Hypoxic changes in gene expression: effect of vhl and p53 genotype

Microarrray experiments were performed in order to identify genes whose expression is altered under hypoxic conditions and to determine whether these changes are dependant on the p53 and HIF tanscription factors. In order to determine HIF dependance, isogenic RCC4 cell lines were used that were either wildtype of null for vhl, the gene responsible for degrading HIF under normoxic conditions. In order to determine p53 dependance, isogenic HCT116 and H1299 cell lines that were wildtype or null for p53 were used. Gene expression was measured after exposure of cells in monolayer culture to hypoxia (<0.01% O2) for various times. Keywords: hypoxia, time course, genetic modification, cell line comparison Affymetrix genechips were used to measure expression in cells exposed to hypoxia in monolayer culture for different times. One replicate was analysed at each time point, except for the control (0 hr) and 18 hr time points in the RCC4 cells, where two independant replicates were performed.本研究开展了微阵列（Microarray）实验，旨在筛选在缺氧条件下表达发生改变的基因，并探究此类表达变化是否依赖于p53与缺氧诱导因子（HIF）转录因子。 为明确该表达变化对HIF的依赖性，本研究采用了同基因RCC4细胞系，此类细胞系分别携带VHL基因的野生型或敲除型——VHL是常氧条件下负责降解HIF的基因。 而为探究该表达变化对p53的依赖性，本研究使用了携带p53野生型或敲除型的同基因HCT116与H1299细胞系。 本研究检测了单层培养细胞暴露于氧气浓度<0.01% O₂的缺氧环境不同时长后的基因表达水平。 关键词：缺氧、时间进程、基因修饰、细胞系对比。 本研究采用Affymetrix基因芯片（Affymetrix genechips）检测不同时长缺氧暴露的单层培养细胞的基因表达水平。除RCC4细胞系的对照组（0小时）与18小时时间点设置了2次独立重复实验外，其余各时间点均仅开展1次生物学重复。