Affects Of MiR-34c And MiR-449b On The Development Of Early Bovine Somatic Cell Nuclear Transfer Embryos

Originally it was thought that sperm deliver paternal haploid chromosome and fertilize the oocyte atferlitization, whereas oocyte provides the regulatory factors that guide embryonic development afterfertilization.The result is that most researchers focus on oocytes when searching for and identifying theregulatory factors in regard to embryonic development and epigenetic reprogramming.With the in-depthunderstanding of sperm, sperm has been shown to deliver a small amount of proteins, mRNAs, miRNAsand other non-coding RNAs, etc, into fertilized oocytes.The substances delivered during fertilization bysperm play an important role in the formation of male and female pronuclei, the temporal order ofblastomere division, the conversion of zygotic genes, embryonic development even offspring phenotypeand so on.After fertilization, oocytes posses the capability of nuclear reprogramming with sperm, in orderto ensure embryogenesis. This form of sperm reprogramming gained from oocyte can be applied to somaticcell nuclear by nuclear transfer (NT).However, the efficiency of somatic cell nuclear reprogramming is lessthan than of the sperm nucleus. The study gives way to induce the sperm kind of chang by inducingexpression, followed by preparation of somatic cell clone embryos through screening the sperm-specificmiRNAs, as the preliminary exploration of the sperm-borne miRNAs in bovine functioning on bovinesomatic cloned embryos in the early development.(1) Comparing the expression of miR-34c and miR-449b in bovine fetal fibroblast cells, sperm andMII oocytes by qRT-PCR, confirming that miR-34c and miR-449b are higher expressed in sperm than thatin somatic cells and oocytes.(2) Construction of miR-34c, and the miR-449b3G Tet-On induced expression vector followed byscreening the positive clones used as donor cells. Doxycycline (Doxycycline, Dox)-induced experimentidentify the feasibility of miRNAs inducible expression vector.(3) Take miR-34c fibroblasts by dox-induced expression as donor cells. The experimental group iscloning embryos by NT technology and the control group is none of Dox.The result indicting that thesperm-detrived miR-34c can improve the ability of embryonic development, nuclear reprogramming andembryo quality by inducing the expression of form.(4) the completion of construction of psiCHECKTM-2-3’ UTR luciferase reporter vector, furtherexploring the impact of miR-34c/miR-449b on the related predicted target genes, causing the decreasingexpression of Myc, HDAC1, E2F5, PDCD2, which needs for further proof yet. The role of miR-34c in SCNT early embryonic development and construction of the miR-449b3GTet-On induced expression vector is preliminary confirmed, followed by the further improving theefficiency of nuclear transfer and clarify the mechanism of epigenetic modification anomalies.