Study On The Culture Of Mouse Embryonic Chimeras Between Male And Parthenogenetic Embryo

This research did a systemic investigation on the critical factors of mouse embryonic chimeras between male and parthenogenetic embryo,by means of studys on the sex indentification of preimplantation embryos,parthenogenetic development of murine oocytes by different chemical simuli and murine embryos aggragate,we established a prelirainary process to make the chimera of male and parthenogentic embryo.The main result are as follows:1.Study on Sex Identification of preimplantation Embryos in mousesduty on the logical of prime rdesign and the influence double pairs primers dual PCR for sex identification system,to resolve the problem of pseudo feminine caused by use single pair primers amplify and the Sry gene didn't amplify as use double pairs primers dual PCR,optimize the sex identification system.(1)Primer design and its logical examination.The special Y chromosome Sry sequences of male mouse and the Zfx sequences exist in all mouse were attained according to the NCBI gene database, and using the software Primer 5.0 designed two primers SRY250U/SRY250L and ZFX399U/ZFX399L,SRY250U/SRY250L is used to amplify the Sry gene and ZFX399U/ZFX399L is used to amplify Zfx gene.Using this two pairs primers amplify male and female's liver genome, the result showed that the primer SRY250U/SRY250L can effectively amplify Sry gene which was the male mouse's liver had,the fragment length is 250 bp,and no homologous fragment to female liver genome,this result is agree with expectation of primer design.The primer ZFX399U/ZFX399L also can effectively amplify Zfx gene in both male and female mouse,and gained a 399 bp fragment, its also agree with expectation of primer design.(2)Study on double pairs primers dual PCR sex identification in mouse's liver organism.In order to avoid the pseudo feminine which caused by Sry gene amplify failure and embryo lost,in this experiment we use double pairs primers dual PCR to do the sex identification with 4 male and 4 female mouse.The result showed that the 4 male mouse samples could amplify two special amplified fragments(250bp,399bp),but 4 female mouse samples only one amplified belts fragment (399bp).This result prove that the double pairs primers dual PCR system we optimized repeatedly could ampilty the special fragment we want successfully in the mouse liver genome,so in the experiments below we use this system to carry out sex indentifcation on mouse preimplantation embyos.(3)Study on the most suitable embryo template quantity of buffalo preimplantation embryos sex identification Applying the optimized sex identification system mentioned above and using the template of four and two embryo blastomeres to identify sex on 8 cells embryos of mouse.The result showed that taking four blastomeres as the template to sex identification,the male and the female embryo ratio(17/19,0.89)approaches 1,which is to say using at least four blastomere as template of the PCR response could ensure the accuracy of sex indentification.2.study on the Parthenogenetic Development of murine Oocytes by different chemical simuli method(1)the influence of routine oocytes teated by single activation agents(7%ethanol,10mmol/L S_rCl₂)in different effect time Oocytes derived in vivo were collected 16h later from the hCG injection,and activated by single activation agents including ethanol(Eth,7%,5min,10min)and strontium(SrCl₂,10mmol/L,4h,6h).The result showed that murine oocytes treated by strontium(10mmol/L SrCl₂)with 6h had higher cleavage(72.38%)and blastocyst(29.52%)rates compared to other treatment groups.(2)the influence of murine oocytes teated by conbination of single agents(7%ethanol, 10mmol/L S_rCl₂)with 6-DMAP in different effect time 6-DMAP is a protine kinase inhibiter,commonly used to achieve a full oocyte activation and increase the diploid rates.The result showed that the ethanol+6-DMAP(4h)treatment group had the highest cleavage(89.58%)and blastocyst(38.54%)rates.3.Study on male and parthenogenetic murine embryo aggragation(1)study on removing of zone pellucid The test adopted Acid Tyrode,0.25%pronase, 0.5%pronase to remove zone pellucide of 8-cell embryo.Then through observing the percentage of obtained naked embryo,the aggregation rate and aggregated embryos' development rate,the effects of four kinds of liquid on the aggregation result were estimated.The result showed that the 0.25% pronase treatment group got the highest aggregation rate(86.36%)and the development rate of aggregation embryos(72.73%),but had the lowest naked embryo rate(32.35%).The 0.5%pronase treated with 20min group had the highest naked rate(50.36%),but their embryo aggregation rate(77.14%)and embryo developemt rate(65.71%)ware both lower than other pronase treated group.Take the concideration of the efficiency and the efficacy,the 0.5%pronas treated 10min was the best choice to remove zone pellucide.(2)study on diverse culture methods of aggregated embryos Two culture methods were compared according as the development rate of aggregated embryos and the blastocyst rate.The results showed that the aggregated embryos cultured in hollowness(30.3%,15.1%)had appreciably higher embryo development ate and blastocyst rate than that of these cultured in culture dish with PHA(16.2%,5.4%),but there was no significant difference between them(P＞0.05).