Construction of recombinant adenovirus vector with SCL gene by AdMax system and transfecting ICC-like cells and expression of adenovirus vector

Purpose To construct recombinant adenovirus vector containing human stem cell leukemia(hSCL) gene and to observe its ability in transfecting Cajal-like interstitial cells and mediating SCL gene expression.Methods SCL gene was amplified from plasmid with SCL gene by PCR and inserted to the polyclonal site of adenovirus shuttle plasmid pDC315-EGFP.HEK293 cells were co-transfected with the constructed recombinant shuttle plasmid pDC315-EGFP/SCL and large adenovirus helper plasmid pBHGlox(delta)E1,3Cre in mediation of liposome.The obtained replication-defective recombinant adenovirus pDC315-SCL was propagated in HEK293 cells,purified pDC315-SCL plasmid and determined for virus titer.The distribution and efficiency of recombinant adenovirus mediated hSCL was observed by the expression of green fluorescence protein(GFP) under the fluorescent microscope.The expression of hSCL mRNA transfected with Cajal-like interstitial cells was measured by RT-PCR method.Results Western blot inspection and PCR analysis confirmed that the hSCL gene was successfully inserted into the adenovirus vector,with titer of the recombinant adenovirus to 1.0×1010 PFU/ml.The adenovirus had a high transfection efficiency up to 98%.RT-PCR analysis showed expression of hSCL mRNA in Cajal interstitial cells transfected by hSCL.Conclusions The recombinant adenovirus containing human hSCL gene is successfully constructed by homogenous recombination in bacteria and it has a high transfection efficiency and can mediate expression of Cajal-like interstitial cells.