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Oocyte genome cloning used in biparental bovine embryo reconstruction

Published online by Cambridge University Press:  05 April 2012

Gabriel Vichera
Affiliation:
Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires, Av. San Martín 4453, C1417 Buenos Aires, Argentina.
Ramiro Olivera
Affiliation:
Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires, Av. San Martín 4453, C1417 Buenos Aires, Argentina.
Daniel Salamone*
Affiliation:
Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires, Av. San Martín 4453, C1417 Buenos Aires, Argentina.
*
All correspondence to: D. Salamone. Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires, Av. San Martín 4453, C1417 Buenos Aires, Argentina. Tel: + 54 11 4524 8000/8196. Fax: + 54 11 4514 8737. E-mail: [email protected]

Summary

Oocyte genome cloning is a method by which haploid maternal embryos are obtained in such a way that parthenogenetic haploid blastomeres from these embryos can be considered as a clone of the original gamete. Our objective was to generate oocyte genome replicates and use them to reconstruct biparental embryos by fusion with haploid male hemizygotes. Furthermore, we generated biparental homogeneous transgene-expressing embryos using parthenogenetic haploid blastomeres that expressed a transgene (EGFP). In the first experiment, parthenogenetic haploid embryos were generated by incubation of oocytes in ionomycin and 6-dimethylaminopurine (DMAP) with a 3 h interval to permit their second polar body extrusion. The cleavage rate was 87.3%. To generate transgene-expressing blastomeres, activated oocytes were injected with pCX–EGFP–liposome complexes 3 h post ionomycin exposure, resulting in a cleavage rate of 84.4%. In the second experiment, haploid parthenogenetic blastomeres that were positive or negative for EGFP expression were used to reconstruct biparental embryos. Cleavage and blastocyst rates for the reconstructed embryos were 78.4% and 61.1% and 10.8% and 8.4%, using EGFP-positive or -negative blastomeres, respectively (P < 0.05). All of the reconstructed embryos showed EGFP expression, with 96.6% of them showing homogenic expression. Oct-4 expression in the reconstructed blastocysts displayed a similar pattern as IVF-blastocyst controls. In conclusion, our results proved that it is possible to use oocyte genome replicates to reconstruct biparental bovine embryos and that this technique is efficient to generate homogeneous transgene-expressing embryos.

Type
Research Article
Copyright
Copyright © Cambridge University Press 2012

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