A unique method to produce transgenic embryos in ovine, porcine, feline, bovine and equine species
F. Pereyra-Bonnet A , R. Fernández-Martín A , R. Olivera A , J. Jarazo A , G. Vichera A , A. Gibbons B and D. Salamone A CA Laboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires, Av. San Martín 4453, C1417 Buenos Aires, Argentina.
B Laboratorio de Reproducción de Rumiantes Menores, Instituto Nacional de Tecnología Agropecuaria, Box 277, EEA Bariloche, Argentina.
C Corresponding author. Email: salamone@agro.uba.ar
Reproduction, Fertility and Development 20(7) 741-749 https://doi.org/10.1071/RD07172
Submitted: 24 September 2007 Accepted: 21 April 2008 Published: 1 August 2008
Abstract
Transgenesis is an essential tool in many biotechnological applications. Intracytoplasmic sperm injection (ICSI)-mediated gene transfer is a powerful technique to obtain transgenic pups; however, most domestic animal embryos do not develop properly after ICSI. An additional step in the protocol, namely assistance by haploid chemical activation, permits the use of ICSI-mediated gene transfer to generate transgenic preimplantation embryos in a wide range of domestic species, including ovine, porcine, feline, equine and bovine. In the present study, spermatozoa from five species were coincubated with pCX-EGFP plasmid and injected into metaphase II oocytes. The chemical activation protocol consisted of ionomycin plus 6-dimethylaminopurine. We detected high proportions of fluorescent EGFP embryos for all five species (23–60%), but with a high frequency of mosaic expression (range 60–85%). To our knowledge, this is the first study to produce exogenous DNA expression in feline and equine embryos. Chemical activation reduces the lag phase of egfp expression in ovine embryos. Our results show that this unique method could be used to obtain ovine, porcine, feline, bovine and equine transgenic preimplantation embryos.
Additional keywords: chemical activation, gene expression, sperm mediated, transgenic mammals.
Acknowledgements
The authors are grateful to Marcela Cueto for critical suggestions and revision of the manuscript. The authors thank Mr. Fernando and Alejandro Gonzales (El Ombu), Dr M. Lloveras (INTA Pergamino), CIALE and Dr San Martín (La Pompeya) for providing the biological material and Dr N. Judewicz (BIOSIDUS) for technical assistance. The authors extend their special thanks in memory of María Muhlmann for her generous assistance with FISH analysis. Part of this work was financed by the Instituto Nacional de Tecnología Agropecuaria (AEGR 3424).
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