High rates of bovine blastocyst development after ICSI - mediated gene transfer assisted by chemical activation

In order to establish conditions for intracytoplasmic sperm injection-mediated gene transfer [ICSI-MGT] in cattle, various aspects of fertilization and embryonic development were assessed after five activation treatments. Spermatozoa were co-incubated with pCX-EGFP [pCX-enhanced green fluorescent pr...

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Detalles Bibliográficos
Otros Autores: Bevacqua, Romina Jimena, Pereyra Bonnet, Federico, Fernández Martín, Rafael, Salamone, Daniel Felipe
Formato: Artículo
Lenguaje:Inglés
Materias:
BOVINE EMBRYOS
DMAP
EGFP
PARTHENOGENESIS
STRONTIUM
ENHANCED GREEN FLUORESCENT PROTEIN
GREEN FLUORESCENT PROTEIN
IONOMYCIN
IONOPHORE
ANIMAL
ANIMAL DISEASE
ANIMAL EMBRYO
BLASTOCYST
CATTLE
CELL COUNT
CELL CULTURE
CYTOLOGY
DRUG EFFECT
EMBRYO CULTURE
EMBRYO DEVELOPMENT
EVALUATION
FEMALE
GENE TRANSFER
GENETICS
INTRACYTOPLASMIC SPERM INJECTION
MALE
PHYSIOLOGY
STIMULATION
TRANSGENIC ANIMAL
ANIMALS
ANIMALS, GENETICALLY MODIFIED
CELLS, CULTURED
EMBRYO CULTURE TECHNIQUES
EMBRYO, MAMMALIAN
EMBRYONIC DEVELOPMENT
GENE TRANSFER TECHNIQUES
GREEN FLUORESCENT PROTEINS
IONOPHORES
SPERM INJECTIONS, INTRACYTOPLASMIC
STIMULATION, CHEMICAL
BOS
BOVINAE
Acceso en línea:http://ri.agro.uba.ar/files/intranet/articulo/2010Bevacqua.pdf
LINK AL EDITOR
Aporte de:Registro referencial: Solicitar el recurso aquí
Biblioteca Central (FAUBA) de Universidad de Buenos Aires
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245 1 0 |a High rates of bovine blastocyst development after ICSI - mediated gene transfer assisted by chemical activation 
520 |a In order to establish conditions for intracytoplasmic sperm injection-mediated gene transfer [ICSI-MGT] in cattle, various aspects of fertilization and embryonic development were assessed after five activation treatments. Spermatozoa were co-incubated with pCX-EGFP [pCX-enhanced green fluorescent protein gene] plasmid and injected into metaphase II oocytes, which were then treated with ionomycin [Io], before further activation with the following agents: 6-dimethylaminopurine [Io-DMAP], additional Io plus DMAP [2Io-DMAP], Io alone [2Io], ethanol [Io-EtOH], or strontium chloride [Io-SrCl2]. Fertilization rates at 16 h after ICSI, presence of a condensed spermatozoon head on Day 4 [Day 0 = ICSI], blastocyst and EGFP expression rates on Day 7, and Oct-4 pattern of Day 8 blastocysts were evaluated. Fertilization rates did not differ significantly among treatments. All [100 percent] of EGFP-positive embryos resulted from ICSI fertilization, whereas at least 60 percent of EGFP-negative embryos [greater than 4 cells] had a condensed sperm head. Blastocyst rates after 2Io-DMAP were not significantly different from Io-DMAP or Io-EtOH, but they were higher than 2Io or Io-SrCl2 treatments [25.9, 18.7, 14.7, 9.4, and 10.9 percent respectively; P less than 0.05]. Transgene expression rates were higher for Io-DMAP, 2Io-DMAP and Io-SrCl2 than for 2Io and Io-EtOH [52.3, 53.0, 42.8, 28.2, and 29.4 percent respectively; P less than 0.05]. Over 80 percent of the blastocysts expressed egfp protein. In conclusion, ICSI-MGT was a powerful technique to produce bovine embryos that expressed the EGFP transgene. Moreover, the actual efficiency of ICSI-MGT could be readily evaluated by this method, which uses a marker expressed early in embryo development. 
653 0 |a BOVINE EMBRYOS 
653 0 |a DMAP 
653 0 |a EGFP 
653 0 |a PARTHENOGENESIS 
653 0 |a STRONTIUM 
653 0 |a ENHANCED GREEN FLUORESCENT PROTEIN 
653 0 |a GREEN FLUORESCENT PROTEIN 
653 0 |a IONOMYCIN 
653 0 |a IONOPHORE 
653 0 |a ANIMAL 
653 0 |a ANIMAL DISEASE 
653 0 |a ANIMAL EMBRYO 
653 0 |a BLASTOCYST 
653 0 |a CATTLE 
653 0 |a CELL COUNT 
653 0 |a CELL CULTURE 
653 0 |a CYTOLOGY 
653 0 |a DRUG EFFECT 
653 0 |a EMBRYO CULTURE 
653 0 |a EMBRYO DEVELOPMENT 
653 0 |a EVALUATION 
653 0 |a FEMALE 
653 0 |a GENE TRANSFER 
653 0 |a GENETICS 
653 0 |a INTRACYTOPLASMIC SPERM INJECTION 
653 0 |a MALE 
653 0 |a PHYSIOLOGY 
653 0 |a STIMULATION 
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653 0 |a EMBRYO CULTURE TECHNIQUES 
653 0 |a EMBRYO, MAMMALIAN 
653 0 |a EMBRYONIC DEVELOPMENT 
653 0 |a GENE TRANSFER TECHNIQUES 
653 0 |a GREEN FLUORESCENT PROTEINS 
653 0 |a IONOPHORES 
653 0 |a SPERM INJECTIONS, INTRACYTOPLASMIC 
653 0 |a STIMULATION, CHEMICAL 
653 0 |a BOS 
653 0 |a BOVINAE 
700 1 |9 67357  |a Bevacqua, Romina Jimena 
700 1 |9 33719  |a Pereyra Bonnet, Federico 
700 1 |9 33720  |a Fernández Martín, Rafael 
700 1 |9 61021  |a Salamone, Daniel Felipe 
773 |t Theriogenology  |g Vol.74, no.6 (2010), p.922-931 
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900 |a ^tHigh rates of bovine blastocyst development after ICSI-mediated gene transfer assisted by chemical activation 
900 |a ^aBevacqua^bR.J. 
900 |a ^aPereyra-Bonnet^bF. 
900 |a ^aFernandez-Martin^bR. 
900 |a ^aSalamone^bD.F. 
900 |a ^aBevacqua^bR. J. 
900 |a ^aPereyra Bonnet^bF. 
900 |a ^aFernández Martín^bR. 
900 |a ^aSalamone^bD. F. 
900 |a ^aBevacqua^bR.J.^tLaboratorio de Biotecnología Animal, Facultad de Agronomía, Universidad de Buenos Aires, CONICET, Av. San Martín 4453, C1417 Buenos Aires, Argentina 
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900 |a ENHANCED GREEN FLUORESCENT PROTEIN 
900 |a GREEN FLUORESCENT PROTEIN 
900 |a IONOMYCIN 
900 |a IONOPHORE 
900 |a ANIMAL 
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900 |a BLASTOCYST 
900 |a CATTLE 
900 |a CELL COUNT 
900 |a CELL CULTURE 
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900 |a DRUG EFFECT 
900 |a EMBRYO CULTURE 
900 |a EMBRYO DEVELOPMENT 
900 |a EVALUATION 
900 |a FEMALE 
900 |a GENE TRANSFER 
900 |a GENETICS 
900 |a INTRACYTOPLASMIC SPERM INJECTION 
900 |a MALE 
900 |a PHYSIOLOGY 
900 |a STIMULATION 
900 |a TRANSGENIC ANIMAL 
900 |a ANIMALS 
900 |a ANIMALS, GENETICALLY MODIFIED 
900 |a CELL COUNT 
900 |a CELLS, CULTURED 
900 |a EMBRYO CULTURE TECHNIQUES 
900 |a EMBRYO, MAMMALIAN 
900 |a EMBRYONIC DEVELOPMENT 
900 |a GENE TRANSFER TECHNIQUES 
900 |a GREEN FLUORESCENT PROTEINS 
900 |a IONOPHORES 
900 |a SPERM INJECTIONS, INTRACYTOPLASMIC 
900 |a STIMULATION, CHEMICAL 
900 |a BOS 
900 |a BOVINAE 
900 |a In order to establish conditions for intracytoplasmic sperm injection-mediated gene transfer [ICSI-MGT] in cattle, various aspects of fertilization and embryonic development were assessed after five activation treatments. Spermatozoa were co-incubated with pCX-EGFP [pCX-enhanced green fluorescent protein gene] plasmid and injected into metaphase II oocytes, which were then treated with ionomycin [Io], before further activation with the following agents: 6-dimethylaminopurine [Io-DMAP], additional Io plus DMAP [2Io-DMAP], Io alone [2Io], ethanol [Io-EtOH], or strontium chloride [Io-SrCl2]. Fertilization rates at 16 h after ICSI, presence of a condensed spermatozoon head on Day 4 [Day 0 = ICSI], blastocyst and EGFP expression rates on Day 7, and Oct-4 pattern of Day 8 blastocysts were evaluated. Fertilization rates did not differ significantly among treatments. All [100 percent] of EGFP-positive embryos resulted from ICSI fertilization, whereas at least 60 percent of EGFP-negative embryos [greater than 4 cells] had a condensed sperm head. Blastocyst rates after 2Io-DMAP were not significantly different from Io-DMAP or Io-EtOH, but they were higher than 2Io or Io-SrCl2 treatments [25.9, 18.7, 14.7, 9.4, and 10.9 percent respectively; P less than 0.05]. Transgene expression rates were higher for Io-DMAP, 2Io-DMAP and Io-SrCl2 than for 2Io and Io-EtOH [52.3, 53.0, 42.8, 28.2, and 29.4 percent respectively; P less than 0.05]. Over 80 percent of the blastocysts expressed egfp protein. In conclusion, ICSI-MGT was a powerful technique to produce bovine embryos that expressed the EGFP transgene. Moreover, the actual efficiency of ICSI-MGT could be readily evaluated by this method, which uses a marker expressed early in embryo development. 
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