Clone And Functional Analysis Of Buffalo7SK/U6Promoter, And Establishment Of Anti-Fmdv Transgenic Mouse Model

Buffalo is a multi-usable livestock with excellent properties such as strong adaptability, resistance to high temperature and high humidity, high disease resistance, easy breeding, the roughest of forage, and long service life etc., which are very suitable for southern villages of our country. Foot-and-mouth disease, caused by foot-and-mouth disease virus (FMDV), is one of the most dangerous diseases of cloven-hoofed animals including buffalo. The disease has been designated by the World Organization of Animal Health, as a transmissible disease that has the potential for serious and rapid spread and is of major socioeconomic importance. To study the feasibility of application of RNAi silencing foot-and-mouth disease virus in transgenic buffalo, and to carried out the research of gene silence induced by buffalo RNA polymerase Ⅲ promoters, we isolated and characterized buffalo7SK and U6promoter, and appled them to construct anti-FMDV multi-promoter RNAi transgenic mouse model. In the following the findings in this thesis were listed:Clone and functional analysis of buffalo7SK and U6promoters:Refer to the bovine genome sequence, the specific primers were designed, and buffalo7SK (Genbank accession No. JN417658) and U6promoters (Genbank accession No. JN417659) with length of430bp and357bp respectively were cloned. To compare polIII promoter activity of different species, we also cloned human, bovine and porcine7SK and U6promoters. And each promoter was ligated at5’ of an EGFP-specific short hairpin RNA (shRNA) double-stranded DNA fragments (shEGFP) respectively, and eight EGFP silencing expression vectors (pbu7SK-shEGFP, pbuU6-shEGFP, ph7SK-shEGFP, phU6-shEGFP, pbo7SK-shEGFP, pboU6-shEGFP, pp7SK-shEGFP and ppU6-shEGFP etc.) were successfully constructed. Transfected into buffalo fetal fibroblasts (BFF), the shEGFP were detected to expressing efficiently in cells72h post-transfection as the known promoters by stem-loop RT-PCR, which could express shEGFP as high as257.17%(±33.79%) and174.67%(±32.94%), compared with h7SK-shEGFP group respectively. Co-transfected each expressing vector and pEGFP-N1respectively in buffalo and mouse cells, the fluorescence reducing was observed by fluorescence microscope at60h post-transfection in both cell lines. Furthermore, by flow cytometry analysis of BFF cells72h post-transfection, The mean fluorescence intensities (MFI) show that EGFP silencing efficiencies were significantly higher than other groups in the pbu7SK-shEGFP and pbuU6-shEGFP groups, which were up to93.82%(±0.16%) and87.45%(±0.52%), respectively. Then, collecting cells to extract total RNA, using QRT-PCR for determination of EGFP expression in cells, the comparison of efficiency of shEGFP expression by different promoters shows that the bu7SK (93.29%±1.30%) and buU6(93.45%±1.25%) promoters induced the greatest level of suppression than other species derived promoters in buffalo cells, but bu7SK promoter showed excellent efficiency among the promoters in mouse cells, approximately91.29%(±1.03%). By sequence analysis on molecular level, detection of function and efficiency on cellular level, we proved that the buffalo7SK and U6promoters cloned from buffalo genome are the efficient pol III promoters with RNAi function.Anti-FMDV multi-shRNAs RNAi vectors construction:First, three shRNAs designed according to the conservative area in3B and3D regions of foot and mouth disease virus, and chemical synthetized as a fragment with the three shRNAs in a series. Buffalo7SK promoter, buffalo U6promoter and bovine U6promoter were ligated by corresponding enzyme site respectively, to construct bu7SK-Consl-buU6-Cons2-boU6-Cons3three tandem shRNA expression cassette. Then the cassette was connected into the lentivirus vector plasmid pSicoR constructing pSicoR-3shRNA FMDV-specific transgenic vector, in which provides a report gene EGFP induced by CMV promoter. The FMDV-specific lentiviral vector was packaged by co-transfecting the three plasmid lentivirus packaging system pNRF, pVSVg and pSicoR-3shRNA into293T cells. Then the transgenic cells were obtained by infecting lentivirus into BHK-21. We further detected the expression of3FMDV-specific shRNAs in tg cells by stem-loop RT-PCR. In vector functional tests, we firstly inoculated O type foot-and-mouth disease virus into BHK-21tg cells and observed and compared the inhibition of foot-and-mouth disease virus replication between normal and tg cells within48h post-infection. The results show that transgenic cells within6h and48h post-infection has obvious inhibition to foot-and-mouth disease virus replication, compared with normal cells, the best inhibiting efficiency in24h post-infection, as high as67.48%; Secondly, we infected FMDV type O strain with1000,100,20, and5LD50titer into3-5days sucking mice respectively, which pre-treated lentiviral vector (LV) or PBS for negative control (NC), observed death time and quantity of sucking mice. According to the results,2mice died of disease under5LD50titer of NC group72h post-infection, and no mouse survived under20LD50titer; in LV group there was no mouse died under5LD50titer, also there were still three mice lentivirus pretreated survived to7days under20LD50titer. Moreover LV group, no matter under which titer, survival time of the dead mice has extended compared with NC group. This study proved that this anti-FMDV multi-shRNAs expressing transgenic vector could improve mice resistance toward foot-and-mouth disease virus, avoid the sucking mice from death under5LD50titer effectively, shows obvious good performance of resistance to the toxicity of foot-and-mouth disease virus.Production of anti-FMDV multi-shRNAs RNAi transgenic mouse model: The purified anti-FMDV multi-shRNAs expressing cassette bu7SK-Consl-buU6-Cons2-boU6-Cons3-CMV-EGFP was micro-injected into mice zygotes. After microinjection and embryo transfer,7positive transgenic mice were obtained by detection of PCR from27born cubs. Choose two of tg parental generation mice which expressed EGFP feeding to the sixth or seventh founders generations respectively. The tg mice per litter in each generation accounted for about50%～60%stably detected by PCR and Southern blotting, which prove that the integrated DNA fragment of our tg mice has good genetic stability. We observed the organs frozen sections of F3offsprings of FO-7and FO-14by laser confocal microscopy, found that in the heart, spleen, kidney tissue of FO-14mice offspring expressed stronger fluorescence, on the contrary, the only EGFP expression of spleen in FO-7mice offspring was slightly stronger than the former. We further determined expression of the three FMDV-specific shRNAs in the two founders, the expression levels of three shRNAs in F0-14’s liver were higher than in the F0-7’s. For the reason, we select offspring of FO-14as a representation of the positive transgenic mice in antiviral detection experiments. In antiviral detection test, sucking mice median lethal dose5,20,100and1000LD50of FMDV serotypes O for tg and normal sucking mice respectively. The FMDV serotypes O was administered the subcutaneous (SQ) neck injection as two0.1ml doses per challenged mouse. Neither tg nor negative sucking mice survived at72h post-infection under1000LD50,100LD50and20LD50titers. However, the death number of tg group was lower than negative group under5LD50titer. To confirm the result,20mice each group (10for20LD50and10for5LD50) were infected by FMDV to compare the survival rates between tg and normal sucking mice. Then the result showed that only3of normal mice had surviving under5LD50titer (30%), while8of tg mice had surviving (80%) under the same titer. The carcass, heart, liver and kidney of the mice were selected for FMDV quantitative detection by Q-PCR post-death or72h post-infection. Results showed that in organs in dead tg mice were no less than negative group, but the viral RNA in the survival mice in tg group seemed slightly less than normal mice.Conclusion, this thesis provided a powerful experimental basis to further generation of anti-FMDV transgenic buffalo. The RNAi stragtage used in the thesis would also be used to improvement reproduction and lactation traits of buffalo in knocking down other functional genes, which could meet the demand of new buffalo breeding plan of Guangxi province.