Experiment Research Of MWOW Cultured Zona-free Morula By Lentivirus Infection

ObjectiveGo through the study of mouse zona-free embryo culture the choice ofmethods Preparation of transgenic mice efficiency of lentiviral infection, andexplore the most suitable for lentiviral zona embryos co-cultured in vitro culture,the initial establishment of a slow virus infection zona pellucida of embryostechnology platform, and lay the foundation for the next phase of lentivirusinfection of the legal system to prepare transgenic mice.MethodsFound that the day of the zygote female vaginal suppository, under sterileconditions, clipping the vas deferens,0.1mlFHM operating fluid flushing the vasdeferens, collect zygotes, hyaluronidase to remove cumulus cells,0.5%chainenzyme protease to remove the zona pellucida, Collected Zona-free zygoticpress mWOW culture method, droplets single zygote+groups zygote culturemethod, droplets single zygote culture method, the droplets groups zygoteculture method were randomly divided into four groups, KSOM-AA mediumtraining4days, per24h half-for medium, early embryonic stages ofdevelopment rate, rate of blastocysts and blastocysts cell number as a measure,study on four kinds of methods in vitro effects on embryonic development oftransparent tape. On this basis, carrying EGFP lentiviral infected to zona-freemorula,48h random infection after blastocyst, single cell nested-PCR fordetecting integration of exogenous genes in embryo. Statistics under thefluorescent microscope fluorescence number of embryos, calculated efficiencyof lentiviral infection. ResultsmWOW culture methods, droplets single zygote+groups zygote culturemethod, droplets single zygote culture method three methods’s blastocystdevelopment rate differences was statistically significant (p <0.05). ComparisonmWOW culture methods, droplets single zygote+groups zygote culture method,droplets single zygote culture method, droplets groups zygote culture method,2-cell growth rate of the four methods were no significant differences (p>0.05),Comparison mWOW culture methods, droplets single zygote+groups zygoteculture method, droplets single zygote culture method,4-cell growth rate of thethree methods were no significant differences (p>0.05). Comparison dropletssingle zygote+groups zygote culture method, droplets single zygote culturemethod,8-cell growth rate of the two methods were no significant difference(p>0.05).Both of them were higher than8-cell growth rate of the mWOWculture methods (p<0.05). morula development rate of mWOW culture methods,droplets single zygote+groups zygote culture method were no significantdifference (p>0.05), were higher than the morula development rate of dropletssingle fertilized egg culture metho (p <0.05). droplets single zygote+groupszygote culture method, droplets single zygote culture method blastocystrecovery rate are100%, blastocyst recovery rate (p <0.05) were higher thanmWOW culture. The cultured cell number of blastocysts of mWOW culturemethods, droplets single zygote+groups zygote culture method, droplets singlezygote culture method were lower than in vivo cultured cell number ofblastocysts (p <0.05). The cultured cell number of blastocysts of roplets singlezygote+groups zygote culture method, droplets single zygote culture methodwere no significant differences (p>0.05),were lower than The cultured cellnumber of blastocysts of mWOW culture methods (p<0.05).A random sample of7blastocyst single cell nested PCR,7samples wereouter primers and inner primers amplified twice were PCR positive.mWOW cultured the zona-free mulberry embryos by Lentiviral infection48h, the blastocyst rate of90.1%(72/80), lentiviral infection rate of78.8%(63/80). Conclusions1、Comparison mWOW culture methods, droplets single zygote+groupszygote culture method, droplets single zygote culture method, mWOW culturemethod is more suitable for co-culture as a Lentiviral and morula2、lentiviral vectors successfully infect mWOW cultured zona-free morula.