Animal Biotechnology Centre, National Dairy Research Institute, Karnal, India.
Theriogenology. 2011 Sep 15;76(5):851-63. doi: 10.1016/j.theriogenology.2011.03.025. Epub 2011 Jun 12.
The main purpose of the experiment was to compare the efficiency of three cell types, namely adult fibroblast, putative embryonic stem (ES) cell, and lymphocyte, as donor cells for somatic cell nuclear transfer by handmade cloning in goats. The outcome clearly shows that putative embryonic stem cells, with a cleavage and blastocyst production rate of 74.69% ± 3.92 and 39.75% ± 3.86, respectively, performs better in comparison to adult fibroblast cell and lymphocyte. Between adult fibroblast cell and lymphocyte no statistically significant difference exists at P < 0.05. An overall cleavage and blastocyst formation rate of 67.41% ± 3.92 and 26.96% ± 3.86 was obtained using adult fibroblast donor cells. The study establishes beyond doubt the reprogrammability of lymphocyte by handmade cloning (HMC) protocol with a cleavage and blastocyst production rate of 56.47% ± 3.92 and 24.70% ± 3.86, respectively. PCR analysis of highly polymorphic 286 bp fragment of MHC II DRB genes of cloned embryos and three donor cells were performed to verify the cloned embryos. The amplified PCR products were subjected to SSCP to confirm their genetic identity. The karyotyping of the cloned embryos showed normal chromosomal status as expected in goat. Significantly, in the second stage of the experiment, the produced cloned embryos were successfully used to derive ntES-like cells. The rate of primary colony formation rate was 62.50% ± 4.62 for fibroblast donor cell derived embryos. The same was 60.60% ± 4.62 for putative ES donor cell derived embryos and 66.66% ± 4.62 for lymphocyte donor cell derived embryos, respectively. The putative ntES colonies were positively characterized for alkaline phosphatase, Oct-4, TRA-1-60, TRA-1-81, Sox-2, and Nanog by Immunocytochemistry and Reverse Transcription PCR. To further validate the stem ness, the produced putative ntES colonies were differentiated to embryoid bodies. Immunocytochemistry revealed that embryoid bodies expressed NESTIN specific for ectodermal lineage; GATA-4 for endodermal lineage and smooth muscle actin-I, and troponin-I specific for mesodermal lineage. The study has established an efficient protocol for putative ntES cell derivation from HMC embryos. It could be of substantial significance as patient specific ntES cells have proven therapeutic significance.
实验的主要目的是比较三种细胞类型——成纤维细胞、胚胎干细胞（ES 细胞）和淋巴细胞——作为供体细胞通过手工克隆进行体细胞核移植的效率。实验结果清楚地表明，胚胎干细胞的卵裂和囊胚生成率分别为 74.69%±3.92%和 39.75%±3.86%，优于成纤维细胞和淋巴细胞。成纤维细胞和淋巴细胞之间在 P<0.05 水平无统计学差异。使用成纤维细胞供体细胞获得的总卵裂和囊胚形成率分别为 67.41%±3.92%和 26.96%±3.86%。该研究通过手工克隆（HMC）方案证实了淋巴细胞的可重编程性，其卵裂和囊胚生成率分别为 56.47%±3.92%和 24.70%±3.86%。对克隆胚胎和三个供体细胞的高度多态性 286bp 片段的 MHC II DRB 基因进行 PCR 分析，以验证克隆胚胎。扩增的 PCR 产物进行 SSCP 以确认其遗传同一性。克隆胚胎的核型分析显示出正常的染色体状态，与山羊预期的一致。值得注意的是，在实验的第二阶段，产生的克隆胚胎成功地用于衍生 ntES 样细胞。原代集落形成率分别为：成纤维细胞供体胚胎衍生的 62.50%±4.62%、假定 ES 供体胚胎衍生的 60.60%±4.62%和淋巴细胞供体胚胎衍生的 66.66%±4.62%。碱性磷酸酶、Oct-4、TRA-1-60、TRA-1-81、Sox-2 和 Nanog 的免疫细胞化学和逆转录 PCR 阳性鉴定 ntES 样细胞。为了进一步验证其干细胞特性，将产生的假定 ntES 集落分化为类胚体。免疫细胞化学显示类胚体表达神经上皮特异性蛋白 NESTIN；GATA-4 用于内胚层谱系；平滑肌肌动蛋白-I 和肌钙蛋白-I 用于中胚层谱系。该研究建立了一种从 HMC 胚胎中衍生假定 ntES 细胞的有效方案。这可能具有重要意义，因为患者特异性 ntES 细胞已被证明具有治疗意义。
