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Production of Cloned Pigs by Whole-Cell Intracytoplasmic Microinjection
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 (nuc...
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Published in: | Biology of reproduction 2003-09, Vol.69 (3), p.995-1001 |
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Main Authors: | , , , , , , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Online Access: | Get full text |
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Summary: | 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. |
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ISSN: | 0006-3363 1529-7268 |
DOI: | 10.1095/biolreprod.103.015917 |