Study On Diferential Expressed Genes And Their Function During Development Of Bovine Preimplan-tation Embryos

Single preimplantation embryo mRNA differential display(SPEDDRT-PCR) was used to select embryonic development related genes of8-cell and blastocyst stages embryos stage bovine embryos cultured in vitro. The overexpression vector and interference vector of selected genes were constructed and transfected into bovine fetal fibroblast cells (BFFCs) by using lipofectamine or electroporation in order to establish the transgenetic cell lines. The transgenetic cells were used for the nuclear donor to construct overexpressed and kncked-down transgenetic embryo models for the advanced study of the function of selected genes.1. Selection of different expression pattern genes in bovine8-cell embryos and blastocystSingle preimplantation embryo mRNA differential display (SPEDDRT-PCR) was used to select embryonic development related genes of8-cell and blastocyst stages embryos stage bovine embryos cultured in vitro. Six specific fragments were selected for further analysis. The results of comparation sequneces in GenBank showed that two fragments had high homology with LYAR gene and RPL31gene respectively.LYAR and RPL31expressed in the8-cell and blastocyst stages embryos by RT-PCR detection; the mRNA of LYAR and RPL31were46and3.2times higher in8-cell stage embryos respectively than blastocysts by real-time quantitative PCR detection.2. The construction of vectors pIRES2-EGFP-LYAR/RPL31and pGCsi-LYAR/RPL31.(1) The LYAR/RPL31coden sequences were cloned from bovine8-cell embryos total RNA by RT-PCR and linked with pIRES2-EGFP between BglⅡ and SalⅠ sites to construct the overexpression vectors pIRES2-EGFP-LYAR/RPL31. Sequencing results and restriction endonucleasa assay showed that the structures of two vectors were consistent with expectation.(2)Based on5'-CAGAGGCCAAGAAGAATAAGA-3'and5'-CACAAG CGCATCCATGGAGTG-3'areas of LYAR/RPL31genes, hairpin interference fragments were synthesized respectively and cloned into pGCsi-DsRed to construct the interference vector pGCsi-LYAR/RPL31.3. LYAR/RPL31gene transfection and identity of transgenetic clonesAttaching tissue explantion was used to isolate bovine fetal fibroblast cells(BFFCs) and cells were cryopreserved after purification and amplification for several passages. The growth curves of5th and10th passages cells were plotted and5st passage cells karyotype were analyzed. Meanwhile, G418toleration of the BFFCs was examined and the optimal screening concentration of G418was800μg/ml. Based on results of above exmainations,3rd passage cells were used for gene transfection.The BFFCs were transfected using the electroporation and lipofection respectively and the effects of transfection were detected by real-time quantitative PCR. Compared with the negative control, the expression of LYAR/RPL31in pIRES2-EGFP-LYAR/RPL31transfected cells were significantly increased(31.23folds and45.99folds respectively, P<0.05); and the expression of LYAR/RPL31in transfection by pGCsi-LYAR/RPL31were significantly decreased (0.047folds and0.068folds respectively, P<0.05).800μg/ml G418was added into the culture medium after48hours last1week. After that, the concerntation of G418decreased to300μg/ml for further screening. The green and red fluorescences were observed respectively.4. The function investigation of selected genes and production of bovine transgenetic embryosThe growth curves of the transgenetic cells and BFFCs were drawed respectively. Compared with BFFCs and pIRES2-EGFP-LYAR/RPL31, pGCsi-LYAR/RPL31transgenetic cells growth speed was slow down significantly. The cell cycle analysis results showed that the portion of G2/M cells increased significantly (P<0.05) in pGCsi-LYAR transgenetic cells.The cloned blastocysts were prepared by somatic cell nuclear transfer, using the transgenic cells as the nuclear donor cells and enucleated oocytes matured in vitro as the recipient cytoplasm. In order to make sure the proper method of activation and development culture of reconstructed embryos, activation and development culture of parthenogenetic oocytes were carried out. The oocytes matured in vitro were activated with7%ethanol for7minutes followed by incubation in SOF+BSA medium containing2mM6-DMAP for4hours. After the activation the embryos were cultured in SOF-BSA medium for36hours, the cleaved embryos (cleaved rate was74.9%) were co-cultured with cumulus cells in SOF medium containing4%FBS for7to9days. The blastocyst formation rate was24.9%. The results indicated the procedure could be used for activation and in vitro development of reconstructed embryos. The results showed that there were no significant difference on blastocyst rates between IRES2-EGFP-LYAR/RPL31cloned embryos and SCNT embryos (26.1/25.0and27.9respectively), however, blastocyst rates of pGCsi-LYAR/RPL31Cloned embryos (10.3/11.7) were significant lower than that of SCNT embryos. Cell number of blastocysts in pGCsi-LYAR/RPL31Cloned embryos were significant lower than that of SCNT embryos(65/73and93, P<0.05), but there were no significant difference between IRES2-EGFP-LYAR/RPL31cloned embryos and SCNT embryos(97/92, respectively). GeXP-PCR assay results showed that cell growth genes TP53, GATA2, CDX2, DNMT3A and OCT4, NANOG expression were decreased owing to down-regulation LYAR or RPL31in blastocysts, howere, BCL-2expression were increased.