| RNA interference (RNAi) is a strong gene silencing technology, providing a new view to investigate the interaction between virus and its host. This technique behaves a wide application prospect in antiviral area. Porcine reproductive and respiratory syndrome (PRRS) has been one of severe diseases which caused huge economic loss in swine industry. It has been indicated that RNAi could exist during the replication of PRRSV. The plasmid and viral vector expressing shRNA targeting PRRSV genome has been constructed successfully, which provide promising compelling new approach for the prevention and treatment of PRRS. With the increasing development of mammalian embryo bio-technology as well as its combining with modern molecular biological technique, it becomes possible to carry out transgenic anti-disease breeding via using genetic modification. Transgenic clone is an advanced technology, with which transgenic somatic cells are used as nuclear donors to produce transgenic embryos or animals. Combination of RNAi with transgenic cloning technology to produce transgenic animals hasd been reported on animal disease models for human medicine or animal anti-disease breeding. To date, there are no reports on the production of either transgenic embryos or transgenic animals with resistance to PRRS. The low efficiency of clone and unstability of transgene expression are the main problems of transgenic cloning. Recently studies suggested that, such as DNA methylation and histone acetylation, could affect directly the cloning efficiency and transgene expression during reprogramming process of somatic cells. In this study, we produced porcine transgenic cloned embryos resisting PRRSV through vector-based shRNA and somatic cell nuclear transfer technology. To decipher inner mechanism of regulation between developmental reprogramming of somatic cells and epigenetic modification, we would study the effects of Trichostatin A on the development and exogenous gene expression of transgenic cloned embryos. The methodology we have set up could obtain high SCNT blastocyst developmental rate, which will establishes a basis for the production of porcine transgenic cloned pigs resisting PRRSV. This study contains the following4parts:Experiment1. Construction and functionality test of transgenic vector expressing shRNA targeting against PRRSVThe purpose of the experiment is to construct the transgenic vector expressing shRNA targeting against PRRSV, and verify its suppressive effect of PRRSV replication in Marc-145cells, which establish the foundation for production of cloned embryo or porcine conferring PRRSV resistance. According to the map of vector, pSUPER-shRNA plasmids were used as a template to produce shRNA-expression cassettes by PCR and to generate EcoO1091restriction enzyme sites (AGGCCCT). And then shRNA-expression cassettes were inserted into the EcoO1091site of pEGFP-N1. The recombinant plasmid pEGFP-G1were identified with restriction enzymes digestion and PCR amplification. After24h the pEGFP-G1transfected into Marc-145cells, transfected cells were infected with PRRSV, the observation of cytopathic effect (CPE)showed that PRRSV replication could be inhibited by pEGFP-Gl in MARC-145cells significantly as compared to the controls.Experiment2. Effects of nuclear donor cells on in vitro production of porcine transgenic cloned embryosThe purpose of this experiment is to compare the effect of different donor cells expressing shRNA on the development competence of porcine cloned embryos, including porcine fetal fibroblast (PFF), porcine ear fibroblast (PEF) and PK-15. The PFF were cultured with tissue explant and collagenase digestion, the results showed that PFF derived form tissue explant could form monolayer formation within5-7days, while only3days were needed with collagenase digestion. Meanwhile, PEF derived form tissue explant would proliferate mostly at5-7days, form monolayer formation within9-10days. Then PFF, PEF and PK-15were transfected with pEGFP-Gl,respectively, cells were selected with G418600μg/mL. G418-resistant colonies expressing shRNA were expanded respectively as donor cells for production of porcine transgenic cloned embryo. Results showed that, the blastocyst rate of clone embryo reconstructed with PFF was significantly higher than other two group (26.6%vs12.9%,0%; P＜0.05). Our study demonstrated that cloned embryo reconstructed with PK-15could not develop at blastocysts, so we concluded that PFF is more suitable as donor cell for production of porcine transgenic cloned embryos.Experiment3. Study on in vitro developmental competence of the porcine transgenic cloned embryosThe purpose of this experiment is to investigate the effects on the development of porcine transgenic cloned embryo with different culture media, activation treatment and Trichostatin A. Results showed, there was no significant difference among the cleavage rates of porcine parthenogenetieally activated embyros cultured with NCSU-23and PZM-3culture media(72.1%vs75.0%, P>0.05), the blastocyst rate of PZM-3was significantly higher than that of NCSU-23(33.3%vs20.9%), PZM-3culture media is more suitable to culture embryos. Comparing activation treatment between electrical activation and electrical activation adding6-DMAP process, there was no significant difference among the blastocyst development rate. The experiment were divided into4groups with TSA treatment, donors treated with50nM TSA(Group A), reconstructed embryos treated with50nM TSA(Group B), both donors and reconstructed embryos treated with TSA(Group C), and no treatment designed as control(Group D). Result showed that blastocyst development rate between Group B and Group C was significantly higher than that of Group A and Group D(respectively,33.0%and35.7%vs15.8%and20.0%;P＜O.05).Experiment4. Effects and mechanisms of Trichostatin A on the developmental competence of porcine transgenic cloned embryosTo assess the effect of epigenetic modification of donor cells and reconstructed embryos on developmental reprogramming of transgenic cloned embryos and explore the mechanism on developmental regulation of transgenic cloned embryos, the experiment were divided into4groups:Donor cells of Group A were treated with50nM TSA for24h before SCNT. Reconstructed embryos of Group B were treated with50nM TSA for24h after activation. Both donor cells and reconstructed embryos in Group C were treated with TSA and Group D were the control without TSA treatment. The acetylation status of H3K9(AcH3K9) and the mRNA expression levels of histone deacetylase gene Hdacl, methylation related gene Dnmtl mRNA, pluripotency-related genes Oct4, anti-apoptotic gene Bcl-2, and transgene EGFP, were analyzed by real-time PCR at different stage of in vitro development in the four groups. Results showed that the effect of TSA on reconstructed oocytes modified the acetylation status at the2-cell stage, which would significantly enhance the development of porcine SCNT embryos in vitro, but not donor cells. The treatment of reconstructed embryo with TSA could increase expression of the genes Oct4at the4-cell and blastocyst stage, stimulate expression of the gene Bcl-2at the4-cell stage and blastocyst stage and decrease expression of gene Dnmtl. All TSA treatment could increase expression levels of EGFP comparing to the control, likely preventing the silence of EGFP. The present experiments showed that TSA could promote development in vitro of porcine transgenic cloned embryos possibly via increasing histone acetylation and anti-apoptotic abilities of those embryos. This is the first report on the production in vitro of porcine transgenic cloned embryos with resistance to PRRSV, which establishes a basis for further pig transgenic anti-desease breeding. |
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