Characterisation of two methods involving double-stranded rna for the functional genetic analysis of the murine embryonic stem cell: antisense gene trapping & rna interference

• Main Author: Chevassut, Timothy J. T
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2006
• Subjects: Morphology

Murine embryonic stem cells are derived from the inner cell mass of a pre-implantation mouse blastocyst. These cells exhibit dual defining characteristics of indefinite self-renewal and pluripotent differentiation. This thesis sought to explore two different methods of functional genetic analysis of embryonic stem cells, namely antisense gene trapping and RNA interference. Antisense gene trapping in embryonic stem cells involves the sequential genomic integration of two vectors: the first to randomly trap active gene promoters and the second to induce functional antisense gene knockout. Clones were selected according to the sequence identity of the trapped locus. Five such gene-trap clones were characterised (Tsix, Catns, Rex2, Nasp, and Ctbp2) and the analyses of these clones are presented. The use of RNA interference to study embryonic stem cell biology is explored including demonstration of knockdown of the maintenance methyltransferase gene, Dnmt1, that results in significant demethylation of genomic DNA. Antisense gene trapping and RNA interference represent two complementary strategies for the functional genetic analysis of the mammalian cell. In both cases, the goal is to correlate genotype with phenotype, or vice versa, through the bi-allelic knockdown of a target gene. By contrast with one another, while gene trapping has the potential to define novel genes according to their function, RNA interference can only be used for known genes whose functions are predicted from their respective sequences. Intriguingly, both methodologies involve double-stranded RNA suggesting a commonality of underlying molecular mechanism: in the case of antisense gene trapping this is generated endogenously, while in the case of RNA interference it is introduced exogenously in the form of short duplexes. In this manner, double-stranded RNA represents a unifying thread to this thesis.