Center of Embryo Engineering and Technology, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.
Theriogenology. 2012 Aug;78(3):583-92. doi: 10.1016/j.theriogenology.2012.03.004. Epub 2012 May 17.
The objective was to investigate the effects of the transgenic donor cell synchronization method, oocyte sources, and other factors, on production of hLF-gene nucleus transfer dairy goats. Three transfected cell lines from ear biopsies from three 3-mo-old Saanen dairy goats (designated Number 1, Number 2, and Number 3, respectively) were selected as karyoplast donors for somatic cell nuclear transfer (SCNT) after detailed identification (including PCR and sequencing of PCR products). In donor cell cycle synchronization studies, the apoptosis rate of hLF transgenic fibroblasts was not different (P > 0.05) after 3 days of serum starvation or 2 days of contact inhibition. Additionally, there was no effect (P > 0.05) on developmental capacity of reconstructed embryos; however, the kidding rate of recipients in the serum starvation group was higher than that in the contact inhibition group (18 vs. 0%, respectively). The production efficiency of the transgenic cloned goats using donor cells from the Number 1 dairy goat cell line was higher than those using the Number 2 and the Number 3 cell lines (kidding rates were 18, 2, and 0%, respectively, P < 0.05). The oocyte source did not significantly affect the pregnancy rate of hLF-transgenic cloned dairy goats, but more fetuses were aborted when using in vitro matured oocytes compared to in vivo matured oocytes. In summary, utilizing transfected 3-mo-old dairy goat fibroblasts as donor cells, seven live offspring were produced, and the hLF gene was successfully integrated. This study provided additional insights into preparation of donor cells and recipient oocytes for producing transgenic cloned goats through SCNT.
本研究旨在探讨转基因供体细胞同步化方法、卵母细胞来源及其他因素对生产 hLF 基因核转移奶山羊的影响。从三只 3 月龄的萨能奶山羊耳组织活检中选择三个转染细胞系（分别编号为 1、2 和 3）作为体细胞核移植（SCNT）的核供体细胞，经过详细鉴定（包括 PCR 和 PCR 产物测序）后使用。在供体细胞周期同步化研究中，hLF 转基因成纤维细胞在血清饥饿 3 天或接触抑制 2 天后的凋亡率无差异（P > 0.05）。此外，对重构胚胎的发育能力无影响（P > 0.05）；然而，血清饥饿组的受体产羔率高于接触抑制组（分别为 18%和 0%）。使用 1 号奶山羊细胞系的供体细胞生产转基因克隆山羊的效率高于使用 2 号和 3 号细胞系（产羔率分别为 18%、2%和 0%，P < 0.05）。供体卵母细胞来源并不显著影响 hLF 转基因克隆奶山羊的妊娠率，但与体内成熟卵母细胞相比，使用体外成熟卵母细胞时会有更多的胎儿流产。总之，利用转染的 3 月龄奶山羊成纤维细胞作为供体细胞，生产了 7 只活产仔，成功整合了 hLF 基因。本研究为通过 SCNT 生产转基因克隆山羊的供体细胞和受体卵母细胞的制备提供了更多的见解。
