[Touchdown PCR and overlap extension PCR for generating CD133(+) cancer stem cell-selective adenovirus vector]

To construct a replication-incompetent adenovirus vector targeting cancer stem cells by modified touchdown PCR and overlap extension PCR and investigate its infection efficiency in CD133(+) SW480 cells in vitro.

The two portions of the fiber gene encoding the Ad5 fiber knob domain with the HI loop deleted were amplified using two pairs of designed primers and then linked by overlap extension PCR. The product obtained was identified by sequencing and inserted into prokaryotic expression vector pEGFP-N1. The product, pEGFP-N1 KNOBδHI, contained a unique EcoRV restriction site in the deleted portion of the sequence encoding the HI loop. The gene sequences of the adenovirus fiber were amplified using both common PCR and overlap extension PCR, then identified by sequencing and inserted into pNEB193, resulting in pNEB-F5. CD133(+) SW480 cells were infected with the generated adenovirus vectors Ad5-GFP and Ad5FHI-GFP to investigate the infection efficiency using fluorescent microscope.

The target fragments of expected sizes were amplified by touchdown PCR and overlap extension PCR, but not by common PCR. Ad5FHI-GFP showed a higher infection efficiency than Ad5-GFP in CD133(+) SW480 cells.

Compared with common PCR, touchdown PCR and overlap extension PCR can significantly improve the specificity and efficiency of the PCR products for constructing CD133(+) cancer stem cell-selective adenovirus type 5 vector, which provides carriers for tumor-targeted gene therapy.