Antiangiogenic therapy is a vascular targeted therapy, allowing local targeting of different targets in the tumor, including genes Eng (endoglin) – CD105 and Mcan (melanoma cell adhesion molecule) – CD146. One way to silence excessively expressed genes is the use of RNAi (small interfering RNA) technology. Since RNAi is rather instable, delivery of the shRNA molecule (short hairpin RNA) on plasmids enables prolonged efficacy and is thus more reasonable. The aim of the present work was the construction a plasmid for silencing genes CD105 and CD146 in antiangiogenic cancer therapy, without antibiotic resistance genes. Using molecular cloning methods, including the ORT technology - operator repressor titration, we successfully prepared a resistance marker-free plasmid encoding two shRNA molecules for silencing CD105 and CD146. The plasmid was introduced into the cells by transfection. The successful silencing of two target genes was confirmed by using RT-PCR – real time polymerase chain reaction. The replacement of the resistance gene with the ORT technology ensures a higher safety level according to the standards of regulatory agencies. Marker-free plasmid functionality is maintained or has even increased and is not affected by the reduction in the expression of cloned genes. Additionally, a cytotoxicity assay was employed to demonstrate how the uptake of the plasmid itself affected the survival of the treated cells.
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