| Eggs have the natural ability to reprogram sperm nuclei with 100%efficiency;however,it shows incomplete reprogramming of the somatic nuclei in the cloning process.It is worth emphasizing that telomerase activity of somatic cell is hardly detectable,that of germ cells yet exists on the temporal variation.Based on this,the study made an in vitro model using adenovirus carrying telomerase,named as hTERT-bMGEs,which telomerase activity of its was identical to those of germ cells.Therefore,hTERT-bMEGs as donor cells for nuclear transfer?NT?,would be investigated whether eggs can improve the efficiency of reprogram and whether epigenetic modification would be affected,when telomerase activity of donor cells are identical to those of germ cells.In a word,this study is to demonstrate the interaction mechanisms between nuclear reprogramming and temporal variation of telomerase activity in somatic cell,and it will have a novel viewpoint to demonstrate the mechanization of the nuclear reprogramming.The results are as follows:1.The recombinant adenovirus AdEasy-1 system was used to construct adenovirus vector containing hTERT gene,and the viral conditions of 293A cells were optimized by transfection,liposome and different serum concentrations.Moreover,telomerase function was verified by infecting mouse fibroblasts isolated and cultured in vitro.Therefore,the results showed that the recombinant adenovirus vector carrying hTERT gene was successfully constructed,and the optimal packaging conditions were as follows:liposome method+15%?v/v?fetal bovine serum,from which the highest viral titer was 1×1011vg/mL.Further RT-PCR and fluorescence detection confirmed that the human telomere gene had functional activity in mouse fibroblasts and could be used to prepare following bovine mammary cell model in vitro.2.Bovine mammary epithelial cells were isolated and purified in vitro and identified their biological characteristics.The results showed that its purity was 100%,the viability was vigorous,and its biological characteristics could be maintained stably in vitro.Moreover,it was further found that the efficiency of monoclonal formation was significantly higher.Therefore,this study provided an effective method for the preparation of mammary cell model in vitro.3.Bovine mammary epithelial cells were infected by recombinant adenovirus carrying human telomere gene,and positive cells were identified.The results showed that hTERT-bMGEs model was successfully prepared in vitro,and the transfection efficiency and positive cloning rate of the eukaryotic expression vectors pCI-neo-hTERT,pEGFP-hTERT and adenovirus vectors carrying human telomere gene were compared.The results showed that the transfection efficiency of adenovirus was significantly higher than that of eukaryotic expression vectors,which was the best way to prepare subsequent cell model.4.Using sperm cells as the control,the optimum cell model was selected to prepare somatic cloned embryos according to telomerase activity of positive cells.Further,the efficiency of somatic cell reprogramming was explored by the detection of cleavage and blastocyst development rate.In addition,epigenetic modification was identified by the expressions of X chromosome inactivation and imprinted genes.The results showed that the13th generation of hTERT-bMGEs was the best nuclear donor model for nuclear transplantation.Moreover,the cleavage and blastocyst development rate of cloned embryos were significantly higher than those of non-trans telomerase gene as donor cells.Moreover,the expression levels of H19,IGF2 and XIST of 4-cell span cloned embryo imprinting genes and X chromosome inactivation gene in hTERT-bMGEsn group were significantly lower than those in non-telomerase modified donor cells group.Therefore,the results showed that the eggs could improve the efficiency of somatic cell reprogramming after modification of telomere when the activity of telomere in donor cells changed similar to that of sperm cells during maturation. |
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