Effect Of Histones And Their Variants On Early Embryonic Development Of Mouse

Histone variants of H2A and H2B were specially expressed in oocytes and spermatid.These proteins have been related to the formation of early embryonic genome,regulation of early embryonic chromosome structures and gene expression,and early embryonic reprogramming.In this study,the mRNAs of H2A/H2B or TH2A/TH2B were microinjected into pronuclear stage embryos and their functions during early development were analysiedFirstly,the full-length of mouse H2A,H2B,TH2A and TH2B gene were amplified by PCR,and cloned into pGEM-T vector,respectively.Subsequently,the cloned genes were confirmed by sequencing analysis and proved that they were fully lenth.To express the genes in cells,the four genes were cloned into pEGFP-C2 vector by replacing the GFP gene and placing the genes under the drive of CMV promoter,respectively.To synthesis of mRNA in vitro,H2A,H2B,TH2A and TH2B genes were inserted into pSP64 poly(A)vector,respectively.After linearization of the pSP64 poly(A)plasmid DNA,the mRNAs of H2A,H2B,TH2A and TH2B genes were synthesized with SP6 RNA polymerase,seperately.The mRNA of H2A/H2B or TH2A/TH2B genes were microinjected into the pronuclear stage of mouse embryo.And the effects of overexpressed H2A/H2B and TH2A/TH2B on the early mouse embryonic development were analyzed.The results show that after 90 hours in vitro,the blastocyst rate of embryos injected of the H2A/H2B mRNAs was 34.37%,which was significantly lower than the control group(43.92%,P<0.05).It indicates that the early mouse embryo development was inhibited by the overexpression of H2A/H2B.In contrast,the blastocyst rate of embryos injected of the TH2A/TH2B mRNAs was 76.12%,which was significantly higher than that of the control group(43.92%,P<0.05)or the group injected of H2A/H2B mRNAs group(34.37%,P<0.05).This result showed that the overexpression of TH2A/TH2B proteins can promote the early mouse embryo development.Combinant the above data,our results support that H2A/H2B and TH2A/TH2B have different function in the early embryo development,which may be useful for somatic cell cloning and derivation of iPSc.