠比较,心、肝、肺、肾、脾、胸腺六种组织中几乎不表达miR-22。Western blot检测出miR-22^(-/-)小鼠组织中的NLRP3蛋白的相对表达量低于野生型小鼠。结论成功构建了miR-22^(-/-)小鼠模型,并得到稳定遗传的纯合miR-22^(-/-)小鼠。miR-22对下游靶基因NLRP3有抑制作用。
Objective To construct micro RNA(miR)-22 gene knockout(miR-22^(-/-))mice using CRISPR/Cas 9 technology,to breed miR-22^(-/-)mice and to identify their genotypes.Methods In this experiment,CRISPR/Cas 9 technology was used to construct miR-22^(-/-)genetically engineered mice.After gene identification,the F0 generation miR-22^(-/-)mice were mated with wild-type mice in the same litter to obtain F1 generation miR-22^(-/-)mice.The miR-22 knockout efficiency was analyzed at the RNA level by real-time fluorescence quantitative polymerase chain reaction(qPCR).Western blot was used to detect the interaction between miR-22 and target genes.Results miR-22^(-/-)mice were successfully constructed using CRISPR/Cas 9 technology,gene identification was performed on the bred mice,and three stable genotypes of miR-22^(+/+),miR-22+/-,and miR-22^(-/-)were identified.The real-time fluorescence quantitative PCR detection results confirmed that miR-22^(-/-)mice showed almost no expression of miR-22 in the heart,liver,lung,kidney,spleen,and thymus tissues compared to wild-type mice in the same litter.Western blot analysis showed that the relative expression level of NLRP3 protein in miR-22^(-/-)mouse tissues was lower than that in wild-type mice.Conclusion A miR-22^(-/-)mouse model is successfully constructed,and stable genetic homozygous miR-22^(-/-)mice is obtained.This indicates that miR-22 has an inhibitory effect on the downstream target gene NLRP3.
作者
王安琪
张慧茹
周园园
刘崇
陈义昭
涂佳杰
Wang Anqi;Zhang Huiru;Zhou Yuanyuan;Liu Chong;Chen Yizhao;Tu Jiajie(College of Pharmaceutical Sciences,Anhui Medical University,Hefei 230032)
出处
《安徽医科大学学报》
北大核心
2025年第6期1052-1058,共7页
Acta Universitatis Medicinalis Anhui
基金
安徽省高校杰出青年科研项目(编号:2022AH020052)。
