The Effect Of PRMT5on Dairy Goat Somatic Cell Reprogramming

Embryonic stem(ES) cells provided a crucial tool for manipulating embryos to studygenetics, development and physiology.With the advent of equivalent human ES cells, havenow opened new vistas for regenerative medicine. While there is a lack of embryonic stemcells for most of mammalian. The goat is an important domesticated animal. Genemodification of goats can improve their production traits, ability to resist disease, and abilityto produce valuable and high-quality proteins. It can be widely applied in the fields ofagriculture and biopharming. However, traditional transgenic techniques result in extremelylow efficiency. It greatly limits the applications of genetically modified goats. Due to littleknowledge of the characteristics and culture conditions of ES cells from goat, it has beendifficulty to isolate and maintain ESCs from the embryos of goats. Pluripotency could beobtained by reprogramming of somatic cells with defined factors. More importantly, mouseinduced pluripotent stem (iPS) cells have been demonstrated as an efficient vehicle for geneticengineering. It give us an idea that goat iPS cells may be used as goat ES cells. In the past fewdecades, no authenticgoat iPS line has been reported, because there have too muchmechanism we do not understand.In cellule, arginine methylation governs important cellular processes. It can impactdifferentiation and development, as well as growth and proliferation. And it has beenpredicted that over1%of genes encode methyltransferases in the mammalian genome. Theyhave the ability to methylation of histone and non-histone proteins, and can be catalyzedsymmetric or asymmetric. Most of them can regulate chromatin structure and expression of awide spectrum of target genes. Deffert from other protein arginine methyltransferase, PRMT5can work with a variety of cellular proteins to induce epigenetic silencing and other functionin vivo and in vitro. Now, there are much of work on PRMT5have shown that it control thegrowth-promoting and pro-survival pathways. As an enzyme, PRMT5is involved in bothepigenetic regulation of anti-cancer target genes and organelle biogenesis. It is shown that PRMT5in combination with classical Yamanaka factors can promotemouse somatic cell reprogramming, consistent with findings from studies of earlydevelopment. In ES cells, knockdown PRMT5leed to pluripotency lost. However, themechanism remains unknown. Recentley, a type I PRMT inhibitor combination with a TGF-βinhibitor was recently identified enabled OCT4-induced mouse embryonic fbroblast (MEF)reprogramming.This showed that type I PRMTs might not enhance the reprogrammingprocess, which suggests that PRMT5and one or more type I PRMTs might have opposingfunctions in somatic cell reprogramming.The PRMT5CDS region (1920bp) was cloned from Guanzhong dairy goat testiculartissues, and identified by double digestion using Xba1/BamHl restriction enzymes. The resultsshown that we successfully constructed pCDH-CMV-PRMT5-EF1-GreenPuro lentiviralvector. Further, the structure of dairy goat PRMT5protein was predicted by the Swiss instituteof bioinformatics online software analysis. The results showed that goat and bos PRMT5protein structure were presented as the same structure.Over expression lentiviral vectors containing four human reprogramming factorsOct4/Sox2/Klf4/C-Myc(OSKM). The AP positive cells were appeared at7days, andgradually formed the bigger, and compact colonies. ES-like colonies were formed and pickedup at30-40days after transduction. The colonies reprogrammed with OSKM werecharacterized by a well defined borders.Within3-4passages, the typical iPS-like cell colonieswere identified by RT-PCR and immunofluorescence analysis. RT-PCR results showed thosecells were positive for Sox2, Klf4, Oct4, C-Myc and Nanog. Immunofluorescence resultsalso conformed that those cells were positive for pluriptent markers:SSEA1, SSEA4, C-Mycand Oct4. To test the potentiality of the iPS-like cell colonies derived POSKM induced, thecells were cultured in suspension for2days, and the cells spontaneously differentiated intotypical embryonic bodies (EBs), then transferred them into a petri dish containing the normalcell culture medium without LIF and bFGF for7days, the differentiated cells expressed thespecific markers of different germ layers including α-Actin(mesoderm marker),NSE(ectoderm marker)，Vasa(germ cell marker) and PDX1(endoderm marker).It is shown that PRMT5can mediates P53methylation,which dispose the cell to arrest.While silencing of p53significantly increased the reprogramming efficiency of humansomatic cells. We infer that PRMT5may through mediates P53methylation enhance thereprogramming. We checked the suppose in goat GEF cells.Here, we explored the PRMT5expression profiles in dairy goat and their effects on the derivation of iPSCs from dairy goatembryonic fibroblasts(GEF). The results showed that PRMT5is a conservative gene andexpressed widely in various tissues and different ages’ testes. PRMT5overexpression in combination with OCT3/4, SOX2, KLF4and c-MYC (POSKM) could significantly increasedthe number of AP positive iPS-like clonies derived-GEF compared with OSKM alone in dairygoat. Moreover, our results demonstrated that PRMT5overexpression stimulated GEFproliferation, and down-regulated P53, P21(a target genes of p53) and apoptoticmarker-Capase3to enhances the somatic cell reprogramming.