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Feasibility of Producing Porcine Nuclear Transfer Embryos by Using G2/M-Stage Fetal Fibroblasts as Donors¹

^a Department of Animal Sciences, University of Missouri, Columbia, Missouri 65211

ABSTRACT

The type of donor cell most suitable for producing cloned animals is one of the topics under debate in the field of nuclear transfer. To provide useful information to answer this question, G2/M- and G0/G1-stage fetal fibroblasts were used as donor cells for nuclear transfer. In vitro-matured oocytes derived from abattoir ovaries were used as recipient cytoplasts. In both groups, nuclear envelope breakdown and premature chromosome condensation were completed within 1–2 h after donor cells were injected into the cytoplasm of oocytes. Microtubules were organized around condensed chromosomes and formed a spindle within 1–1.5 h after activation. Decondensation of chromosomes could be seen within 2–4 h after activation. Reformation of the new nuclear envelope occurred 4–6 h after activation and was followed by nuclear swelling and formation of a pronucleus-like structure (PN) 8–12 h after activation. Most (80.6%) of the reconstructed oocytes derived from G2/M cells extruded polar body-like structures (PB). However, a much lower frequency of PB (21.7%) was observed in the reconstructed oocytes derived from G0/G1 donors. A variety of PN and PB combinations were observed in reconstructed oocytes derived from G2/M-stage donors, including 1PN+0PB, 1PN+1PB, 1PN+2PB, 2PN+0PB, 2PN+1PB, 2PN+2PB, and 3PN+1PB. Chromosomes of most embryos (10/13) derived from G2/M stage were diploid. The percentage of cleavage and blastocysts and the average nuclear number of blastocysts in the G2/M and G0/G1 groups were not different. These results demonstrate that the G2/M stage can be morphologically remodeled by cytoplasm of MII oocytes in pigs. To maintain normal ploidy, the extra chromosomes derived from G2/M-stage cells could be expelled by oocytes as a second polar body. G2/M-stage fibroblast nuclei could direct reconstructed embryos to develop to the blastocyst stage.

FOOTNOTES

First decision: 26 December 2000.

¹ This study was supported by Food for the 21st Century and the National Institutes of Health (NCRR 13438) and is a contribution from the Missouri Agricultural Experiment Station journal series 13 091.

² Correspondence: Randall S. Prather, Department of Animal Science, Lab 162, University of Missouri, Columbia, MO 65211. FAX: 573 882 6827;pratherr{at}missouri.edu

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