Kishi M, Itagaki Y, Takakura R, Imamura M, Sudo T, Yoshinari M, Tanimoto M, Yasue H, Kashima N
Embryo Transplantation Laboratory, Snow Brand Milk Products Co Ltd Tomakomai, Hokkaido, Japan.
Theriogenology. 2000 Sep 15;54(5):675-84. doi: 10.1016/S0093-691X(00)00382-4.
To assess the developmental potential of nuclear transfer embryos in cattle using mammary gland epithelial (MGE) cells derived from the colostrum, we compared the effectiveness of cloning using those cells and fibroblast cells derived from the ear. The fusion rate of the enucleated oocytes with fibroblast cells (75 +/- 4%) was significantly higher than that with MGE cells (56 +/- 7%, P<0.05). There were no significant differences in the cleavage rate (85 +/- 3% vs. 91+/- 2%) or in the developmental rate to the blastocyst stage (35 +/- 6% vs. 35 +/- 5%) using MGE cells vs. fibroblast cells as donor nuclei (P>0.05). After transfer of blastocysts derived from nuclear transfer embryos produced using MGE cells and fibroblast cells, 13% (4/31) and 16% (6/37) of recipient heifers were pregnant on Day 42 as assessed by ultrasonography, respectively. Two of the 4 and 4 of the 6 recipients of embryos with MGE cell- and fibroblast cell-derived nuclei, respectively, aborted within 150 days of pregnancy. Four live female calves were obtained from MGE cells or fibroblast cells. However, one died from internal hemorrhage of the arteria umbilicalis. The other three calves were normal and healthy. There were no differences in the pregnancy rate or calving rate when using MGE cells vs. fibroblast cells. Microsatellite DNA analyses confirmed that the cloned calves were genetically identical to the donor cows and different from the recipient heifers. We conclude that colostrum-derived MGE cells have the developmental potential to term by nuclear transfer, and the efficiency of development of those cloned embryos was the same as that of embryos obtained using fibroblast cells as donor nuclei, although there was a significant difference in the fusion rate. This method using MGE cells derived from colostrum, which is obtained easily and safely from live adult cows, is more advantageous for cloning with somatic cells.
为了评估利用初乳来源的乳腺上皮（MGE）细胞进行牛核移植胚胎的发育潜能，我们比较了使用这些细胞和耳源成纤维细胞进行克隆的效率。去核卵母细胞与成纤维细胞的融合率（75±4%）显著高于与MGE细胞的融合率（56±7%，P<0.05）。以MGE细胞与成纤维细胞作为供体核时，卵裂率（85±3%对91±2%）或发育至囊胚阶段的发育率（35±6%对35±5%）均无显著差异（P>0.05）。将使用MGE细胞和成纤维细胞产生的核移植胚胎来源的囊胚移植后，通过超声检查评估，在第42天时，分别有13%（4/31）和16%（6/37）的受体母牛怀孕。分别有4头和6头接受MGE细胞和成纤维细胞来源核胚胎的受体母牛中，各有2头和4头在怀孕150天内流产。从MGE细胞或成纤维细胞中获得了4头存活的雌性犊牛。然而，其中1头死于脐动脉内出血。另外3头犊牛正常且健康。使用MGE细胞与成纤维细胞时，怀孕率或产犊率没有差异。微卫星DNA分析证实，克隆犊牛在基因上与供体母牛相同，与受体母牛不同。我们得出结论，初乳来源的MGE细胞通过核移植具有发育至足月的潜能，尽管融合率存在显著差异，但这些克隆胚胎的发育效率与使用成纤维细胞作为供体核获得的胚胎相同。这种使用从成年活牛中轻松安全获取的初乳来源MGE细胞的方法，在体细胞克隆方面更具优势。
