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Department of Animal Science, Laval University, Charny, Quebec, Canada 1
The possibility of modifying the porcine genome by early embryo manipulations has become a powerful tool to modify farm animals. However, microinjection is severely impaired by a low yield of transfected embryos. The aim of this project was to use repetitive homologous sequences associated with our gene of interest to induce a homologous recombinaison event. We added a satellite sequence from the family of (dG-dT)n(dA-dT)n, at both ends of our control construct which consists of the green fluorescent protein(GFP) gene coupled to the b-actin promotor and the human cytomegalovirus (CMV) enhancer. Embryos used for microinjection were produced by IVM-IVF method. Briefly, the oocyte-cumulus complexes were obtained by aspiration of the follicules between 3 and 6 mm in diameter from prepubertal gilt ovaries. The oocytes were first matured in NCSU37 supplemented with gonadotrophins for a period of 24 hours. Then, the oocytes completed their maturation with an additional period of 24 hours, without hormonal supplement. Once maturation was achieved, 30 to 40 denuded oocytes were inseminated with frozen thawed semen for 6 hours. Between 18 and 24 after insemination, the embryos were injected with the two different constructs (control and control+satellite) at a final concentration of 1 ng of DNA/ml. After 7 days of culture in NCSU37, the embryos were exposed and observed under an inverted fluorescent microscope with an excitation peak of 490nm and an emission of 510 nm which permits the visualization of the GFP. The addition of the microsatellites allowed for a great increase of the number of green fluorescent transgenic pig embryos (n=186) (44% versus 7,2 % ) compared to our control construct. Furthermore, five embryos out of eleven who have reached the morula and blastocyst stage expressed the GFP.
This abstract is being presented on Tuesday, August 3 at 4:45 PM at CUB 2nd Floor Ballroom.