Oocyte genome cloning used in biparental bovine embryo reconstruction
Oocyte genome cloning is a method by which haploid maternal embryos are obtained in such a way that parthenogenetic haploid blastomeres from these embryos can be considered as a clone of the original gamete. Our objective was to generate oocyte genome replicates and use them to reconstruct biparental embryos by fusion with haploid male hemizygotes. Furthermore, we generated biparental homogeneous transgene-expressing embryos using parthenogenetic haploid blastomeres that expressed a transgene [EGFP]. In the first experiment, parthenogenetic haploid embryos were generated by incubation of oocytes in ionomycin and 6-dimethylaminopurine [DMAP] with a 3 h interval to permit their second polar body extrusion. The cleavage rate was 87.3 percent. To generate transgene-expressing blastomeres, activated oocytes were injected with pCX-EGFP-liposome complexes 3 h post ionomycin exposure, resulting in a cleavage rate of 84.4 percent. In the second experiment, haploid parthenogenetic blastomeres that were positive or negative for EGFP expression were used to reconstruct biparental embryos. Cleavage and blastocyst rates for the reconstructed embryos were 78.4 percent and 61.1 percent and 10.8 percent and 8.4 percent, using EGFP-positive or -negative blastomeres, respectively [P less than 0.05]. All of the reconstructed embryos showed EGFP expression, with 96.6 percent of them showing homogenic expression. Oct-4 expression in the reconstructed blastocysts displayed a similar pattern as IVF-blastocyst controls. In conclusion, our results proved that it is possible to use oocyte genome replicates to reconstruct biparental bovine embryos and that this technique is efficient to generate homogeneous transgene-expressing embryos.
| Main Authors: | , , |
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| Format: | Texto biblioteca |
| Language: | eng |
| Subjects: | BOVINE, CLONING, TRANSGENIC, DRUG DERIVATIVE, ENHANCED GREEN FLUORESCENT PROTEIN, GREEN FLUORESCENT PROTEIN, IONOMYCIN, N[6],N[6] DIMETHYLADENINE, N[6],N[6]-DIMETHYLADENINE, OCTAMER TRANSCRIPTION FACTOR 4, ANIMAL, ANIMAL EMBRYO, BLASTOCYST, BLASTOMA, CATTLE, DIPLOIDY, DRUG EFFECT, EMBRYO TRANSFER, FEMALE, GENETICS, GENOME, HAPLOIDY, HEMIZYGOTE, MALE, MOLECULAR CLONING, OOCYTE, PARTHENOGENESIS, PHYSIOLOGY, POLAR BODY, PREGNANCY, TRANSGENE, ADENINE, ANIMALS, ANIMALS, GENETICALLY MODIFIED, BLASTOMERES, CLONING, MOLECULAR, EMBRYO, MAMMALIAN, GREEN FLUORESCENT PROTEINS, LIPOSOMES, OCTAMER TRANSCRIPTION FACTOR-3, OOCYTES, POLAR BODIES, TRANSGENES, BOVINAE, GAMETE, LIPOSOME, TRANSGENIC ANIMAL, |
| Online Access: | http://ceiba.agro.uba.ar/cgi-bin/koha/opac-detail.pl?biblionumber=46980 |
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