Construction of mouse Slfn3 recombinant adenovirus vector and determination of transfection efficiency in EPCs
Objective To develop and evaluate a recombinant adenoviral vector expressing mouse Schlafen3(Slfn3)in endothelial progenitor cells(EPCs).Methods The mouse Slfn3 gene sequence was retrieved from GenBank and amplified using PCR with primers designed by VectorNTI software.The amplified gene was inserted into the pcADV-EF1-mNeonGreen-CMV-MCS-3xFLAG shuttle vector.Following plasmid extraction from positive clones,construct validation was performed using EcoRI/BamHI restriction digestion and DNA sequencing.The recombinant adenovirus was generated using the Admax system in HEK293 cells.Viral titer was determined and Slfn3 expression was confirmed by Western blot analysis.Mouse spleen-derived EPCs were cultured to 70％confluence and transfected with the recombinant virus.Transfection efficiency was assessed by fluorescence microscopy after 48 hours.Results The recombinant adenoviral vector was successfully constructed as confirmed by DNA sequencing.The viral titer reached(3.16×1010)ifu/mL.The vector effectively transfected HEK293 cells,producing a 57 kDa Slfn3-EGFP-3xFLAG fusion protein.EPC transfection efficiency was(71.43±2.58)％.Conclusion A functional mouse Slfn3 adenoviral expression vector has successfully been generated,and demonstrated high transfection efficiency in EPCs.
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维普
