223 ISOLATION OF RABBIT PLURIPOTENCY GENES TO GENERATE RABBIT INDUCED PLURIPOTENT STEM CELLS

Abstract

Induced pluripotent stem cells (iPSC) technology, which allows direct reprogramming of somatic cells to a pluripotent state, is a promising tool for gene-function studies disease modelling, drug screening, toxicology tests and to generate knockout animal models. The goal of the current work was to close the gap in knowledge with regard to the molecular biological background for rabbit iPS work by isolating the putative pluripotency genes from the rabbit, based on the sequences published for other species. The sequence of known pluripotency genes (Oct4, Sox2, Klf4, c-Myc, Nanog) were analysed and primers designed based on the similarity of sequences. Sequences of each individual rabbit pluripotency gene was compared to other vertebrates (e.g. human, mouse, bovine) phylogenetically. Rabbit ESCs and blastocyst stage embryos were collected from superovulated rabbits to isolate total RNA. Genes of interest were amplified using RT-PCR and electrophoretically separated for cDNA fragment isolation. Isolated and subcloned cDNA fragments were sequenced and analysed. Our results showed that after restriction digestion the size of amplified and cloned rabbit Oct4, Sox2, Klf4, c-Myc and Nanog gene fragments correspond to the expected amplicon size. Furthermore, sequence confirmation by DNA sequencing has been completed in the case of Oct4, c-Myc, Klf4 and Nanog. The homology of these genes to that of their mouse and human orthologs were as follows: Oct4: at Na level 79% homologue to mouse, 85% homologue to human, at Aa level 81% homologue to mouse, 90% homologue to human; Klf4: at Na level 98% homologue to mouse, 85% homologue to human, at Aa level 95% homologue to mouse, 84% homologue to human; c-myc: at Na level 88% homologue to mouse, 92% homologue to human, at Aa level 91% homologue to mouse and 94% homologue to human; Nanog: at Na level 71% homologue to mouse, 78% homologue to human, at Aa level 55% homologue to mouse, 66% homologue to human. In conclusion, we have revealed differences at both Na and Aa level in all four major rabbit pluripotency gene sequences in comparison to their mammalian orthologs which might partially explain difficulties in generation of rabbit iPSC capable of germline transmission. Our further goal is to apply rabbit specific pluripotency genes to reprogram somatic cells and generate iPSC more efficiently than by using mouse or human genes.

This work was supported by grants from Plurabbit, OMFB-00130/2010 ANR-NKTH; NKTH-OTKA-EU-7KP HUMAN-MB08-C-80-205; EU FP7 (AniStem, PIAP-GA-2011-286264PartnErS, PIAP-GA-2008-218205; InduStem, PIAP-GA-2008-230675; InduHeart, PEOPLE-IRG-2008-234390; InduVir, PEOPLE-IRG-2009-245808; PluriSys, HEALTH-2007-B-223485).