Comparison of different methods for exogenous DNA uptake by bovine spermatozoa

  • Renata Simões Universidade Federal do ABC
  • Weber Beringui Feitosa Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnica
  • Marcella Pecora Milazzotto Universidade Federal do ABC, Centro de Ciências Naturais e Humanas (CCNH)
  • Alessandra Corallo Nicacio Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnica
  • Flavia Regina Oliveira de Barros Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnica
  • José Sergio de Arruda Gonçalves Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnica
  • Mariana Groke Marques Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnica
  • José Antônio Visintin Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnica
  • Mayra Elena Ortiz D'Ávila Assumpção Universidade de São Paulo, Faculdade de Medicina Veterinária e Zootecnica
Keywords: Spermatozoa, Cattle, Animal transgenesis, In vitro fertilization, Embryos

Abstract

Although genetic manipulation of farm animals is of great interest for animal production and the pharmaceutical industry, its efficiency remains far from satisfactory. Pronuclear injection, which is the most widely used technique for such modification, mainly in mice, remains limited for this species. Some alternatives have been developed such as sperm mediated gene transfer, in which the spermatozoa are used as vectors for DNA delivery during in vitro fertilization. Mature sperm cells are able to spontaneously bind exogenous DNA molecules which may be internalized into sperm nuclei. Given the potential of sperm mediated gene transfer for livestock animals transgenesis, the aim of this study was to evaluate four methods of DNA uptake for sperm mediated gene transfer in bovine: incubation with DNA, plasma membrane alteration induced by calcium ionophore followed by incubation with DNA, electroporation and lipofection. Spermatozoa not exposed to exogenous DNA were used as control group. Cleavage, blastocyst and hatching rates were recorded at 72 hours post insemination (hpi), days 9 and 12 of embryo culture, respectively. Exogenous DNA-positive embryos were evaluated by PCR. No effect of treatment was observed on cleavage, blastocyst and hatching rates. In addition, percentage of DNA positive blastocysts did not differ among experimental groups. In spite of the low number of positive embryos, our results show that all treatments presented similar efficiencies for DNA delivery during in vitro fertilization. In conclusion, although the development rates were similar and constant in all groups, other factors such as exogenous DNA sequence, size and concentration should be considered to improve sperm mediated gene transfer.

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Published
2015-04-13
How to Cite
Simões, R., Feitosa, W., Milazzotto, M., Nicacio, A., Barros, F. R., Gonçalves, J. S., Marques, M., Visintin, J., & Assumpção, M. E. (2015). Comparison of different methods for exogenous DNA uptake by bovine spermatozoa. Brazilian Journal of Veterinary Research and Animal Science, 52(1), 78-86. https://doi.org/10.11606/issn.1678-4456.v52i1p78-86
Section
ARTICLES