Comparison of different methods for exogenous DNA uptake by bovine spermatozoa
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 specie...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Universidade de São Paulo
2015-04-01
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| Series: | Brazilian Journal of Veterinary Research and Animal Science |
| Subjects: | |
| Online Access: | http://www.revistas.usp.br/bjvras/article/view/82459 |
| _version_ | 1811331021074857984 |
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| author | Renata Simões Weber Beringui Feitosa Marcella Pecora Milazzotto Alessandra Corallo Nicacio Flavia Regina Oliveira de Barros José Sergio de Arruda Gonçalves Mariana Groke Marques José Antônio Visintin Mayra Elena Ortiz D'Ávila Assumpção |
| author_facet | Renata Simões Weber Beringui Feitosa Marcella Pecora Milazzotto Alessandra Corallo Nicacio Flavia Regina Oliveira de Barros José Sergio de Arruda Gonçalves Mariana Groke Marques José Antônio Visintin Mayra Elena Ortiz D'Ávila Assumpção |
| author_sort | Renata Simões |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-13T16:12:35Z |
| format | Article |
| id | doaj.art-3c0edb031821482f8737cdfa9505ecf1 |
| institution | Directory Open Access Journal |
| issn | 1413-9596 1678-4456 |
| language | English |
| last_indexed | 2024-04-13T16:12:35Z |
| publishDate | 2015-04-01 |
| publisher | Universidade de São Paulo |
| record_format | Article |
| series | Brazilian Journal of Veterinary Research and Animal Science |
| spelling | doaj.art-3c0edb031821482f8737cdfa9505ecf12022-12-22T02:40:09ZengUniversidade de São PauloBrazilian Journal of Veterinary Research and Animal Science1413-95961678-44562015-04-0152110.11606/issn.1678-4456.v52i1p78-8687785Comparison of different methods for exogenous DNA uptake by bovine spermatozoaRenata Simões0Weber Beringui Feitosa1Marcella Pecora Milazzotto2Alessandra Corallo Nicacio3Flavia Regina Oliveira de Barros4José Sergio de Arruda Gonçalves5Mariana Groke Marques6José Antônio Visintin7Mayra Elena Ortiz D'Ávila Assumpção8Universidade Federal do ABCUniversidade de São Paulo, Faculdade de Medicina Veterinária e ZootecnicaUniversidade Federal do ABC, Centro de Ciências Naturais e Humanas (CCNH)Universidade de São Paulo, Faculdade de Medicina Veterinária e ZootecnicaUniversidade de São Paulo, Faculdade de Medicina Veterinária e ZootecnicaUniversidade de São Paulo, Faculdade de Medicina Veterinária e ZootecnicaUniversidade de São Paulo, Faculdade de Medicina Veterinária e ZootecnicaUniversidade de São Paulo, Faculdade de Medicina Veterinária e ZootecnicaUniversidade de São Paulo, Faculdade de Medicina Veterinária e ZootecnicaAlthough 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.http://www.revistas.usp.br/bjvras/article/view/82459SpermatozoaCattleAnimal transgenesisIn vitro fertilizationEmbryos |
| spellingShingle | Renata Simões Weber Beringui Feitosa Marcella Pecora Milazzotto Alessandra Corallo Nicacio Flavia Regina Oliveira de Barros José Sergio de Arruda Gonçalves Mariana Groke Marques José Antônio Visintin Mayra Elena Ortiz D'Ávila Assumpção Comparison of different methods for exogenous DNA uptake by bovine spermatozoa Brazilian Journal of Veterinary Research and Animal Science Spermatozoa Cattle Animal transgenesis In vitro fertilization Embryos |
| title | Comparison of different methods for exogenous DNA uptake by bovine spermatozoa |
| title_full | Comparison of different methods for exogenous DNA uptake by bovine spermatozoa |
| title_fullStr | Comparison of different methods for exogenous DNA uptake by bovine spermatozoa |
| title_full_unstemmed | Comparison of different methods for exogenous DNA uptake by bovine spermatozoa |
| title_short | Comparison of different methods for exogenous DNA uptake by bovine spermatozoa |
| title_sort | comparison of different methods for exogenous dna uptake by bovine spermatozoa |
| topic | Spermatozoa Cattle Animal transgenesis In vitro fertilization Embryos |
| url | http://www.revistas.usp.br/bjvras/article/view/82459 |
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