Liu L, Shin T, Pryor J H, Kraemer D, Westhusin M
Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A & M University, College Station, Texas 77843, USA.
Cloning. 2001;3(2):51-8. doi: 10.1089/15204550152475554.
Regenerated bovine fetal fibroblast cells were derived from a fetus cloned from an adult cow and passaged every 2-3 days. Serum starvation was performed by culturing cells in DMEM/F-12 supplemented with 0.5% FCS for 1-3 days. In vitro matured bovine oocytes were enucleated by removing the first polar body and a small portion of cytoplasm containing the metaphase II spindle. Cloned embryos were constructed by electrofusion of fetal fibroblast cells with enucleated bovine oocytes, electrically activated followed by 5 h culture in 10 microg/mL cycloheximide + 5 microg/mL cytochalasin B, and then cultured in a B2 + vero-cell co-culture system. A significantly higher proportion of fused embryos developed to blastocysts by day 7 when nuclei were exposed to oocyte cytoplasm prior to activation for 120 min (41.2%) compared to 0-30 min (28.2%, p < 0.01). Grade 1 blastocyst rates were 85.1% and 73.3%, respectively. The mean number of nuclei per grade 1 blastocyst was significantly greater for 120 min exposure (110.63 +/- 7.19) compared to 0-30 min exposure (98.67 +/- 7.94, p < 0.05). No significant differences were observed in both blastocyst development (37.4% and 30.6%) and mean number of nuclei per blastocyst (103.59 +/- 6.6 and 107.00 +/- 7.12) when serum starved or nonstarved donor cells were used for nuclear transfer (p > 0.05). Respectively, 38.7%, 29.4%, and 19.9% of the embryos reconstructed using donor cells at passage 5-10, 11-20 and 21-36 developed to the blastocyst stage. Of total blastocysts, the percentage judged to be grade 1 were 80.9%, 79.2%, and 54.1%, and mean number of nuclei per grade 1 blastocysts, were 113.18 +/- 9.06, 100.04 +/- 6.64, and 89.25 +/- 6.19, respectively. The proportion of blastocyst percentage of grade 1 blastocysts, and mean number of nuclei per grade 1 blastocyst decreased with increasing passage number of donor cells (p < 0.05). These data suggest that regenerated fetal fibroblast cells support high blastocyst development and embryo quality following nuclear transfer. Remodeling and reprogramming of the regenerated fetal fibroblast nuclei may be facilitated by the prolonged exposure of the nuclei to the enucleated oocyte cytoplasm prior to activation. Serum starvation of regenerated fetal cells is not beneficial for embryo development to blastocyst stage. Regenerated fetal fibroblast cells can be maintained up to at least passage 36 and still support development of nuclear transfer embryos to the blastocyst stage.
再生牛胎儿成纤维细胞源自一头从成年母牛克隆而来的胎儿，并每2 - 3天传代一次。通过在补充有0.5%胎牛血清的DMEM/F - 12培养基中培养细胞1 - 3天来进行血清饥饿处理。体外成熟的牛卵母细胞通过去除第一极体和一小部分含有中期II纺锤体的细胞质来进行去核。克隆胚胎通过将胎儿成纤维细胞与去核牛卵母细胞电融合构建，电激活后在10微克/毫升放线菌酮 + 5微克/毫升细胞松弛素B中培养5小时，然后在B2 + vero细胞共培养系统中培养。与激活前细胞核暴露于卵母细胞细胞质0 - 30分钟（28.2%，p < 0.01）相比，当激活前细胞核暴露于卵母细胞细胞质120分钟时，到第7天发育成囊胚的融合胚胎比例显著更高（41.2%）。1级囊胚率分别为85.1%和73.3%。与激活前暴露0 - 30分钟（98.67 ± 7.94，p < 0.05）相比，激活前暴露120分钟时每个1级囊胚的平均细胞核数显著更多（110.63 ± 7.19）。当使用血清饥饿或未血清饥饿的供体细胞进行核移植时，在囊胚发育（37.4%和30.6%）以及每个囊胚的平均细胞核数（103.59 ± 6.6和107.00 ± 7.12）方面均未观察到显著差异（p > 0.05）。分别使用第5 - 10代、11 - 20代和21 - 36代供体细胞构建的胚胎中，发育到囊胚阶段的比例分别为38.7%、29.4%和19.9%。在所有囊胚中，判定为1级的百分比分别为80.9%、79.2%和54.1%，每个1级囊胚的平均细胞核数分别为113.18 ± 9.06、100.04 ± 6.64和89.25 ± 6.19。随着供体细胞传代次数增加，1级囊胚的囊胚百分比和每个1级囊胚的平均细胞核数比例下降（p < 0.05）。这些数据表明，再生胎儿成纤维细胞在核移植后支持高囊胚发育率和胚胎质量。激活前细胞核长时间暴露于去核卵母细胞细胞质可能有助于再生胎儿成纤维细胞核的重塑和重编程。再生胎儿细胞的血清饥饿对胚胎发育到囊胚阶段并无益处。再生胎儿成纤维细胞至少可维持传代至36代，并且仍能支持核移植胚胎发育到囊胚阶段。
