Investigation of mRNA ablation strategies in ES cells

• Main Author: Lee, Sonia Anita
• Published: University of Edinburgh 2002
• Subjects: 572.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.653772

The aim of the work presented in this is to investigate mRNA ablation systems for assignment of gene function in ES cell self renewal and differentiation. Such a technology could be used to determine the potential role of candidate genes or to identify new genes in a functional library screen. My strategy was to express antisense RNA, ribozymes or double stranded hairpin RNA using a high efficiency episomal transduction and expression system. Antisense RNAs were targeted against the 5’ and 3’ UTRs of <i>LIFR, STAT3 </i>and <i>Oct4</i> genes required for the differentiation phenotype when cells were maintained in the presence of LIF. Ribozyme ablation was also ineffective when targeting the coding region of the p300 gene target, using a ribozyme reported previously to the effective in embryonal carcinoma cells. In contrast the operation of RNAi in ES cells was established. This was demonstrated by the specific suppression of luciferase expression from reporter plasmids co-transfected with 21 nt specific short interfering RNAs, siRNAs. The feasibility of an RNAi strategy by in situ production of dsRNA from stable electroporation of an episomally maintained vector harbouring long inverted repeats to the coding regions of eGFP, LIFR and SOCS3 targets was then investigated. dsRNA to eGFP appeared to produce sequence specific RNAi against a chromosomal eGFP gene randomly integrated in ES cells, where up to a 33% reduction of eGFP expression was observed. However, I found that the cells expressing reduced eGFP were dying as determined by staining with Annexin V and 7-AAD, so that reduced fluorescence may be a secondary consequence. Cell death could be attributable to activation of an interferon response as it is known that long dsRNAs in mammalian cells are capable of inducing apoptosis. Evidence of ablation of the other targets was not observed, as there was neither phenotypic ablation of the LIFR target nor a reduction of SOCS3 mRNA.