| DNA methylation is one of the most important epigenetic markers; it has beenwell characterized and demonstrated to be involved in many biological processes,including transposable element silencing, genomic imprinting and X chromosomeinactivation. However, the mechanism underlying DNA demethylation is largelyunknown. Recently, the functions of the Ten-eleven translocation (Tet) family ofmethyldioxygenases (Tet1, Tet2and Tet3) have been implicated in DNAdemethylation via converting the5-methylcytosine (5mC) of DNA to5-hydroxymethylcytosine (5hmC). iPSCs exhibit many similarities with ESCs.Despite their undoubted promise in regenerative medicine, many obstacles must beovercome before using these cells as a source of transplant material; there is anespecially strong need for animal models whose anatomy and physiology betterresemble these of humans than do those of the mouse to test the efficacy and thesafety of the iPSCs transplantation.The process of reprogramming somatic cells to iPSCs is accompanied bychanges in DNA methylation and certain epigenetic changes. Thus piPSCs provide afavorable system in which to study the epigenetic features that are prerequisite forpluripotency. In this study, we derived piPSCs and investigated whether Tet1playedan important role in the epigenetic regulation of these cells. The main results are asthe follow:1. Porcine somatic cells were reprogrammed into iPS cells.(1) Fetuses were firstobtained from Yorkshire pigs that pregnanted35day, and then embryonic fibroblaswere isolated and establishedt from the fetus;(2) pMXs retroviral vectors encodingmouse Sox2, Klf4, Oct4and c-Myc (SKOM) cDNAs were individually used toco-transfect the293T cells with Vsvg, and then retroviruses that specially expressingembryonic factors were obtained.(3) When fibroblasts were rerogrammed usingretroviruses, colonies appeared on approximately the9th day after transduction.However these colonies exhibited a dispersed and amorphic appearance. On day14, the typical ESC-like colonies appeared. The iPS cells were obtianed after colony werepicked mechanically.(4) The iPSCs were characterized using common PCR, and theresults indicated the expression of the pluripotency genes Nanog, Oct4, Sox2andLin28. Immunofluorescence staining demonstrated the expression of pluripotentmarkers OCT4, SOX2, NANOG, REX1and SSEA4in piPSC colonies.(5) Usingdrop culture, iPSCs can form EBs. When EBs were differenatiated for a longer time,they attached to the substratum, began to spread, and displayed overt signs ofdifferentiation. The expression of three embryonic layers markers (Endoderm, Afp;Mesoderm, Bmp4; Ectoderm, Gfap) were detected.(6) Teratoma formed after5×106piPSCs were injected into NOD/SCID mice after4weeks, and tumors containedtissues derived from three germ layers, including ectoderm-derived neural epithelium,mesoderm-derived striated muscle and blood vessel, and endoderm-derivedcryptae-like structures.2. Effects of Tet1on genes expressions in iPS were studied.(1) After RNAs ofiPS were extracted and inverse transcripted into cDNA, common PCR were used todetected the expression of Tet1, and its expression was further demonstrated withsquencing and NCBI blast; Immunofluorescence staining that conducted using specialantibody for TET1demonstrated that its expression on protein levels; Western blotthat conducted using special antibody for TET1also demonstrated its expression onprotein levels;(3) Transfecion conditions were optimized using FAM-labeled siRNAafter screened for FAM-positive cells. It was determined that2ul:30pmol siRNA usingES mediam in a well of24well plates had the highest efficiency.(3) Four smallinterference RNA were chemically synthesized and transfected into iPS, and the resultshowed that two siRNA can significantly down-regulated the expression of Tet1, andone siRNA had the highest efficiency.(4) Striking morphological changes wereobserved after the down-regulation of Tet1. The iPS lost its self-renewing status andbegun to differentiate.(C), whereas no similar results were obtianed in other groups (5)After the down-regulation of Tet1, some genes expression was affected in iPS.RT-PCR assays demonstrated that the expression of differentiation-related genes suchas Pitx2, Hand1, Gata6and Lef1were up-regulated, whereas pluripotency-relatedgenes such as Lefty2, Klf2and Sox2were down-regulated, and Oct4, c-Myc, Klf4andNanog were not down-regulated.3. Effects of Tet1on methylation status in iPS were studied.(1) Using glycosylation, digestion and PCR, it was demonstrated that5mC,5hmC andunmodified C existed together in promoters of Oct4, Sox2, Klf4and c-Myc. Thereforeit demonstrated the coexistence of3types of C in promoter regions of genes.(2) Themethylation status in iPS were detected with the method of bisulfite treated PCR. Theresults indicated that methylation status in promoters of pluripotent genes wereaffected after down-regulation of Tet1. by siRNA, but the effect was locus-specific.The levels of methylation in the promoters of Oct4and c-Myc increased obviously,whereas it was barely changed for Sox2and Nanog, and slightly increased for Klf4.(3)The total5mC and5hmC on genome-scale were measured using Dot Blot, and thetotal5mC and5hmC on genome-scale were affected after down-regulation of Tet1bysiRNA. The5hmC levels of the whole genomes decreased after Tet1siRNAinterference, whereas the5mC levels of the whole genomes increased... |
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