人Mirk基因短发夹RNA慢病毒Tet-on表达载体构建与RNA干扰效率的鉴定

发布时间：2018-06-10 10:31

本文选题：人Mirk基因 + 短发夹RNA　；参考：《华中科技大学》2013年硕士论文

【摘要】：目的：构建人Mirk基因的短发夹RNA (shRNA)慢病毒Tet-on表达载体并在横纹肌肉瘤RD细胞上鉴定其沉默效率。方法：设计合成靶向人Mirk基因的shRNA序列，构建于慢病毒pTRIPZ表达载体，克隆筛选出有效shRNA慢病毒载体，重组质粒经双酶切和DNA测序。重组质粒与包装质粒在293T细胞中磷酸钙共沉淀法包装成病毒颗粒,，然后感染横纹肌肉瘤RD细胞，DOX诱导RNA干扰，普通PCR方法检测mRNA水平靶基因的沉默效率。结果：构建pTRIPZ-Mirk-shRNA的酶切鉴定和测序正确，包装病毒后滴度达到5x106TU/ml。shRNA慢病毒颗粒感染横纹肌肉瘤RD细胞后在DOX诱导下Mirk基因的mRNA表达量较阴性对照组明显下降，差异有统计学意义。结论：成功构建靶向人Mirk基因的shRNA慢病毒Tet-on表达载体，，DOX诱导下该载体能够在核酸水平有效沉默靶基因。
[Abstract]:Aim: to construct human Mirk gene short hairpin RNA shRNA-lentivirus Tet-on expression vector and identify its silencing efficiency in rhabdomyosarcoma Rd cells. Methods: the shRNA sequence targeting human Mirk gene was synthesized and constructed into lentivirus pTRIPZ expression vector. The effective shRNA lentivirus vector was cloned and screened, and the recombinant plasmid was digested by double enzyme and DNA sequenced. Recombinant plasmids and packaging plasmids were packaged into viral particles by calcium phosphate coprecipitation in 293T cells, and then infected with rhabdomyosarcoma R D cells induced RNA interference. Results: pTRIPZ-Mirk-shRNA was identified correctly by restriction endonuclease digestion and sequencing. After packaging virus titer reached 5x106 TUP / ml. ShRNA lentivirus particles infected Rd cells of rhabdomyosarcoma, the mRNA expression of Mirk gene decreased significantly after DOX-induced infection compared with negative control group. Conclusion: the expression vector of shRNA lentivirus Tet-on targeting human Mirk gene can effectively silence the target gene at nucleic acid level.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R3416

【共引文献】