| Background and objectiveSince Dolly was born in 1997, rapid progress has been made in somatic cell nuclear transfer (SCNT). So far, SCNT has succeeded in live animals in many species, including cattle, mice, goats, pigs, cats, horses, rats and dogs. As a technology of vegetative propagation, SCNT plays a significant role in scientific research and animal husbandry. SCNT can contribute to agriculture by copying excellent breeding stock and endangered mammals. Combined with transgenic and gene knockout technology, it could be used to produce animal models for studying some human diseases. Rabbit is a commonly used animal in scientific research, its short reproductive cycle, low-cost, closer to humans in development than rodents, moderate size, make it have a lot of advantages in surgery, blood and metabolism research than rodents and is more suitable to be used in studying the cardiovascular system, eye diseases, drug toxicity testing and production of antibodies. Compared with monkey, the application of rabbits in scientific research is not only with low cost, but also easily acceptable in ethics. Developing a clone rabbit technology using the long passage cultured cells by SCNT is a hot point in rabbits cloning research. Chesne obtained the world’s first cloned rabbit using granulosa cells in 2002, Feikun Yang and Qinggang Meng, respectively, in 2007 and 2008 obtained cloned rabbit using granulosa cells, Li shangang, from Shanghai Jiaotong University in China in 2006 obtained the world’s first cloned rabbit using adult fibroblasts, and in 2008 using fetal fibroblasts as nuclear donor obtained cloned rabbit. But till now the efficiency of cloning rabbits is depressing, the survival rate of cloned rabbit is very low, usually 1%-3%, and most of the cloned rabbits were died in three weeks after its birth, only a small number of cloned rabbits can survive to adulthood and have the breeding ability.Therefore, the purpose of the study is to explore cloning rabbit technology with high survival rate and matured cloned rabbits production system by SCNT using granulosa cells and fetal fibroblast cells as nuclear donors respectively. Our laboratory, cooperated with Guangzhou Institute of Biomedicine and Health Research Institute, conducts the project "the prevention of AIDS, viral hepatitis and other major infectious diseases in human" from the national Ministry of Science and technology to establish an animal model suited for HIV Research:transgenic rabbits with Human CD4 and CCR5 gene as HIV-1 infectious animal model, in which the basic work is successfully cloning rabbits.Methods1. Superovulation and oocyte collectionOocytes were collected from New-Zealand White rabbits treated with PMSG & HCG. The rabbits were superovulated by intramuscular injection of 80IU of PMSG, 96h later, by intravenous injection of 150IU HCG to induce ovulation. The ovulated oocytes were recovered at 13h post HCG injection by flushing the oviducts with pre-warmed M199. Cumulus cells were removed by exposure to 100 IU hyaluronidase in M199 medium for 3 min, and then pipetted. Denuded oocytes were collected and maintained in medium in a humidified atmosphere until use.2. Fetal fibroblasts preparation and cumulus collectionFetal fibroblasts were prepared from the fetuses (15 days) of New Zealand White rabbits. The tissue of fetuses was cut into cubes, digested by collagenase, cultured at 38.5℃in 5% CO2. About 2 days later, most of the fibroblasts were frozen in liquid nitrogen for future use, the rest were used for SCNT. Contemporary cumulus cells were obtained from the superovulated rabbit mentioned above. They were collected from the cumulus-oocyte complexes, centrifuged at 1500 rpm for 5min and then keep in M199 at 4℃before cell insertion.3o Affection of different culture medium on the development of rabbit parthenogenetic embryosThe oocytes were activated by three 20μsec 3.0kVcm-1DC pulses and cultured in vitro. The parthenogenetic rabbit embryos were cultured in M199/mRD and EBSS-complete medium, and compared their development at 16h/72h and 120h after activation.4. Setting up a system for the embryo transferSuperovulated rabbits were used for hybridization to get fertilized embryos. After hybridization, to make sure the rabbits ovulate, anintravenous injection of 150IU HCG to induce ovulation was used. The embryos were flushed from oviducts 18-24h later, and incubated in EBSS-complete medium with 5% CO2 in air at 38.5℃for 30 min, and subsequently were surgically transplanted into invivo from oviduct of synchronous estrus rabbits.5. Setting up a system of cumulus cell-cloned rabbitsThe cumulus cells were used as nuclear donors. After nuclear transferred, the SCNT embryos were cultured in EBSS-complete medium with 5% CO2 in air at 38.5℃. Notes were made on the development in 16h/72h and 120h after activation. 16h later, when the embryos were all at the two-to four-cell stage, the SCNT embryos were surgically transplanted into invivo from oviduct of synchronous estrus rabbits.6. Setting up a system of the cloned rabbits from fetal fibroblastsThe fetal fibroblasts were used as nuclear donors. After nuclear transferred, the SCNT embryos were cultured in EBSS-complete medium at the same situation as described. Notes were made on the development in 16h/72h and 120h after activation. 16h later, when the embryos were all at the two-to four-cell stage, the SCNT embryos were surgically transplanted into invivo from the oviduct of synchronous estrus rabbits.Results1. SuperovulationTwenty to thirty oocytes from each rabbit were collected after superovulation. The female rabbits were used for superovulation when they were sexual maturity. Compared to the rabbits with body maturation, both of them have no difference in the number of oocytes collected from each rabbit, the former sometimes was more, but the quality of the latter were better than the former, with thicker zona pellucida, more gap legth perivitelline space and stronger granular sensation of the cytoplasmic.2. The preparation of fetal fibroblasts of rabbitsThe culture and preservation system for the fetal fibroblasts of rabbits was set up during the study of culturing the fibroblasts in vitro, freezing and thawing. The cells were cultured up to 16 passages, and grew well.3. The development of the rabbit parthenogenetic embryos in different culture mediumThe parthenogenetic embryos of rabbits were cultured into blastocysts in vitro (rate of success 90%). Compared with different culture medium, there was a significantly higher percentage of blastocyst cultured in EBSS-complete than in M199 and mRD when the parthenogenetic embryos were activated 72h later, but no statistical differences in cleaved stage and blastocyst in 120h. Therefore EBSS-complete medium is suitable for culturing rabbit parthenogenetic embryos.4. Embryo transferA total of 48 fertilized embryos without transport were transferred into the oviducts of three recipients, all the three recipients became pregnant and carried the pregnancy to term and delivered 19 live of 21 pups. In all, the pregnant rate was 100%, the birth rate (the number of full-term fetus from tansferred fertilized embryos) was 43.75%, and the rate of live fetus (the number of live fetus from tansferred fertilized embryos) was 39.58%.5. Setting up a system of cumulus cell-cloned rabbitsThe cumulus cell-oocytes constructs were cultured to blastocyst in vitro after SCNT. A total of 105 SCNT embryos with two-to four-cell stage were transfer into the oviducts of 6 asynchronous estrus rabbits, all of them became pregnant, and carried the pregnancy to term and delivered 28 pups, which the pregnant rate was 100%. All the pups developed to term, and the rate (the number of fetus from transferred embryos) was 26.67%, ten pups were alive which the rate was 9.52%. Three recipients gave natural birth 14 progenies with 7 alive; the others delivered 14 progenies with 3 alive. Most of the alive pups died within three days, only one is surviving to adulthood.6. Setting up a system of fetal fibroblast-cloned rabbitsThe fetal fibroblast cell-oocytes constructs were cultured to blastocyst in vitro after SCNT. A total of 1186 SCNT embryos with two-to four-cell stage were transfer into the oviducts of 54 asynchronous estrus rabbits, nine of them became pregnant, and three recipients carried the pregnancy to term and delivered 5 pups, and the other six can carried the pregnancy to about 20 days, which the pregnant rate was 16.67%.ConclusionsIn summary, these findings indicate that the female rabbits were efficiently superovulated by using different doses of PMSG & HCG, A suitable medium for culturing rabbit embryos was found, which can get higher blastocyst rate and the efficient system of cumulus cell-cloned rabbits, and a system of fetal fibroblast-cloned rabbits had been established. |
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