Culture Of Pig Fibroblasts Cells And Histone Modifications Of Aged Donor Cells After Somatic Cell Nuclear Transfer

The local pig breeds in Jiangsu are various and famous with their high reproductive performance.Although the local agricultural department has made a significant progress in conservation work,the main method is still traditional method,which contains a lot drawbacks including uniformity of means and uncontrollability of outburst epidemic resulting in high pressure of preserving local breed.Therefore,the developed cryopreservation of cells for pig genetics should be performed.1 Culture and cryopreservation fibroblasts of pigThrough the cultivation of pig fibroblasts of different ages,results showed that cells derived from fetal was growing faster than derived from old pig ear tissue.In addition to fibroblasts,primary cultured cells also contain a small amount of other cells,which can be isolated and purified two to three times to obtain a single fibroblast cell line.Afterβ-galactosidase staining,results showed that the positive rate of cells in the elderly(O,≥9 years old)was significant higher than the youth and fetus,which indicated that the staining deepened and the proportion of aged cells increased with age(P<0.05),but there was no significant difference in middle age pig ear fibroblasts(M)and fetal fibrils(F).In this experiment,fibroblasts from 4 tissues and 31 individual pigs were cultured and cells further were frozen for conservation.2 The modification of cells status by rapamycin and Torin1 treatmentThe porcine fibroblasts and fetal fibroblasts were treated with rapamycin and Torin1.After 48 hours,the drugs were washed and the cells were cultured continually for 72 hours,then samples were collected for LC3 protein level detection.Results showed that drug treatment can result in significant increase in LC3 protein levels in the aged pig ear fibroblast group but no significant change was observed in the fetal fibroblast group.At the same time,reduced the number of cells in both groups of cells.In addition,β-gal level of aged cells was significantly decreased when rapamycin and Torin1 performed(P<0.05).Finally,the mitochondria in cells were evaluated by jc-1 staining,results showed that the mitochondrial membrane potential of aged pig fibroblasts could be improved,and the distribution of mitochondria also changed.3 The detection of the developmental efficiency of cloned embryos and histone modificationIn this experiment,the cloned embryos were produced by handmade cloning(HMC),porcine ear fibroblasts from aged and newborn Shawutou pigs were used as donors for theconstruction of cloned embryos.The histone methylation of H3K9me2 and H3K27me3 were detected in cell and cloned embryos by immunofluorescence staining.At 0 h,12 h,24 h stages of the cloned embryo,no significant difference was observed between the old and the newborn pigs,but the nucleus of the cloned embryos in the early stage of embryonic development had the different status.The developmental competence of the cloned embryos varies between 13.04%and 36.65%,while the average blastocyst rate is 27.04%±4.08%.In conclusion,the present study indicates that aged pig ear fibroblast is suitable for cryopreservation of pig genetic resource.Once needed,the cloned embryos could be produced efficiently.