| Transgenic rabbits have been found to be excellent animal models for inherited and acquired human diseases including hypertrophic cardiomyopathy, perturbed lipoprotein metabolism and atherosclerosis. The transgenic rabbit system is very crucial because it fills an important gap between the lab mouse and larger domestic mammals. Size wise it is the smallest domesticated animal that can be used to create recombinant protein in its milk both on an experimental and a commercial level. The rabbit appears particularly flexible for the preparation of human antibodies, and recombinant human proteins for replacement therapies have also been produced in rabbit milk. However,until recently, transgenic rabbits were produced exclusively by pronuclear microinjection which results in additive random insertional transgenesis. In order to establish high efficiency system for producing gene-modified rabbit, we performed the present study to establish rabbit somatic cell nuclear transfer system , which combined with transgenic technology will be suitable for producting transgenic rabbits; we also investigated if nuclear reprogramming (DNA methylation) and nuclear status in fused one cell SCNT embryos in rabbits could explain the difference observed in cloning outcomes when using fetal fibroblasts and cummulus cells as donors; in addition, we obtained transgenic rabbits and knockout rabbit by virus infection and zinc finger nuclease technology.Establishment of a system for producing somatic cell nuclear transfer rabbits:(1)First we compared the results of two protocols for hormone-induced superovulation: PMSG&HCG and FSH&HCG. Two protocols had similar results,20 to 30 oocytes could be obtained from each rabbit. We also found that the female rabbits which were sexual maturity but not be body maturation (4 to 5 months old) had more oocytes, but the quality of which was woerse than that of oocytes from female rabbits were body maturation (≥6 months old);(2)Next, we compared the efficiency of blind enucleation, chemically-assisted enucleation and spinde view-assisted enucleation for enucleating rabbit oocytes. The enucleation rate of blind enucleation was 26.7%, although the encleation rate of chemically-assisted enucleation after membrane protrusion was high the rate for inducing membrane protrusion was not high, therefore the overall rate of encleation was 67.7%, we achieved 100% encleation rate by spinle view-assisted encleation, which therefore should be the most suitable method for rabbit oocytes encleation;(3)We compared the effect of three embryo culture medium (M199, mRD, EBSS-complete) on the development of rabbit parthenogenetic embryos. 5 days after activation, the blastocyst rate of parthenogenetic embryos in all medium could achieve about 90%, while, 72h post activation the blastocyst rate of parthenogenetic embryos in EBSS-complete medium was significantly higher than that of the other groups, therefore EBSS-complete was used as rabbit embryo culture medium in follow-up experiments.(4)A total of 48 fertilized embryos were transferred into three recipients, all recipients became pregnant and delivered 21 pups, the birth rate was 43.75%;(5)Fresh cumulus cells were used as donor cells for rabbit somatic cell nuclear transfer. 105 NT embryos were transferred into 6 recipients, all recipients became pregnant, the pregnant rate was 100% and delivered 28 cloned rabbits, of which 10 pups were alive, the birth rate of alive pups was 9.52%。2 The effect of donor cell type on nuclear remodeling in rabbit somatic cell nuclear transfer embryos was investigated in this study:(1)First, we established rabbit fetal fibroblast cells line and transfected the cells with green fluorescent protein (GFP) by electroporation method, after drug screening, we obtained transgenic cells;(2)We used transgenic cells as donor cells for nuclear transfer, the blastocyst rate of transgenic NT embryos was similar with that of non-transgenic NT embryos, these results indicated that transgenic process does not affect the efficiency of rabbit nuclear transfer;(3)The effect of donor cell type on the in vitro development of rabbit SCNT embryos was investigated, the blastocyst rate from CC-embryos was significantly higher than FF-embryos (79.0% vs. 39.3%). We also found that the in vitro growth of FF-embryos was slower than CC-embryos;(4)Around 90% of donor cells, regardless of cell type, were at G0/G1 phase and the percentages in fresh cumulus cells and fetal fibroblasts following serum starvation were not significantly different, so we can rule out the cell cycle as a major cause for the different in developmental potential between the two groups;(5)Dynamics of DNA demethylation in rabbit zygotes and one-cell embryos following somatic nuclear transfer were studied, we found that the absence of genome-wide DNA active demethylation in early rabbit one-cell stage embryos and NT embryos;(6)All donor nuclei happened premature chromosome condensation (PCC) in NT embryos after fusion. Most PCC was manifested by the formation of an M phase plate, however, in the majority of cases, a misaligned metaphase plate was associated with the spindle, and sometimes individual chromosomes were localized near the spindle poles. The proportion of abnormal PCC in FF-embryos was significantly higher than that in CC-embryos and the proportion of irregular pronuclei in CC-embryos was significantly lower in FF-embryos;(7)After embryos transfer, we obtained cloned rabbits from both groups, while the birth rate of the CC group was significant higher than that of the FF group. These results suggest that the rabbit fetal fibroblast cells are suitable for transgenic, but as donor cell, the development capacity of FF-embryos is inferior to CC-embryos, The nuclear remodeling abnormality of donor cells might be an important factor determining the developmental success of the two groups of rabbit cloned embryos.3 Transgenic and knockout rabbits were producted by virus-transfected and pronuclear microinjection technology in this study:(1)We obtained human CD4&CCR5 gene transgenic rabbits by pronuclear microinjection technology. Embryos after injected were transfer into 8 recipients,17 pups were born,of which 7 were transgenic positive rabbits(.2)Virus which contained transgenci vectors were injected under the zona pellucida of rabbit fertilized embryos, after embryo transfer, ATX80Q and A53T transgenic rabbits were born. We futher obtained F1 A53T transgenic rabbits ; The results indicated that the transgenic efficiency of virus-transfected was higher than that of pronuclear micronjection.
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