Off-target effects of VEGF-A regulation by shRNAs. Mus musculus

The aim of this project was to investigate how genome-wide gene expression patterns change when the expression of VEGF-A is modulated using different lentivirally delivered shRNA molecules that are complementary to VEGF-A promoter region. To study these changes, 12 gene expression array experiments were conducted with mouse endothelial cell line (C166). Overall design: The C166 cells were transduced (20% confluency, MOI 10) with lentiviral vectors that all express GFP marker gene and two of them express additional shRNA molecule that is complementary to different regions in the VEGF-A promoter and either up-regulate (VEGF-up) or downregulate (VEGF-down) VEGF-A expression. Fresh medium was changed 72h after transduction. Cells were collected at day 7.

本项目旨在探究：当采用靶向血管内皮生长因子A（VEGF-A）启动子区域的不同慢病毒递送型短发卡RNA（shRNA）分子调控VEGF-A的表达时，全基因组基因表达模式的变化规律。为研究此类表达变化，本研究以小鼠内皮细胞系（C166）为实验模型，共计开展12次基因表达芯片实验。

实验整体设计如下：以细胞汇合度20%、感染复数（Multiplicity of Infection, MOI）为10的条件，将C166细胞与慢病毒载体进行转导处理。所有载体均表达绿色荧光蛋白（GFP）标记基因，其中2种载体额外表达靶向VEGF-A启动子不同区域的shRNA分子，分别可上调（VEGF-up）或下调（VEGF-down）VEGF-A的表达。转导后72小时更换新鲜培养液，于转导后第7天收集细胞。