Production of transgenic porcine blastocysts by hand-made cloning
P. M. Kragh A C D , G. Vajta A , T. J. Corydon C , S. Purup B , L. Bolund C and H. Callesen AA Section of Reproductive Biology, Department of Animal Breeding and Genetics, Danish Institute of Agricultural Sciences, DK-8830 Tjele, Denmark.
B Section for Metabolism, Growth and Lactation, Department of Animal Nutrition and Physiology, Danish Institute of Agricultural Sciences, DK-8830 Tjele, Denmark.
C Department of Human Genetics, University of Aarhus, DK-8000 Aarhus, Denmark.
D To whom correspondence should be addressed. email: peterm.kragh@agrsci.dk
Reproduction, Fertility and Development 16(3) 315-318 https://doi.org/10.1071/RD04007
Submitted: 19 January 2004 Accepted: 2 March 2004 Published: 26 April 2004
Abstract
Recently, a zona-free technique for bovine somatic cell nuclear transfer (NT) with no requirement for micromanipulation (i.e. hand-made cloning (HMC)) has been described. The present study demonstrates the application of the HMC technique in the production of transgenic porcine blastocysts. In vitro-matured zona-free porcine oocytes were bisected manually using a microblade and halves containing no chromatin (i.e. the cytoplasts) were selected. Two cytoplasts were electrofused with one transgenic fibroblast expressing enhanced green fluorescent protein and reconstructed embryos were activated in calcium ionophore (A23187) followed by 6-dimethylaminopurine. Subsequently, embryos were cultured in NCSU-23 medium supplemented with 4 mg mL–1 bovine serum albumin for 7 days. In five replicates, 93.0 ± 7.0% (mean ± s.e.m.) of attempted reconstructed embryos fused and survived activation (31/31, 15/23, 28/28, 37/37 and 28/28). On Day 7 after activation, the respective blastocyst rates (per successfully reconstructed embryos) were 6% (2/31), 7% (1/15), 7% (2/28), 3% (1/37) and 7% (2/28), resulting in an average of 6.0 ± 0.8%. Enhanced green fluorescent protein was expressed in all cells of all eight developing blastocysts. Efforts are now directed towards the production of offspring from such transgenic NT blastocysts.
Acknowledgments
The authors thank Anette M. Pedersen, Klaus Villemoes, Ruth Kristensen, Anette K. Nielsen and Tina F. Hindkjaer for their excellent technical assistance.
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