Study On Expression Of Imprinted Genes,DNA Methylation Binding Protein And Histone Acetylation Enzyme In Transgenic Cloned Goats

Transgenic cloned goats have broad application prospects in the field of medicine,animal husbandry and biological materials.Somatic cell nuclear transfer(SCNT)is one of the most commonly methods of making transgenic cloned animals,which provided a powerful tool to make genetic modification and positive-selecting on the cellular level.In recent years,our laboratory obtained a few batch of cloned goats using SCNT.However,transgenic cloned fetuses were prone to miscarriage during embryo implantation or perinatal period.Even if part of the cloned animals were alive at birth,but they would eventually die from dysplasia.Consequently,the efficiency of transgenic cloning was not ideal.At present,it is generally believed that transgenic cloning inefficiency is mainly due to incomplete reprogramming of donor cells.Therefore,this study proceed from epigenetic reprogramming and was specifically performed on analysis of imprinted genes,DNA methylation binding protein and histone acetylation enzyme in transgenic cloned goats in order to provide theoretical reference for improving the efficiency of transgenic cloning.1.Expression of development related genes in lung tissue of transgenic cloned goatsThe abnormal development of lung tissue is one of main reasons for the failure of most transgenic cloned animals development.In this study,we observed the lung tissue morphology using HE staining,and examined expression of 15 development related genes in lung tissue of deceased transgenic cloned and normal goats of various ages in order to investigate molecular background of lung developmental problems in transgenic cloned goats.The results showed that lung tissues of dead transgenic cloned goats have wide range of inflammatory cells infiltration.Compared with control group?,expression of 13 genes(BMP4,FGF10,GHR,HGFR,PDGFR,RABP,VEGF,H19,CDKNIC,PCAF,MeCP2,HDAC1,and Dnmt3b)decreased in transgenic cloned goats that died after birth(P<0.05);Expression of 8 genes(FGF10,PDGFR,RABP,VEGF,PCAF,HDAC1,MeCP2,and Dnmt3b)decreased in abortion stage of transgenic cloned goats(P<0.05).Moreover,expression of two epigenetic transcription genes(PCAF and Dnmt3b)decreased in disease death of transgenic cloned goats(death in 1-5 months old)(P<0.05).These findings showed that lung tissues of abortion and new born death transgenic cloned goats appeared obvious pathological changes,meanwhile,the abnormal expression of development related genes may associated with lung development defection of transgenic cloned goats.2.Expression of imprinted genes in different tissues of transgenic cloned goatsGenomic imprinting is an important epigenetic mechanism,which have vital effects on growth and development of the fetus.In this study,we observed four tissues morphological features of dead transgenic cloned goats by HE staining.As well,eight imprinted genes were selected to RT-PCR analysis in heart,liver,spleen and kidney of dead transgenic cloned and normal goats.HE staining results of abortion group showed that heart and spleen did not appear obvious morphological changes,while the inflammatory cell infiltration and the characteristics of glomerular nephritis were respectively observed in liver and kidney.In death group,there were inflammation cells in liver,but no apparent structural changes in heart,spleen and kidney.Compared with the control group,in heart tissue,CDKN1C,H19,IGF2R and SNRPN were significantly over-expressed(P<0.05),while XIST was significantly reduced in the abortion group(P<0.05),moreover expression of SNRPN and XIST decreased in the death group(P<0.05);In liver tissue,CDKN1C and DLK1 decreased and GNAS,H19,IGF2R,PEG3 and XIST increased significantly in the abortion group(P<0.05);CDKN1C expression was descendent and expression of GNAS and PEG3 was adscendent in the death group(P<0.05);In spleen tissue,expression of DLK1 was increased while GNAS,H19,IGF2R,PEG3,SNRP,and XIST were decreased in the abortion group(P<0.05),meanwhile,DLK,H19,IGF2R,PEG3,SNRPN and XIST also were decreased in the death group(P<0.05);In kindey tissue,CDKN1C,DLK1,GNAS,IGF2R and PEG3 expression were increased while H19 and XIST were decreased in the abortion group(P<0.05);PEG3 expression was decreased in the death group(P<0.05).Above all,expression of imprinted genes was abnormal in different tissues of transgenic cloned goats.Moreover,the degree of abnormal genomic imprinting was severer in abortion group than death group.These results suggested that abnormal expression of imprinted genes may cause developmental defection of transgenic cloned goats.At the same time,there were abnormal epigenetic modifications existing in reprogramming of transgenic donor cell.3.MBD1 and MeCP2 expression in embryos and different tissues of transgenic cloned goatsDNA methylation as an important epigenetic modification,which plays important roles on the regulation of gene function and maintenance of genome stability.MBD1 and MeCP2 are two members of the MBD subfamily of proteins that bind methylated CpG to maintain the silencing effect of DNA methylation.Given their important roles in linking DNA methylation with gene silencing,this study aimed to characterize the coordinated mRNA expression and protein localization of MBD 1 and MeCP2 in embryos and different tissues and analysis the effect of MBD1 and MeCP2 on abnormal transgenic cloned goats.Compared with the corresponding different development period of the fertilized egg,MBD1 expression of transgenic cloned embryo increased significantly at 2-4 and 8-16 cell stage(P<0.05),then decreased at the morula and blastocyst stages(P<0.05);MeCP2 expression in transgenic cloned embryo was significant decreased at 2-4 cell stage and increased at 8-16 cell stage(P<0.05).The placenta morphology analysis showed that the cotyledon number of death group was significantly lower than the control group and living group(P<0.05).MBD1 and MeCP2 were obviously detectable in the placental trophoblastic binucleate cells and gastrointestinal mucosa epithelial cells by immunohistochemical staining.Moreover,expression of MBD1 and MeCP2 in death group was significant higher than the control group and living group(P<0.05).Compared with the control group,MBD1 expression increased significantly in heart and liver of dead transgenic cloned goats(P<0.05),while MeCP2 expression reduced significantly in liver,spleen,rumen,abomasum,small intestine,colon and rectum of death group(P<0.05).In summary,aberrant expression of methylation CpG binding protein MBD1 and MeCP2 was detected in embryonic development and multiple tissues,which reflected abnormal transcription regulation and DNA methylation involved in MBD1 and MeCP2.This may responsible for abortion and death of cloned transgenic goats.4.HDAC1,HDAC2 and HDAC3 expression in embryos and different tissues of transgenic cloned goatsClass I histone deacetylases(HDAC1,HDAC2,and HDAC3)are recruited by cognate corepressor proteins into specific transcriptional repression complexes that target HDAC activity to chromatin resulting in chromatin condensation and transcriptional silencing.This study aimed to characterize the coordinated mRNA expression and protein localization of HDACs in embryos and different tissues with the purpose of investigating effects of HDACs on development and growth of transgenic cloned goat.Compared with the corresponding different development period of the fertilized egg,expression of HDAC1 and HDAC3 in transgenic cloned embryo reduced significantly at 2-4 cell,morula and blastocyst stage(P<0.05),while raised at 8-16 cell stage(P<0.05).The immunohistochemical analysis showed that HDAC1,HDAC2 and HDAC3 were localized in the placental trophoblastic cells and gastrointestinal mucosa epithelial cells.Moreover,expression of HDAC1 and HDAC3 in death group was significant higher than the control group and living group(P<0.05).Compared with the control group,HDAC1 expression of death group was significantly increased in heart,while decreased in liver,spleen,rumen,reticulum,omasum,duodenum,jejunum and colon(P<0.05);HDAC2 expression of death group was significantly increased in heart and cecum,while decreased in liver,spleen,reticulum,omasum,duodenum and colon(P<0.05);HDAC3 expression of death group was significantly decreased in heart,liver,spleen,rumen,omasum,duodenum,jejunum,colon and rectum,while increased in reticulum and cecum(P<0.05).These results suggested that aberrant expression of HDAC1 and HDAC3 may responsible for alloplasia of transgenic cloned goats.