Development and expression of the green fluorescent protein in porcine embryos derived from nuclear transfer of transgenic granulosa-derived cells

Nuclear transfer (NT) techniques have advanced in the last few years, and c loned animals have been produced from somatic cells in several species incl uding pig. In this study we examined the feasibility of using granulosa-der ived cells (GCs) as donor cells combined with a microinjection procedure to transfer those nuclei. In vitro matured oocytes were enucleated by aspirat ing the first polar body and adjacent cytoplasm. Mural GCs infected with an enhanced green fluorescence protein (EGFP) gene were serum-starved (0.5% s erum, 7 days), injected directly into cytoplasm of enucleated oocytes and t he oocytes were electrically activated. The reconstructed embryos were cult ured for 7 days and stained with Hoechst 33342 to determine the number of n uclei. Non-manipulated oocytes were electrically activated and cultured as controls. At 9 h post-activation, the pronuclear formation rates were 78.7 +/- 3.7% in NT and 97.4 +/- 4.4% in controls at 9 h post-activation. After 7 days culture, the cleavage rates were 24.5 +/- 7.2% in NT and 79.3 +/- 5. 6% in controls. The blastocysts formation rates were 4.9 +/- 2.4% in NT and 26.8 +/- 3.8% in controls. To examine the effect of activation time on dev elopment of NT embryos, oocytes were activated at 0-0.5, 1-2, or 3-4 h post -injection. At 18 h post-activation the pronuclear formation rates were hig her (62.5 +/- 7.3%) in the 3-4h group as compared to the 0-0.5h (22.0 +/- 1 2.5%) or 1-2h (44.5 +/- 6.3%) groups (P < 0.05). However, the cleavage rate s (9.6 +/- 4.6 to 10.7 +/- 4.2%) and the blastocysts formation rates (1.2 /- 2.4 to 4.9 +/- 3.7%) were not different among treatments (P > 0.05). The mean cell number of blastocysts was 15.7 +/- 5.7 in NT and 25.3 +/- 24.7 i n controls. Green fluorescence was observed in roughly half of the embryos from the one-cell to the blastocyst stage. These results indicate that gran ulosa-derived cell nuclei can be remodeled in the cytoplasm of porcine oocy tes, and that the reconstructed embryos can develop to the blastocyst stage . In addition, EGFP can be used as a marker for gene expression of donor nu clei. (C) 2001 Elsevier Science B.V All rights reserved.