Lee Jang-Won, Wu Shin-Chih, Tian X Cindy, Barber Michele, Hoagland Thomas, Riesen John, Lee Kun-Hsiung, Tu Ching-Fu, Cheng Winston T K, Yang Xiangzhong
Department of Animal Science and Connecticut Center for Regenerative Biology, University of Connecticut, Storrs, Connecticut 06269, USA.
Biol Reprod. 2003 Sep;69(3):995-1001. doi: 10.1095/biolreprod.103.015917. Epub 2003 May 28.
Cloning by somatic cell nuclear transfer has been successfully achieved by both fusing of a donor cell with and injecting an isolated donor cell nucleus into an enucleated oocyte. However, each of the above methods involves extended manipulation of either the oocytes (fusion) or the donor cells (nucleus isolation). Additionally, cloning efficiency can be reduced by low fusion rate of the cell fusion method, and specialized micromanipulation equipment and exacting nucleus isolation techniques are required for the nucleus injection method. Here we report a whole-cell injection technique for nuclear transfer in pigs and the production of cloned piglets with comparable, if not higher, efficiency than the other two nuclear transfer procedures. First, we tested the feasibility of this technique with three types of frequently used donor cells (cumulus, mural granulosa, and fibroblasts) and obtained the optimal nuclear reprogramming conditions for these cells. We further improved our protocol by avoiding ultraviolet exposure during enucleation and achieved a 37% blastocyst rate. We then conducted whole-cell injection using skin fibroblasts from the ear of a sow transgenic for two genes, the porcine lactoferrin and the human factor IX, and produced four live-born cloned transgenic piglets from three recipients. The present study demonstrated the applicability of producing normal, cloned piglets by the simple and less labor-intensive whole-cell intracytoplasmic injection.
通过将供体细胞与去核卵母细胞融合以及将分离出的供体细胞细胞核注入去核卵母细胞，体细胞核移植克隆均已成功实现。然而，上述每种方法都涉及对卵母细胞（融合）或供体细胞（细胞核分离）的长时间操作。此外，细胞融合法的低融合率会降低克隆效率，而细胞核注射法则需要专门的显微操作设备和精确的细胞核分离技术。在此，我们报告一种用于猪核移植的全细胞注射技术，以及利用该技术生产克隆仔猪的效率，即便不比其他两种核移植程序更高，至少也与之相当。首先，我们用三种常用的供体细胞（卵丘细胞、壁颗粒细胞和成纤维细胞）测试了该技术的可行性，并获得了这些细胞的最佳核重编程条件。我们通过避免去核过程中的紫外线照射进一步改进了方案，囊胚率达到了37%。然后，我们使用来自一头转染了猪乳铁蛋白和人凝血因子IX这两个基因的母猪耳部的皮肤成纤维细胞进行全细胞注射，从三只受体母猪中产出了四只存活的克隆转基因仔猪。本研究证明了通过简单且劳动强度较小的全细胞胞质内注射来生产正常克隆仔猪的适用性。
