| Summary: | 碩士 === 中興大學 === 動物科學系所 === 95 === The purposes of this study were to isolate and characterize the presumptive rabbit embryonic stem (rES) cell lines. The efficiencies of establishing rES cell lines using different feeder layers and culture procedures were also compared. In Experiment 1, the embryo outgrowths and cell colonies were disaggregated with trypsin-EDTA assisted with a fine glass pipette. No rES cell lines were derived in culture on either mouse embryonic fibroblast (MEF) or STO (SIM mouse embryo-derived thioquanine and ouabain resistant) feeder layers. Also, no ES cell lines were isolated from the whole-blastocyst and immunosurgical inner cell mass (ICM) culture on STO. In Experiment 2, proliferating ICM cells after removing trophoblast cells (Tb) from the day 4 or 5 embryo outgrowths were mechanically disaggregated. The efficiency of establishing rES cells on MEF feeder layer was significantly higher than on STO feeders (86% vs. 15%, P<0.001) and no differences between the whole-blastocyst and ICM groups on MEF (67% vs. 67%) were observed. The rES cells expressed the pluripotent markers including AP, Oct4, SSEA-4, and formed the tissues of three germ layers in embryoid bodies. These results suggest that disaggregation of ICM by the mechanical method is an efficient method to isolate rES cell lines.
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