| dc.description.abstract | Embryonic stem cells (ESC), derived from cultured blastocyst embryos, have the potential to provide insight into developmental and medical issues. The cellular transition, however, from blastocyst to ESC has not been well characterized. The purpose of this study was to identify transcriptional differences between blastocysts and ESC through use of suppression subtraction hybridization (SSH) in murine CD1 blastocyst and 129SvEv ESC. Comparisons between the following groups: blastocyst - blastocyst, trophectoderm - blastocyst, blastocyst - ESC, and ESC - fibroblast feeder cell, resulted in the cloning, sequencing, and identification of uniquely expressed sequences. The second objective was to compare expression levels of three selected transcripts through real time PCR in blastocysts, 129SvEv ESC, C57BL/6 ESC, and fibroblast feeder cells. Using SSH, 33 unique clones were identified and 10 had strong ribosomal homologies while 23 had either gene specific homologies or else were unknown. Prdx1, Ywahz, and Vdacs3 were analyzed through real time PCR. For Prdx1, fibroblast feeder cells had the lowest expression level, blastocysts had a 4.81 fold increase, 129SvEv ESC had a 7.51 fold increase, and the C57BL/6 ESC showed a 27.28 fold increase. Expression levels were significantly different between the blastocyst and C57BL/6 samples as well as the fibroblast feeder cells and both ESC samples based on ?CT statistical analysis. For Ywhaz, the fibroblast feeder sample showed the lowest expression level, blastocysts had a 7.65 fold increase, 129SvEv ESC showed a 6.49 fold increase, and C57BL/6 ESC showed a 16.56 fold difference. The two ESC values were significantly different as was the fibroblast feeder cell value from all other samples. For Vdacs3, the blastocyst sample had the lowest level of expression, fibroblast feeder cells had a 2.16 fold difference, 129SvEv ESC had a 68.07 fold difference, and C57BL/6 ESC had a 161.04 fold difference. Statistically, the blastocyst sample was different from both of the ESC samples and the fibroblast sample from either ESC sample. Prdx1 and Vdacs3 both demonstrated significant upregulation in comparison between blastocyst stage embryos and ESC and function to enhance cell survival and continued cell reproduction. | |
