Rictor基因特异性RNA干扰表达载体的构建和筛选

目的构建针对Rictor基因的特异性RNA干扰质粒,稳定转染人膀胱癌T24细胞,观察其对目的基因Rictor表达的影响,为探讨沉默Rictor基因对人膀胱癌细胞生物学行为的影响奠定基础。方法根据GENEBANK提供Rictor基因系列,设计并化学合成三对Rictor-RNAi和一对阴性对照寡核苷酸序列,定向克隆入真核质粒载体pGCSIL-PUR中,构建pGCSIL-RICTOR-RNAi表达载体。各质粒载体瞬时转染到T24细胞,Western-Blot检测筛选载体。筛选出的载体稳定转染,Realtime-PCR和Western-Blot筛选干扰效果明显的稳定株。结果重组质粒pGCSIL-RICTOR-RNAi经酶切鉴定及DNA测序证实序列完全正确,成功构建Rictor-RNAi载体。瞬时转染T24细胞,RICTOR蛋白表达明显降低。稳定转染T24细胞,Rictor基因表达在mRNA水平和蛋白水平均明显降低。结论成功构建Rictor-RNAi表达载体,转染T24细胞后可有效抑制Rictor表达。

Construction and Identification of RNAi Expressing Vector for Rictor

Objective To construct RNA interference vectors specific for Rictor gene,stably transfect human bladder cancer cell line T24,and explore the effect of silencing Rictor gene by RNAi.Methods Three pairs of siRNA and one pair of negative control oligonucleotides were designed and synthesized based on the sequence of Rictor mRNA in GENEBANK.These oligonucleotides were annealed and cloned into pGCSIL-PUR vector.The expression of Rictor in transient transfected cells was examined by western blot.The recombinant plasmids vector that displaying optimal interference effect were stably transfected to T24.Realtime PCR and Western Blot were performed to examine the inhibitory effect at the mRNA and protein level respectively.Results The recombinant plasmids were analyzed by restriction endonuclease analysis and DNA sequencing.Three Rictor-RNAi vectors and negative control vector were constructed.The expression of Rictor in transient transfected T24 cell apparently decreased.The expression of Rictor in stably transfected T24 cell clones was significantly down regulated at the mRNA and protein level respectively.Conclusion The Rictor-RNAi expression vectors were constructed successfully and they can inhibit the expression and translation of Rictor gene effectively in T24 cells.