针对小鼠VEGFA基因的siRNA干扰载体的构建和慢病毒包装

目的 针对NM_001025250.3,NM_001025257.3,NM_009505.4,构建4个针对靶基因小鼠VEGFA(血管内皮生长因子A)的小干扰RNA(siRNA)干扰载体,并将其重组转成慢病毒载体.方法 根据靶基因设计并合成4对siRNA干抚序列,将siRNA干扰序列克隆到pcDNA6.2-GW/EmGF...

A recombinant lentiviral vector carrying siRNA targeting the vascular endothelial growth factor a gene in mice

Objective For NM₀01025250.3,NM₀01025257.3,NM₀09505.4,We built 4 small interfering RNA（siRNA） interference vector for the target gene in mice VEGFA（vascular endothelial growth factor A）,and reorganizated them into a lentiviral vector carrier.Methods 4 pairs of siRNA interference sequences were designed and synthesized based on target genes,siRNA interference sequence was cloned into pcDNA6.2-GW/EmGFP miR carrier.4 interference plasmids were constructed and transformed into competent cells DH5α.Interference carrier transiently transfected into target cells,meanwhile,negative control plasmid group and non transfection group were also set up and the interference effect of interference carrier against target genes was detected by qPCR methods.Lentiviral vector carrier pLentii6.3-VEGFA-MR2 was built using the interference plasmid pcDNA TM6.2-GW/EmGFPmi-MR2 by Gateway recombination technology.Constructed entiviral vector carrier and packaging plasmids（packaging mix） were cotransfected into 293T cells,then packaging virus,collecting the virus stock solution,ultra-centrifugating,condensing,and detecting the titer.Results Four interference plasmids for target gene were successfully constucted and were following: pcDNA TM 6.2-GW/EmGFPmi-MR1,pcDNA TM 6.2-GW/EmGFPmi-MR2,pcDNA TM 6.2-GW/EmGFPmi-MR3 and pcDNA TM 6.2-GW / EmGFPmi-MR4,After interference plasmid transfected into mouse lung cancer cells LLC,silencing effect of interference vectors on target gene of pcDNA TM 6.2-GW/EmGFPmi-MR2 was 58.43%,Lentiviral titer was 7.5×108TU/ml.Conclusion The construction and screening the specific siRNA Lentiviral virus for mice VEGFA silencing were success.