Abstract

Reprogramming somatic cells to induced pluripotent stem cells involves the over-expression of four transcription factors, Oct4, c-Myc, Klf4 and Sox2. Although this phenomenon has been demonstrated to be reproducible in a spectrum of cell types and animal species, molecular mechanisms behind the acquisition of pluripotency are unclear.

Elucidation of factors that replace or enhance the initial defined set of four genes allows us to take a mechanistic peek at the pathways behind the plasticity of cellular pluripotency. Of the four transcription factors, Oct4 has been most recalcitrant to substitution by family members. Moreover, Oct4 alone can act as an impetus for the conversion of neural stem cells to pluripotent derivatives. Substitutes for the exogenous requirement of Oct4 have been exemplified through the use of directed genetic screens and the analysis of markers in reprogramming intermediates. To further define the roles of Oct4, we conducted an unbiased genetic screen in mouse embryonic fibroblasts to search for genes that can replace the function of ectopic Oct4 in the reprogramming process.

Transposon mediated mutagenesis entails the mobilisation of a genetic cassette into random fractions of the genome, disrupting normal gene function with either a splice acceptor or splice donor site. In our study, we constitutively expressed piggyBac transposase together with a piggyBac trapping cassette consisting of a splice donor site, in addition to CAG promoter driven c-Myc, Klf4 and Sox2. This generated a mutagenised library of mouse embryonic fibroblasts. After 20 days, colonies which were activated at the endogenous Oct4 promoter were selected for through the addition of puromycin. DNA from these colonies was pooled and integration sites were sequenced. In summary, 275 integration sites were identified and the candidate genes were categorised according to their functions. Analysis of gene targets may shed light on the intricacies behind the molecular circuitry involving Oct4 and perhaps uncover novel pathways in the maintenance of pluripotency, hence deepening our understanding of the reprogramming process.

Biography
Jolene graduated from the National University of Singapore with a degree in Life Sciences and subsequently worked at the Institute of Molecular and Cell Biology in Singapore, where she assisted in projects involved in cell signalling and cancer genetics. In 2008, Jolene embarked on a PhD in Molecular Biology at the University of Cambridge, with the primary aim of using piggyBac transposon mutagenesis to conduct various genetic screens. Funded by the Agency of Science, Technology and Research in Singapore, her PhD project is a joint collaboration between Dr. Pentao Liu at the Wellcome Trust Sanger Institute and Drs. Neal Copeland and Nancy Jenkins at the Institute of Molecular and Cell Biology.

Host: Dr. Jonathan Loh (IMCB) and Dr. Shawn Hoon (SCEI)