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Production of Cloned Pigs by Whole-Cell Intracytoplasmic Microinjection¹

Department of Animal Science and Connecticut Center for Regenerative Biology,⁴ University of Connecticut, Storrs, Connecticut 06269 Animal Technology Institute Taiwan,⁵ Mialoli, Taiwan, Republic of China Department of Animal Science,⁶ National Taiwan University, Taipei, Taiwan, Republic of China

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.

¹ This manuscript is a scientific contribution (No. 2101) to the Storrs Agricultural Experimental Station at the University of Connecticut. J.-W.L. and S.-C.W. contributed equally to this work.

² Correspondence: Xiangzhong Yang, Dept. of Animal Science, University of Connecticut, 1390 Storrs Road, Extension U-4163, Storrs, CT 06269-4163. FAX: 860 486 0534; jyang{at}canr.uconn.edu

³ Correspondence: Winston T.K. Cheng, National Taiwan University, 50 Lane 155, Section 3, Keelong Road, Taipei 10764, Taiwan, ROC. FAX: 02-2732 4070; wtcheng{at}ccms.ntu.edu.tw