Cho Seong-Keun, Kim Jae-Hwan, Park Jong-Yi, Choi Yun-Jung, Bang Jae-Il, Hwang Kyu-Chan, Cho Eun-Jeong, Sohn Sea-Hwan, Uhm Sang Jun, Koo Deog-Bon, Lee Kyung-Kwang, Kim Teoan, Kim Jin-Hoi
Division of Applied Life Science, College of Agriculture and Life Science, Gyeongsang National University, Jinju, GyeongNam, South Korea.
Dev Dyn. 2007 Dec;236(12):3369-82. doi: 10.1002/dvdy.21308.
Somatic cell nuclear transfer (scNT) is a useful way to create cloned animals. However, scNT clones exhibit high levels of phenotypic instability. This instability may be due to epigenetic reprogramming and/or genomic damage in the donor cells. To test this, we produced transgenic pig fibroblasts harboring the truncated human thrombopoietin (hTPO) gene and used them as donor cells in scNT to produce first-generation (G1) cloned piglets. In this study, 2,818 scNT embryos were transferred to 11 recipients and five G1 piglets were obtained. Among them, a clone had a dimorphic facial appearance with severe hypertelorism and a broad prominent nasal bridge. The other clones looked normal. Second-generation (G2) scNT piglets were then produced using ear cells from a G1 piglet that had an abnormal nose phenotype. We reasoned that, if the phenotypic abnormality of the G1 clone was not present in the G2 and third-generation (G3) clones, or was absent in the G2 clones but reappeared in the G3 clones, the phenotypic instability of the G1 clone could be attributed to faulty epigenetic reprogramming rather than to inherent/accidental genomic damage to the donor cells. Blastocyst rates, cell numbers in blastocyst, pregnancy rates, term placenta weight and ponderal index, and birth weight between G1 and G2 clones did not differ, but were significantly (P < 0.05) lower than control age- and sex-matched piglets. Next, we analyzed global methylation changes during development of the preimplantation embryos reconstructed by donor cells used for the production of G1 and G2 clones and could not find any significant differences in the methylation patterns between G1 and G2 clones. Indeed, we failed to detect the phenotypic abnormality in the G2 and G3 clones. Thus, the phenotypic abnormality of the G1 clone is likely to be due to epigenetic dysregulation. Additional observations then suggested that expression of the hTPO gene in the transgenic clones did not appear to be the cause of the phenotypic abnormality in the G1 clones and that the abnormality was acquired by only a few of the G1 clone's cells during its gestational development.
体细胞核移植（scNT）是创建克隆动物的一种有用方法。然而，scNT克隆表现出高度的表型不稳定性。这种不稳定性可能归因于供体细胞中的表观遗传重编程和/或基因组损伤。为了验证这一点，我们制备了携带截短型人血小板生成素（hTPO）基因的转基因猪成纤维细胞，并将其用作scNT中的供体细胞来生产第一代（G1）克隆仔猪。在本研究中，将2818个scNT胚胎移植到11头受体母猪体内，获得了5头G1仔猪。其中，一头克隆猪面部外观呈二态性，有严重的眼距过宽和宽阔突出的鼻梁。其他克隆猪看起来正常。然后，使用来自具有异常鼻表型的G1仔猪的耳部细胞生产了第二代（G2）scNT仔猪。我们推断，如果G1克隆的表型异常在G2和第三代（G3）克隆中不存在，或者在G2克隆中不存在但在G3克隆中再次出现，那么G1克隆的表型不稳定性可归因于表观遗传重编程错误，而非供体细胞的固有/偶然基因组损伤。G1和G2克隆之间的囊胚率、囊胚中的细胞数量、妊娠率、足月胎盘重量和 ponderal 指数以及出生体重没有差异，但显著低于对照年龄和性别匹配的仔猪（P < 0.05）。接下来，我们分析了用于生产G1和G2克隆的供体细胞重建的植入前胚胎发育过程中的全基因组甲基化变化，未发现G1和G2克隆之间的甲基化模式有任何显著差异。实际上，我们在G2和G3克隆中未检测到表型异常。因此，G1克隆的表型异常可能是由于表观遗传失调。进一步的观察表明，转基因克隆中hTPO基因的表达似乎不是G1克隆中表型异常的原因，并且这种异常是在G1克隆的少数细胞在其妊娠发育过程中获得的。
