Transfection, Culture And Construction Of Non-intact Zona Embryos Of Mouse

With development of embryonic application technology, it more and more showsolicitude for the application of zona-free embryos due to its better value on animalcloning, embryos mosaic, embryos development and transgenic. Since culture methodsof zona-free embryos were cumbersome, and the development rate, pregnancy rate andrepeatability of zona-free embryos were lower so that its application was limits.Therefore, improving the development of zona-free embryos or finding other zonatreatment approach would become an important research topic. The overall objectives ofthe present study enhance efficiency of gene transfer based on the embryo development,which depends on the improvement of medium of zona-free embryos or obtainingnon-intact zona embryos. The experimental material in the study was early embryo ofKunming mouse, which was obtained by superovulation. Zona-free embryos andnon-intact zona embryos were obtained with zona pellucida digested by Pronase E. Thezona-free embryos and non-intact zona embryos were transfected by adenovirus vectorand obtained the following results.The digestion time of pronase E significantly effected transfection rate, embryosdevelopment and proportion between zona-free embryos and non-intact zona embryos(P<0.05). And the largest proportion of non-intact zona embryo could be obtained indigestion medium containing0.35%pronase E, temperature22℃and4min; theoptimum conditions of the prevent digestion was serum concentrations20%and4-6min.The minimum time that the embryos could be transfected by adenovirus vector wasabout3h, and minimum adsorption time between adenovirus vector and embryo is about120min, so adenovirus receptor on embryo was present before embryonic cleavage. Thebest titer of the adenovirus vector was1×106-7PFU/mL. The transfection rate ofnon-intact zona embryos was lower than that of zona-free embryos (non-intact zona52.6±2.24%; zona-free100%), but the embryos development rate was higher non-intactzona embryos (71.7±0.42%) than zona-free embryos (56.8±0.92%, P<0.05). Thedevelopment rate of zona-free embryos and non-intact zona embryos were significantlyenhanced in the medium containing trehalose (1.2-2.0mg/mL), which was benefit ofearly embryo development (P<0.05). The development rate of zona-free embryos andnon-intact zona embryos were significantly improve in the medium containing hyaluronic acid (0.8-1.6mg/mL), which the viability of zona-free embryos was betterthan that of non-containing trehalose (P<0.05)The transfected rate of non-intact zona embryos was decreasing containing trehalose orhyaluronic acid in the transfected medium; however, the transfected rate of zona-freeembryos was no significance different for non-containing or containing trehalose orhyaluronic acid. Moreover, the transfected rate of zona-free embryos was no significancechanging the order of addition of trehalose and hyaluronic acid with adenovirus vector intransfected medium. However, the transfected rate of non-intact zona embryos wassignificantly decreasing (P<0.05) when trehalose or hyaluronic acid was added intotransfected medium and then adenovirus vector was supplemented; If adenovirus vectorwas first added into transfected medium and then trehalose or hyaluronic acid wassupplemented, it was not significantly low (P>0.05). Although a toxicity of adenovirusvectors could damage embryo development, the damage of zona removal was greaterthan that of toxicity of adenovirus vectors (1×106PFU/mL) on embryonic developmentpotential.The formation process and transfected mechanism of the non-intact zona embryo werecreated based on the experiments data, which think that zona pellucida of mice was madeof iDGF (indigestible glycoproteins filaments) and DGF (digestible glycoproteinsfilaments). Non-intact zona embryo was an embryo that DGF was digested by Pronase E.Zona pellucida still existed under optical microscope, but it had become penetrationstructure, which could be traversed by the adenovirus vector or other biologicalmacromolecules and they transfected embryos.In summary, non-intact zona embryos can be obtained and transfected base on aboveresults. Moreover, the development rate of non-intact zona embryos transfected issignificantly higher than that of the zona-free embryos, which can improve the efficiencyof transgenic embryos. Moreover, it was created that was the formation process andtransfected mechanism of the non-intact zona embryo. The new research ideas ofembryonic gene transfer are explored in view of non-intact zona embryo transfected,which has certain reference and application in transgenic animal research.