Comparison Of The Processes About Bovine Oocytes H1foo Incorporation Into Between Male Pronucleus And Nuclear Transfer Donor Nuclei

The birth of Dolly sheep in1997, is considered a milestone in mammalian cloningtechnology, with the continuous development of cloning technology innovation, then thereare a variety of mammals have been successfully cloned, however, this technique, still havemany problems, such as low efficiency of cloning large proportion of fetal malformations, thereasons for these problems so far no thorough study clearly, but now universally acceptedcause cloning inefficient main reason is due to the process of nuclear transfer oocyte nucleusfor nuclear somatic incomplete or abnormal caused by reprogramming, H1foo, as a kind ofoocytes specific linker histones, that exist only in period part of oocytes and early embryo,involved in chromosome formation and inhibit the differentiation process, and was thought toresponsible during donor cells reprogramming.Based on the previous for H1foo gene research, the H1foo effects on bovine oocytesmaturation for further data analysis, finally, real-time live cell imaging techniques were usedto observation the process of H1foo embedded in nuclear transfer embryos and bovinefertilized egg pronuclei, and compare two of its schedule, The results of the project are asfollows:1. We use the heated hot plate and insulation to avoid the mask as a support to build anoocyte culture environment outside the incubator, and analysis the oocytes maturation ratewhich were cultured in the incubator and heated hot plate, the extrusion of first polar body asa reference to oocyte maturation, the results showed that the excretion rate of oocytes firstpolar body which were cultured both inside and outside the incubator was not significantlydifferent, since then, we monitor the oocytes which was cultured outside of the incubatorthrough live cell imaging technology. Interestingly, a very interesting phenomenon was found.Oocytes cytoplasm when cultured for18to20hours, there will be a sudden enlargement andthen gradually retraction process. Through the literature search, we found a aquaporin namedAQP3. And through the experiment we confirm this swelling phenomenon indeed inconnection with AQP3. In our opinion, however, the most direct cause of this phenomenon ishypotonic environment.2. At the first, we constructed H1foo gene mutants by site-directed mutagenesistechniques, to guarantee its nucleotide sequence changes but the amino acid sequence remains unchanged, in order to the mutant sequence escape specific siRNA interference, Thewild-type/the mutant H1foo and somatic H1E mRNA, and interference H1foo expressionspecific siRNA fragments in different combinations injected into the GV stage bovine oocytes.The results show that the H1foo significant role in promoting the maturation of bovineoocytes.3. This study with nuclear transfer and ICSI for technical support, respectively, bovineoocytes as experimental material, compared to the two processes of both H1foo embedded ofbovine fertilized egg sperm nucleus and embedded nuclear transfer embryos, fetal bovineskin fibroblasts was first isolated and subcultured. Over-expression H1foo oocytes as areceptor, fetal bovine fibroblasts stained nuclear as donor cells for nuclear transfer, Inaddition, thawed bovine sperm as donor for ICSI. The results show that25min after ICSI,H1foo to appear on the male pronucleus,75min at a peak, while50min after electric fusioncan vaguely see the emergence of H1foo, obviously found its accumulation in the nucleus ofthe donor after100min,200min at a peak, in the other words, the process of H1fooembedded into the male pronucleus and expression after ICSI significantly faster than theprocess of embedded in the somatic cell type nuclear embryo after nuclear transfer.