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

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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
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Aporte de:Registro referencial: Solicitar el recurso aquí
Biblioteca Central (FAUBA) de Universidad de Buenos Aires
Descripción
Sumario: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.
ISSN:0093-691X

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