Generation Of EGFP Transgenic Mouse And Dynamic Changes Of Cytoskeleton In Pronuclear Microinjected Embryos

Pronuclear microinjection is a commonly used method for the preparation of transgenicmice, and this technique is important for analyzing gene in vivo quickly and precisely. Inorder to set up a mouse zygotic microinjection system, we conducted systematic study on thekey procedures of constructing transgenic animals via pronuclear microinjection in this study.After digesting the plasmid pEGFP-C1with ApaL I and Mlu I, the1.6kb DNA fragment waspurified and recovered and diluted into a final concentration2.5ng/uL. The purified DNAfragment was injected into the male pronuclear. When developed to2-cell, they weretransplanted into the oviducts of pseudopregnant ICR female mice.19~21days later,46offspring were born，including11positive transgenic mice. So we have initially set up themicroinjection system to generate transgenic mice. To observe the dynamic changes ofα-tubulin and F-actin, during the embryonic development in KM mouse embryos afterpronuclear microinjection, and lay the foundation for improving the developmental capacityof the pronuclear microinjected embryos, immunofluorescence and laser confocal microscopywere used to detect the location of α-tubulin and F-actin in the first meiotic division of themicroinjected zygotes. To investigate the dynamic changes of α-tubulin, γ-tubulin and F-actin,and study the expression of cytoskeleton-associated genes and proteins in mouse pronuclearmicroinjected zygotes, immunofluorescence and laser confocal microscopy were used todetect the location of α-tubulin, γ-tubulin and F-actin, real-time PCR and western blottingwere performed to analyze the expression of α-tubulin, β-actin, γ-tubulin, Aurora A, Aurora Band Tpx2in the first mitosis of the microinjected zygotes.Following results have been obtained:1. In this study,859zygotes were injected,645of which had normal morphology anddeveloped into2-cell embryos after in vitro cultured, the survival rate was75.11%. After24h,we got about512normal2-cell, the cleavage rate was79.70%. Some were cultured in vitro toobserve the expression of EGFP transgene and the developmental capacity, we finally got70blastocysts, the blastocyst rate was45.45%. Then the other2-cell embryos were transferred to20ICR foster mothers, finally we observed11mice were pregnant with birth of46offspring (including the unborn embryos). In conclusion, the pregnant rate was30%, the birth rate was11.86%, and the tranegene integration rate was23.91%.2. The results indicated that: the effects of PI on the microtubule network mainlyconcentrated at the pronuclear stage and mitotic prometaphase. While the effects on themicrofilament network concentrated from the pronuclear stage to the beginning of the firstmitosis;3. The results of Real time PCR and western blotting analysis indicated that: Inpronuclear microinjected embryos, α-tubulin and F-actin gathering around male and femalepronuclear might participate in the repair of the mechanical damage to the normal pronuclearmigration. Rearranged γ-tubulin might relate to the correct spindle pole distribution; thecombined action of Tpx2and Aurora A might contribute to maintain the correct MTsorganization and spindle assembly, also the spindle pole separation, Aurora B appeared to bedominant in the regulation of cytokinesis.