奶山羊转基因供核细胞的再饥饿对核移植胚胎发育的影响

为提高转基因奶山羊体细胞核移植胚胎早期发育率，将经转染外源基因的山羊胎儿成纤维细胞经饥饿培养(含0．5％FCS的DMEM)5天后分成两部分：第一部分细胞-80℃或液氮冻存，试验前复苏后直接用作供核细胞(试验组Ⅰ)，或复苏后恢复培养(含10％FCS的DMEM)2-5天后再饥饿5天用作供核细胞(试验组Ⅱ)；第二部分细胞作传代培养(含10％FCS的DMEM)2天后再饥饿5天用作供核细胞(试验组Ⅲ)。将上述不同处理的供核细胞进行细胞周期与存活率的检测，并将该供核细胞移入去除遗传物质的山羊MⅡ期卵母细胞的卵周隙内，经电融合、化学激活后，将核移植(NT)胚胎经0．8％琼脂糖包理后移入临时寄母输卵管内，培养6天后回收并观察NT胚胎的早期发育。结果，试验组Ⅱ所用供核细胞中G0／G1期细胞所占比例及其存活率分别为95．68％、99．9％，均显著地高于试验组Ⅰ(88、66％、80％)；试验组Ⅱ的桑椹及囊胚期NT胚胎的发育率(66．09％)显著地高于试验组Ⅰ(22．00％)与试验组Ⅲ(50．5l％)。将以上发育的NT胚胎分别移入同步发情的受体后，35天作B超妊娠诊断，试验组Ⅱ的受体妊娠率为45．83％，显著地高于试验组Ⅰ(20．00％)与试验组Ⅲ(29．58％)。流式细胞仪分析结果表明，饥饿后的供核细胞经冷冻，复苏后恢复培养2-5天，再经饥饿处理，能显著地提高G0／G1期细胞的比例及细胞存活率；应用该细胞所组建的NT胚不仅具有较高的桑椹与囊胚期发育率，而且具有较高的受体妊娠率。

Effect on development in NT embryos after transplantation of nuclei derived from transfected goat fetal fibroblasts suffering different treatments into enucleate eggs]

In order to improve the development rate of preimplantation nuclear transfer embryos (NT embryos) after transplanting nuclei derived from transgenic goat fetal cells, the donor fetal fibroblasts starved for 5 days in DMEM containing 0.5% FCS were divided into three groups and treated with different methods respectively before using as donor cell. Group 1 was frozen at -80 degrees C or in liquid nitrogen for several days or months. Group 2 was at first treated as the same as group 1, then cultured for 2-5 days in DMEM containing 10% FCS and starved for another 5 days subsequently. Group 3 was cultured for 2-5 days in DMEM containing 10% FCS and starved for another 5 days subsequently. The rate of G0/G1 phase cells from group 2 was 95.68% and significantly different from group 1's 88.66%. The rate of survival cells from group 2 was 99.9% and significantly different from group 1's 80.00% (P < 0.05).The morula- blastocyst stage NT embryos development rate of group 2 was 66.09% and significantly different from group 1's 22.00% and group 3's 50.51% (P < 0.05). All NT embryos of above three groups were transferred into synchronous oestrus recipients and the pregnant status of recipients was checked by B-mode ultrasound diagnosis after 35 days. The recipient pregnancy rate of group 2 was 45.83%, much higher than that of group 1(20.00%) and group 3 (29.58%). The result of this experiment showed that donor cells treated with freezing and two times starvation could significantly improve the rate of G0/G1 phase cells, the rate of survival cells, the NT embryos development rate and the recipient pregnancy rate.