Shah Riaz A, George Aman, Singh Manoj K, Kumar Dharmendra, Chauhan Manmohan S, Manik Radhaysham, Palta Prabhat, Singla Suresh K
Embryo Biotechnology Laboratory, Animal Biotechnology Center, National Dairy Research Institute, Karnal-132001, India.
Cloning Stem Cells. 2008 Dec;10(4):435-42. doi: 10.1089/clo.2008.0033.
Hand-made cloning (HMC) has proved to be an efficient alternative to the conventional micromanipulator-based technique in some domestic animal species. This study reports the development of an effective culture system for in vitro culture of zona-free cloned buffalo (Bubalus bubalis) embryos reconstructed using adult skin fibroblast cells as nucleus donor. Cleavage and blastocyst rates observed were 52 and 0% in modified Charles Rosenkrans 2 (mCR2), 61 and 4.6% in modified Synthetic Oviductal Fluid (mSOF), and 82 and 40.3% in Research Vitro Cleave (RVCL; Cook, Australia) medium, respectively. Similarly, higher blastocyst rates (24.5 +/- 4.1%) were observed when zona-free parthenotes were cultured in RVCL medium. Culturing zona-free cloned buffalo embryos on flat surfaces (FS) yielded significantly higher (p < 0.05) blastocyst rates than Well of the Wells (WOW) or microdrops (MD). Furthermore, development in WOW was found to be significantly better than MD culture. The quality of HMC blastocysts was examined using differential staining. This study establishes the application of zona-free nuclear transfer procedures for the production of hand-made cloned buffalo embryos and the development of efficient culture system and appropriate media requirements for enhancing their preimplantation development.
手工克隆（HMC）已被证明在一些家畜物种中是一种替代传统基于显微操作技术的有效方法。本研究报告了一种有效的培养系统的开发，用于体外培养使用成年皮肤成纤维细胞作为核供体构建的无透明带克隆水牛（Bubalus bubalis）胚胎。在改良的查尔斯·罗森克兰斯2（mCR2）培养基中观察到的卵裂率和囊胚率分别为52%和0%，在改良的合成输卵管液（mSOF）中为61%和4.6%，在研究体外卵裂（RVCL；澳大利亚库克公司）培养基中为82%和40.3%。同样，当无透明带孤雌胚在RVCL培养基中培养时，观察到更高的囊胚率（24.5±4.1%）。在平面（FS）上培养无透明带克隆水牛胚胎产生的囊胚率显著高于孔中孔（WOW）或微滴（MD）培养（p<0.05）。此外，发现WOW培养中的发育明显优于MD培养。使用差异染色检查HMC囊胚的质量。本研究确立了无透明带核移植程序在生产手工克隆水牛胚胎中的应用，以及开发高效培养系统和合适的培养基要求以促进其植入前发育。
