Functional genomics analysis in human embryonic stem cells using RNA interference.

Abstract

To fully understand self-renewal and pluripotency in hESCs, it is necessary to unravel genes that are crucial for these defined characteristics. Despite previous discovery of NANOG, OCT4 and SOX2 as key pluripotency associated genes, many genes and molecular mechanisms involved in pluripotency and self-renewal remains largely unknown. Since limited genes have been identified so far, through the use of RNAi, 153 genes were transiently knockdown to elucidate their effect on pluripotency. To assess this effect, NANOG-luciferase and internal control CMV-Luciferase constructs were transfected in concert with siRNA and assess by luciferase assay. Of these 153 genes, 65 genes were found to downregulate NANOG luciferase activity. Next, based on secondary luciferase screening and their expression levels in hESCs and EBs, 24 genes with enriched gene expression in hESCs were narrowed to 6 putative genes, BRD4, SIRT1, HDAC5, FLH32384 (HEXIM2), SLC38A1 and ASNS. Accuracy and specificity of these luciferase assays were validated by viability and promoter tests respectively. Their expression in hESC and EBs were also verified by qRT-PCR. Moreover, a slight decrease in these knockdown, NANOG and OCT4 gene expression were observed using qRT-PCR. Together, this preliminary RNAi screen for novel factors uncovers a potential novel role of these genes in hESC pluripotency.