Li Y X, He X G, Yao Y G, Wang Y
Department of Otorhinolaryngology Head and Neck Surgery, the First Affiliated Hospital of Kunming Medical University, Kunming, 650032, China.
Key Laboratory of Animal Models and Human Disease Mechanisms,Kumming Institute of Zoology,Chinese Academy of Sciences.
Lin Chuang Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 2017 Apr 20;31(8):627-632. doi: 10.13201/j.issn.1001-1781.2017.08.014.
The aim of this study was to construct and identify a lentiviral vector for RNA interference of mouse GATA-3.The coding region of the GATA-3 gene sequences were analyzed for a single-stranded oligo design and synthesis.PCR was performed for splicing reaction of synthetic oligo,which was synthesized into the lentiviral vector and transformed into the competent cell DH5α.An expression vector shRNA-GATA-3 was constructed after sequencing and identifying the gene sequence of the recombinant clones.Accoding to the target gene sequence ,four pairs are as following: siRNA-685 group(LV3-GATA-3-Mus-685),siRNA-1152 group (LV3-GATA-3-Mus-1152),siRNA-1615 group(LV3-GATA-3-Mus-1615) and control group ,which were designed and syntheized and builded into the lentiviral carrier.The shRNA vectors were cotransfected with expression vectors into the 293T cell line,and the optimal interference sequence was screened by QPCR. The 293T cells were cotranfected with the constructed lentiviral interference vectors and packing plasmids,the viruses were packed,the virus stock was collected and concentrated by ultracentrifugation,and the titer of the viruses was determined. Gene sequence proved that the recombinant clone gene sequence was correct,and the lentiviral vector was successfully constructed. The relative expression of GATA-3 mRNA in siRNA-1615 group(0.004) was significantly reduced after RNA interference comparing with that in the control group(0.022),siRNA-1152 group(0.009) and siRNA-685 group(0.009). The screened LV3-GATA-3-Mus-1615 ,as the most effective interference sequence,was constructed into the lentiviral vector.The titer of the concentrated virus of LV3-GATA-3-Mus-1615 was 5×108 TU/ml in the viral supernate of 293T cells collected at 48 hours after co-transfection. Alentiviral RNAi expression vector targeting the GATA-3 gene (LV3-GATA-3-Mus-1615) was successfully constrcted and identified.
本研究旨在构建并鉴定一种用于小鼠GATA-3基因RNA干扰的慢病毒载体。对GATA-3基因序列的编码区进行分析，以设计并合成单链寡核苷酸。进行PCR以对合成的寡核苷酸进行剪接反应，将其合成到慢病毒载体中并转化到感受态细胞DH5α中。对重组克隆的基因序列进行测序和鉴定后，构建了表达载体shRNA-GATA-3。根据靶基因序列，设计并合成了四对，如下所示：siRNA-685组（LV3-GATA-3-Mus-685）、siRNA-1152组（LV3-GATA-3-Mus-1152）、siRNA-1615组（LV3-GATA-3-Mus-1615）和对照组，并构建到慢病毒载体中。将shRNA载体与表达载体共转染到293T细胞系中，通过QPCR筛选最佳干扰序列。将构建的慢病毒干扰载体与包装质粒共转染293T细胞，包装病毒，收集病毒原液并通过超速离心浓缩，测定病毒滴度。基因序列证明重组克隆基因序列正确，成功构建了慢病毒载体。RNA干扰后，siRNA-1615组（0.004）中GATA-3 mRNA的相对表达量与对照组（0.022）、siRNA-1152组（0.009）和siRNA-685组（0.009）相比显著降低。筛选出的LV3-GATA-3-Mus-1615作为最有效的干扰序列，构建到慢病毒载体中。共转染48小时后收集的293T细胞病毒上清液中，LV3-GATA-3-Mus-1615浓缩病毒的滴度为5×108 TU/ml。成功构建并鉴定了靶向GATA-3基因的慢病毒RNAi表达载体（LV3-GATA-3-Mus-1615）。
