| Background:Rat enhancer of split-and hairy-related protein-2(SHARP-2,also referred to as the Stra13 or DEC1)is a basic helix-loop-helix transcriptional repressor which plays important roles in the transcriptional regulation of cellular proliferation,differentiation,and oncogenesis.Our preliminary studies has shown that TGF-βcan promote SHARP-2 expression,and SHARP-2 transcription factor could promote the secretion of IL-2.besides in Stra13 knockout mice INF-γand IL-2 expression has decreased,thus we speculate if SHARP-2 gene will play an important role in organ transplantation.In order to investigate SHARP-2 gene funtion,we drew assistance from RNA interfering to induce SHARP-2 gene silencing.RNA interference (RNAi)is induced by double-stranded RNA(dsRNA)and results in gene silencing through sequence-specific cleaved by Dicer into 21-23bp siRNA duplexes which are separated by a multi-protein complex called RISC,the antisense strand which remains bound to the RISC (RNA-induced silencing complex)complex will locate homologous mRNA sequences within the cytoplasm and induce cleavage of mRNA,thereby preventing its translation into protein. For RNAi to be effective and elicit gene silencing response,the double strand RNA molecules must be delivered into the target cell.Using vectors to express short hairpin RNAs(shRNAs) that are processed by Dicer into siRNA molecules can also induce target gene silencing.An adenoviral vector-based approach has now been developed for RNAi,which can achieve long-term suppression of gene expression in mammalian cells.We have won the NSFC fund, and this research as a basis for the SHARP-2 gene in organ transplantation research to construct the SHARP-2 gene-specific RNA interference adenovirus vector systems.Objective:To construct recombinant adenovirus(Rad-hSHARP)expressing short hairpin RNA (SHARP-2-shRNA).Methods: 1.A couple of SHARP-2-shRNA template DNA sequences were designed and synthesized. The annealed shRNA templates were inserted into PDC316-EGFP-U6 Shuttle Plasmid to construct the recombinat Plasmids(PDC316-SHARP-shRNA).2.The PDC316-SHARP-shRNA Shuttle Plasmids and the Backbone Plazmid pBHGloxdelE13cre were cotransfected into 293 cells,to construct the reconbined adenovirus(Rad-hSHARP).3.The DNA extracted from the recombined adenovirusand was verified by PCR..4.The SHARP-2 gene silencing with vector of recombinant adenovirus was determined by RT-PCR in NRK cells.Results:1.The successful cloning of SHARP-2-shRNA template DNA sequences into PDC316-EGFP-U6 Shuttle Plasmid was confirmed by the sequencing technique.2.The PDC316-SHARP-shRNA Shuttle Plasmid and the Backbone Plazmid pBHGloxdelE13cre were cotransfected into 293 cells,and the reconminant adenovirus was constructed sucessfully with high titer.3.The analysis of PCR indicated that the recombinant adenovirus Rad-hSHARP containing SHARP-2-shRNA template sequence.4.The success of silencing the SHARP-2 gene with vector of recombined adenovirus was determined by RT-PCR in NRK cells.Conclusion:The recombinant adenovirus which expressing short hairpin RNA targeting SHARP-2 gene had been constructed successfully,which may be used for further investigation of the funtion of SHARP-2 gene silencing with adenoviral vector in poliferation and differentiation of T cells. |
