Title

Murine embryonic stem cell fusion-mediated reprogramming of primary mouse embryonic fibroblasts

Date of Completion

January 2008

Keywords

Biology, Molecular|Biology, Genetics|Biology, Cell

Degree

Ph.D.

Abstract

We fused J1 129/SvJae puromycin resistant embryonic stem cells (ESCs) with BALB/c ROSA26βgeo primary mouse embryonic fibroblasts (MEFs) in effort to reprogram the somatic-sourced genome present within hybrids. We purposefully used cells that differed in genetic backgrounds to allow for the detection of the chromosomal source of expression in hybrid cells as no other fusion studies had utilized this strategy which would allow us to develop a tractable system of studying reprogramming events initiated by fusion. Polyethylene glycol 1500 was used to combine the membranes of the two cell types and resulted in the establishment of hybrid lines A3, A4, and A7. Hybrids resembled stem cells in morphology and growth characteristics. Hybrid cell lines were characterized by detecting the presence of somatic-sourced ROSA26βgeo in hybrids and by assaying the chromosomal compliments present within hybrid cells. Gene expression profiles of hybrid cell lines were evaluated by reverse-transcriptase PCR and Affymetrix DNA microarrays which resulted in similar but not identical expression patterns of hybrids as compared to ESCs. Hybrid cells shared many properties of ESCs including pluripotentcy as hybrid cells formed embryoid bodies and expressed markers of all three primordeal germ layers. Evidence of somatic reprogramming was collected by RNA fluorescence in situ hybridization which detected four sites of transcription of the stem cell pluripotency factor Oct4 in hybrid cells. Sequencing of expressed single nucleotide polymorphisms between 129 and BALB/c detected bi-allelic expression of chromatin remodeling genes in hybrid cells and together these results indicate that reprogramming of the somatic genome does indeed take place when fused with ESCs.^