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目的建立针对MYOC基因RNAi慢病毒载体的优化及筛选方法,为研究后续突变基因的功能奠定基础。方法 Western Blot筛选RNAi有效靶点,Real-time PCR检测小梁细胞MYOC基因Knock Down效率。结果 Western blotting结果显示含有敲减质粒的组别其蛋白表达明显减少;Real-time PCR结果显示在荧光显微镜下观察,与对照组相比含有敲减质粒的组别其MYOC基因的表达明显下降,1#、2#、3#具有确切的干扰效果。结论经过Western blotting及Real-time PCR方法检测所构建的4个干扰靶点的敲减效率,证实RNAi慢病毒干扰效果可靠,且两种方法结果一致,可随机选择1#、2#、3#中的任一位点作为后续干扰靶点。
Objective To establish a method for optimization and screening of RNAi lentiviral vector for MYOC gene and lay a foundation for the study of the function of subsequent mutated gene. Methods Effective targets of RNAi were screened by Western Blot and Knock Down efficiency of trabecular meshwork cells was detected by Real-time PCR. Results Western blotting showed that the expression of MYOC gene was significantly decreased in the control group compared with the control group. The results of Real-time PCR showed that the expression of MYOC gene was significantly decreased compared with the control group, 1 #, 2 #, 3 # has the exact interference effect. Conclusion The knockdown efficiency of the four constructed interference targets was verified by Western blotting and Real-time PCR. The interference effect of RNAi lentivirus was confirmed, and the results of two methods were consistent. The results of 1 #, 2 #, 3 # In any one point as a follow-up interference target.