| Production of transgenic mice is one of the models for researching the regulation of gene expression and gene integration. Other applications were to aid our fundamental understanding of biologic mechanisms and approach to human disease. In this study the in vitro culture system of Kunming mice was established, then green fluorescent protein gene was induced the male pronuclear of mouse zygotes through microinjection, following the process of embryo development expression of GFP was observed and analyzed. The main object of this study is to examine foreign gene integration and expression in preimplantation embryos in mice.1. This study mainly established a kind of culture system to improve in vitro development of Kunming mouse pronuclear embryos. Mouse pronuclear embryos were obtained through superovulation, then the nomal morphologic zygotes were selected and cultured in M16,CZB and KSOM respectively, or co-cultured with mice oviduct epithelia. The cell number of blastocysts achieved from in vivo/in vitro were counted. The ratio of blastocyst formation were improved in KSOM and CZB by addition of fetal bovine serum(50.71% vs. 85.74%, 0% vs. 16.67%). Co-culture system increased percentage of cleavage and blastocyst formation, and was advantageous for embryos quality and synchronized development. Mouse pronuclear embryos developed in vitro highly in KSOMpes cocultured with oviduct epithelia.2. Crystallization changes of various cryoprotectants sections were analyzed at different seeding temperature using programmed freezing procedure. When seeded at -5 C, PBS was freezed completely within 2 min, PBS+ethylene glycol(PEF), PBS+ glycerol(PGF) and PBS+ethylene glycoH-glycerol(PEGF) were not crystallized entirely although the process lasted for 15 min; When seeding temperature was -5.5 C, PEF and PEGF were crystallized entirely in 10 min, but PGF was not in 15 min; While seeding temperature reduced -6 C and -6.5 C, the three kinds of cryoprotectants were solidified in 8 min, 10 min and 10 min vs 5 min, 8 min and 5 min respectively. Mouse embryos were exposed to the three cryopreservative compounds and seeded at -5.5 C and -6 C followed lauching into liquid nitrogen at -35 C,after thawing and in vitro culture, percentage of hatching embryos were 81.7%,68.5% and 87.5% vs 80%,76.5% and 90.9% ,they both had higher developmental abilities than that seeded at -6.5C(10%,13.3% and 38.4%). Compared with PEF and PGF, PEGF was better effect on the freezing of mice embryos.3. construction of fused gene of bcl-2 and green fluorescent protein provides the scientific basis for clarifying the foreign gene expression in animal tissues. The promoter of this vector is CMV which has strong startup effect. Bcl-2 cDNA was mutant at the begin point by PCR for correct transcribe reading. Finally the vector was enzymed and purified for transgenic research.4. The lined bcl/GFP expressed vector is about 2.3 kb and dissolved 1-3 ug/mL in special TE medium. The pronuclear zygotes of mice were collected at morning or afternoon, microinjection procedure were finished at 10:00 and 16:00 respectively. We found that the percentage of obvious PN formation is high about 16:00 and the developmental ability as so.5. When embryos were exposed in ultraviolet(UV) for different times, the blastocysts formation percentage were various obviously. Comparison of without UV irradiation, exposed for 10 s, 20 s and 40 s the percentage of development were 88.46, 85.14, 78.33 and 56.67 respectively. Thus the time of exposing tinder UV irradiation was benefit less than 20 s otherwise UV maybe hurtful to embryos.6. After microinjection the zygotes were cultured in vitro for observing green fluorescence. GFP promoted by CMV appeared at 2-cell period, but the early expression embryos were difficult to develop continuously. At 8~16 cells periods the number of embryos expressed GFP was most, percentage of positive expression was 26.5 in all blastocysts. This result could illuminate the possibility of transgenic production thr... |
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