The Fundamental Research Of De-aging Project Taking Mice As The Imitative Objects

This thesis is about the fundamental research for the first step on the de-aging project taking Kunming mice as the imitative objects. The research gains some data on doing the research of de-aging under our laboratory's condition, which lays a foundation for carrying on the de-aging project in the future. The studies show that under the ordinary rearing condition, the mean life-span of the Kunming male mice (FuZhou) are 508 ?62 days, and the mean life-span of the female mice (FuZhou) are 618?0 days. The mean life-span of the female is longer than that of the males, but this is no statistically significant(P>0.05). The studies also reveal that the Kunming female mice which body weigh 30g or so are the most suitable for superovulation and 40-50 of postovulatory oocytes can be usually collected. CZB medium is the best culture medium for the mouse embryo culture in vitro, and the developmental block of 2-cell stage can also be broken to some extent (54.2%) by using M16 medium after supplemented 40 percent new born bovine serum. By trial and error, the procedure of preparing medium is worked out. The procedure can prevent sedimentation and the change of osmalarity, in which the method of gradual adding NaHCO3 is the better way to not only adjust pH but also maintain the stabilization of composition in the medium. Additionally, there are certain spontaneous parthenogenetic ratio of Kunming female mice in vivo (about 20 percent of total oocytes), and the ratio can be increased to a great extent with an intraperitoneal injection of pentobarbital sodium (about 42 percent of total oocytes, P<0.05), in which there are high ratio of embryos developed to the morula-blastocyst stage(about 60 percent of parthenogenetic embryos). Though the parthenogenetic embryos can not develop to term ( no born parthenogenetic mice are found), a few of female mice are parthenogenetic gestation obviously. Histological observations show that some of the spontaneous parthenogenones can develop to the cylinder stage at least. These results indicate that both the effects of the spontaneous parthenogenones and the enhanced effects of the anaesthetic pentobarbital sodium should be considered when the nuclear or embryo transplantation in Kunming female mice are carried on. So ethyl ether may be the better anaesthetic than pentobarbital sodium to Kunming mice when nuclear or embryo transplantation are carried on. The simple micromanipulating instruments fitted the ordinary laboratory are worked out in this experiment. With these instruments, the first use of intracytoplasmic microinjection with pronuclei transplantation is carried out, and the heterozygous diploid androgentic embryos are prepared successfully and they develop to the morula in vitro. Meanwhile,-steps the procedure fitted nuclei transplantation in Kunming female mouse, are worked out. The procedure of ?-steps?is that after ensuring penetration of the plasma membrane and success of the prenuclei transplantation, the injecting pipette is withdrawn by three steps. Firstly, the pipette is kept in the original place for 30 seconds. Secondly, the pipette is withdrawn carefully to the upper side of inclined plane and stayed there for 5 seconds. Thirdly the pipette is withdrawn carefully to near the tip of pipette and stayed there for 10 seconds. Finally the pipette is withdrawn thoroughly. Together with the treatment of cytochalasin B, i.e. the fertilized eggs are placed in the embryo medium containing cytochalasin B (5 u g/ml) and incubated for 30...