Study On The Efficiency Of Nuclear Reprogramming By HTERT-modified Donor Cells

Objective: telomerase can not only prolong telomere to maintain the integrity of chromosome structure,but also promote cell proliferation and reprogramming in vitro.In order to further explore the regulatory role of telomerase in cell reprogramming,a donor cell model in vitro was optimized and prepared by recombinant adenovirus carrying telomerase h TERT gene.Furthermore,the effect of h TERT modified donor cells on reprogramming efficiency was analyzed based on epigenetic and RNA-Seq data.Methods:firstly,the recombinant adenovirus containing the target gene h TERT was constructed by adenovirus vector Ad-Max,and the positive cell model was screened by infecting bovine mammary epithelial cells with different virus titers(MOI),and the prepared cell model was detected by biological characters,and then efficiency of somatic cell reprogramming were identified by changes in epigenetic modification patterns,including histone acetylation level,DNA methylation,X chromosome inactivation gene and Imprinted gene.Finally,the h TERT overexpression transcriptome library was constructed,the differentially expressed genes,GO terms and KEGG pathway were analyzed at cellular and embryonic level to elucidate the regulatory mechanism of telomerase on somatic cell nuclear reprogramming.The main results are as follows:1.Adenovirus expressing h TERT gene was constructed by adenovirus Ad-Max system and infected bovine mammary epithelial cells.The telomerase activity of positive cells was detected atdifferent MOI values.The results showed that the telomerase activity of positive cells was the highest in MOI=80,and cell cycle analysis showed that the number of cells in G0 / G1 phase was lower in telomerase overexpression positive cells group,while the number of cells in S phase was significantly higher.It was confirmed that the-donor cell model of h TERT was successfully established and telomerase could stimulate cell proliferation in vitro.2.The expression levels of DNMT1,Dnmt3 a and DNMT3 b genes were significantly down-regulated in telomerase-modified donor cells group.Meanwhile,the expression of Tet1 was significant up-regulated in telomerase-modified donor cells group,but that of Tet2 and Tet3 was not significant.Furthermore,the expression levels of imprinted genes(H19,IGF2,IGF2 R,Peg3,SNRPN)and X-chromosome inactivation gene Xist in the telomerase-modified donor group were significantly lower than those in the control group.Bisulfite sequencing analysis(BSP)showed that the methylation level of H19 DNA in telomerase-modified donor cells group was lower than that in the control group,but there was no significant difference between Xist and IGF2.Therefore,it was confirmed that changing of epigenetic modification in telomerase modified donor cells could promote the process of cell reprogramming3.The acetylation levels of histone H3K18 and H4K16 were analyzed by Westernblot and immunofluorescence(l F).The results showed that compared with control group,the acetylation levels of H3K18 and H4K16 increased in the telomerase modified donor cells group,in which histone modification might promote nuclear reprogramming.4.The RNA-seq of donor cell transcriptional group showed that 250 genes were up-regulated and 740 genes were down-regulated in telomerase modification group.Go enrichment and KEGG pathway enriched analysis showed that the differential genes VEGF,PGF,IGF2,IGFBP3,FYN were mainly related to all levels of cells,tissues,organs and ontogeny,which could activate PI3K-Akt and Wnt signaling pathway.It is confirmed that telomerase modified donor cells might play a positive regulatory role in the process of cell reprogramming.5.In the cloned embryo transcriptome sequencing,it was found that the differentially expressed genes in the telomerase modified donor cells group compared with in vitro fertilized embryos were not significant,only 60 genes up-regulated and 75 genes down-regulated;however,there were 377 genes up-regulated and 988 genes down regulated in the telomerase modified group compared with untreated group.Moreover,screening and analysis of differentially expressed genes showed that telomerase modification could significantly activate the expression of TRF1 and POT1 in SCNT embryos.In summary,the gene expression pattern of telomerase modified donor cells in somatic cell nuclear reprogramming process was basically the same with that of normal fertilized embryos,and could activate many genes related to the development process,thus promoting cell reprogramming.