Hiendleder S, Schmutz S M, Erhardt G, Green R D, Plante Y
Department of Animal Breeding and Genetics, Justus- Liebig- University, Giessen, Germany.
Mol Reprod Dev. 1999 Sep;54(1):24-31. doi: 10.1002/(SICI)1098-2795(199909)54:1<24::AID-MRD4>3.0.CO;2-S.
To assess the extent of cytoplasmic genetic variability in cloned cattle produced by nuclear transplantation procedures, we investigated 29 individuals of seven male cattle clones (sizes 2-6) from two different commercial sources. Restriction enzyme and direct sequence analysis of mitochondrial DNA (mtDNA) detected a total of 12 different haplotypes. Transmitochondrial individuals (i.e., animals which share identical nuclei but have different mitochondrial DNA) were detected in all but one of the clones, demonstrating that mtDNA variation among cloned cattle is a very common phenomenon which prevents true genetic identity. The analyses also showed that the cytoplasmic genetic status of some investigated individuals and clones is further complicated by heteroplasmy (more than one mtDNA type in an individual). The relative proportions of different mtDNA-types in two animals with mild heteroplasmy were estimated at 2:98% and 4:96% in DNA samples derived from blood. This is in agreement with values expected from karyoplast-cytoplast volume ratios. In contrast, the mtDNA haplotype proportions observed in six other heteroplasmic animals of two different clones ranged from 21:79% to 57:43%, reflecting a marked increase in donor blastomere mtDNA contributions. These results suggest that mtDNA type of donor embryos and recipient oocytes used in nuclear transfer cattle cloning should be controlled to obtain true clones with identical nuclear and cytoplasmic genomes.
为了评估通过核移植程序产生的克隆牛中细胞质遗传变异的程度，我们调查了来自两个不同商业来源的7个雄性牛克隆（2 - 6号）的29个个体。对线粒体DNA（mtDNA）进行限制性酶切和直接序列分析，共检测到12种不同的单倍型。除了一个克隆外，在所有克隆中都检测到了线粒体转移个体（即具有相同细胞核但线粒体DNA不同的动物），这表明克隆牛之间的mtDNA变异是一种非常普遍的现象，它阻碍了真正的基因同一性。分析还表明，一些被调查个体和克隆的细胞质遗传状态因异质性（个体中存在不止一种mtDNA类型）而进一步复杂化。在来自血液的DNA样本中，两只轻度异质性动物中不同mtDNA类型的相对比例估计分别为2:98%和4:96%。这与核质体 - 细胞质体积比预期的值一致。相比之下，在两个不同克隆的其他6只异质性动物中观察到的mtDNA单倍型比例范围为21:79%至57:43%，这反映出供体卵裂球mtDNA贡献明显增加。这些结果表明，在核移植牛克隆中，应控制用于供体胚胎和受体卵母细胞的mtDNA类型，以获得具有相同核基因组和细胞质基因组的真正克隆。
