STAT1干扰慢病毒载体的构建及其基因沉默效应测定

How to construct lentiviral vectors of STAT1 RNAi and detect the gene silencing

目的 :构建STAT1基因RNA干扰(RNAi)慢病毒载体并测定其基因沉默效应。方法 :根据人STAT1基因序列,设计干扰序列,将Oligo退火成双链并连接穿梭载体p LKO-1-EGFP-puro-sh RNA,构建sh RNA慢病毒载体质粒,并转化至感受态细胞DH5α,进行测序验证。在脂质体介导下转染293T细胞,包装生产慢病毒。慢病毒载体转染人肝癌细胞MHCC97L的STAT1过表达瘤株(MHCC97L-stat1),用Real-time PCR和Western Blot检测RNAi组(MHCC97L-stat1-sh RNA-1)和对照组(MHCC97L-stat1)中STAT1基因的表达情况,确定其干扰STAT1表达的有效性。结果:测序证实慢STAT1基因RNAi病毒载体质粒构建成功;慢病毒载体经293T细胞包装成功;慢病毒载体转染人肝癌细胞MHCC97L-stat1瘤株48 h后,STAT1基因在m RNA水平和蛋白质水平上表达明显降低。结论:STAT1基因RNAi慢病毒载体构建成功,能够在人肝癌细胞MHCC97L-stat1瘤株中表达,并具有显著的基因沉默效果。

Objective： To construct the lentiviral vectors of RNA interference （RNAi） of STAT1 gene and detect the effect of gene silencing. Method： Based on the mRNA sequence of human STAT1 gene, interference sequence was designed to make oligo anneal into double chains connected with shuttle vector pLKO-1-EGFP-puro-shRNA. Then lentiviral vector plasmid of shRNA was constructed and transformed into competent cells DH5 cc which was verified by sequencing. Under the liposome mediation 293T cells were transfected to achieve the lentivirus packaging production. STAT1 of MHCC97L in human hepatoma cells which were transfected by lentiviral vectors showed overexpression of tumor cell strains （MHCC97L-stat1）. By Real-time PCR and Western Blot, the expression of STAT1 was detected in both the RNAi group （MHCC97L-stat1-shRNA-1） and the control group ~MHCC97L-stat1） in order to determine its validity of interferencing the STAT1 ex- pression. Result： It was confirmed by sequencing that lentiviral vector plasmid of STAT1 RNAi was constructed successfully.The packaging production of lentiviral vectors transfected by 293T cells was successful.48 h after the lentivirus transfected MHCC97L of human hepatoma cells,the expression of STAT1 decreased significantly at both the mRNA level and the protein level. Conclusion： The lentiviral vectors of STAT1 RNAi were constructed successfully, which can achieve their expression of MHCC97L in human hepatoma cells and also have obvious effect of gene silencing.