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 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.
| Main Authors: | , , , |
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| Format: | Texto biblioteca |
| Language: | eng |
| Subjects: | 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, |
| Online Access: | http://ceiba.agro.uba.ar/cgi-bin/koha/opac-detail.pl?biblionumber=46780 |
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