Interspecies Nuclear Transfer Of Bengal Tiger And Study Of Histone Acetylation

Extinction of wildlife genetic resources imply that basic material with which we do cellular and molecular biological scientific research will lost forever.If these genetic resources are not preserved by anay way,we will lost the gene resources forever,lost search on cellular and molecular biological mechanism of the extincted wildlife species and recovery those species by somatic cell cloning technology,also lost worldwide Genetic resourses and treasure house of life scientific theory.Bengal tiger at the distribution of our country is a narrow,extremely scarce quantity of rare and endangered wild animals.Tibet Yarlung Zangbo Grand Canyon region of wild Bengal tigers in China are the main areas of habitat.So,in this research,we study the establishment of cell line and biological characteristic research on the purpose of long-term preservation and sustainable utilization of the valuable genetic resources by somatic cell and provision of valuable experimental material for cell biology,medicine,genomics,post genomics,genetic engineering and embryo engineering research.The Bengal tiger group ear marginal tissue fibroblast cell bank(BTEM2/2) And analyzed their biological characteristics,chromosomes,cell cycles and exogenous gene expression,to provide not only nuclear donors with good morphology,but also reliable data for further studying.The biological analysis results showed that,population doubling time(PDT) for revival cells was approximately 28h,there was no cross-contamination among the cells by measure Lactic dehydrogenase(LDH) and Malic dehydrogenase(MDH) isoenzymes. Chromosome analysis showed that the frequency of cell chromosome number of 2n=38 was 98.6%.The test results of the bacteria,fungi,virus,and mycoplasma were negative.In order to study exogenous gene expression,six fluorescent proteins were transfected into the BTEM cells.The transfection efficiency of these genes was between 4.4%～31.9%.The corresponding fluorescence was distributed throughout the cytoplasm and nucleus 24 h after transfection except for some cryptomere vesicles.Every index of the Bengal tiger cell line meets all the standard quality controlsof ATCC(American type Culture Collection).Not only had the germplasm resources of Bengal tiger been preserved at the cell level,but also valuable material had been provided for the research of genome,postgenome and somacloning.Establishment of somatic cell banks using low emperature biological techniques is new effective approach to conserve and maintain the diversity of wild animals.Donor cells with different treatments were used to verify how donor cells influence nuclear-transfer reconstructed embryos development in the Bengal tiger.The results showed: there also was no significantly difference(P＞0.05) between the cleavage rates and the blastula ratios of the nuclear-transfer embryos reconstructed by using donor cells with 5 time passages after revival;The Bengal tiger cells after treatments have good morphology,10ng/mL TSA make the cell cycle stop at the G0/G1 and the highest cleavage and blastula rate(P＜0.01).The Bengal tiger fibroblast cells deal with 10ng/mL TSA not only have a high level of acetylation and better morphology change compare with the other groups,but also the highest percent of blastula,so it must be more suitable method to deal with the donor cells.Comparitive study of level of histone acetylation in IVFand cloned embroyo after activation with different time shows:the histone fluorescence signal at 0 hours is cloned embroyo higher than IVF embroyo,and there were not significant differenences.The histone fluorescence signal at 2 hours is cloned embroyo lower than IVF embroyo.The histone fluorescence signal at 4 hours is cloned embroyo higher than IVF embroyo,and there were not significant differenences.The histone fluorescence signal at 6 hours is cloned embroyo lower than IVF embroyo.The histone fluorescence signal at 8 hours is gradually increased too, cloned embroyo lower than IVF embroyo.The histone fluorescence signal at 10 hours is gradually increased too,cloned embroyo higher than IVF embroyo.The histone fluorescence signal at 12 hours is gradually increased too,and at this time the aeetylation level reach to Maximum,and there were not significant differenences.