ORIGINAL_ARTICLE The aim of this study was to construct two plasmid-specific shRNA transcripts of the bcl-2 gene in order to prepare for reverse of cell apoptosis. The plasmid was designed according to a previously published sequence of interfering RNA following an appropriate reference, using appropriate software. By annulling complementary oligonucleotides, doublestranded inserts were formed. Recombinant shRNA-encoding plasmids were constructed by digestion of psiRNAx7SKGFPzeo plasmid (psiRNA-x7SKGFPzeo, with restrictive endonuclease BbsI electrophoresis in ultra-pure agarose with low melting point (LMP-Agarose). For each of the constructs, a suitable double-stranded insert downstream of x7SK (strong RNA III promoter) with T4 DNA ligase was cloned. The control plasmid psiRNAScr was used directly for transformation. The PC-3 cell lines were transfected with 2 plasmids, psiRNA-Bcl-2 and psiRNAScr to suppress the bcl-2 gene construct. The results have shown that the lowest level of bcl-2 genes was 48 h, and even lower 72 h after the transfer, and the mRNA levels returned to normal in 120 h. An increase in the percentage of cells with spontaneous apoptosis has been observed with successful inhibition of the bcl-2 gene. The induction of apoptosis in transfected cells increased the percentage of necrotic cells proportionally. The percentage of apoptotic cells transfected with psiRNA-bcl-2 plasmid increased proportionally to the increase of hydrogen peroxide concentration. The transfection of the PC-3 cell line from prostate cancer with constructed shRNA plasmid has induced suppression of bcl-2 gene expression versus control Scr plasmid. Suppression of bcl-2 gene expression significantly increased cell sensitivity to apoptosis induction. https://macvetrev.mk/Files/Article/2022/10.2478/macvetrev-2022-0028/macvetrev-2022-0028.pdf 2022-10-15 187 199 10.2478/macvetrev-2022-0028 shRNA plasmid Bcl-2 apoptosis cytotoxicity prostate cancer Igor Esmerov iesmerov@gmail.com false 1 Faculty of Veterinary Medicine – Skopje, Ss Cyril and Methodius University in Skopje, Lazar Pop Trajkov 5-7, 1000 Skopje, North Macedonia LEAD_AUTHOR Branko Atanasov false 2 Faculty of Veterinary Medicine – Skopje, Ss Cyril and Methodius University in Skopje, Lazar Pop Trajkov 5-7, 1000 Skopje, North Macedonia AUTHOR Aleksandra Angeleska false 3 Faculty of Veterinary Medicine – Skopje, Ss Cyril and Methodius University in Skopje, Lazar Pop Trajkov 5-7, 1000 Skopje, North Macedonia AUTHOR Radmila Crceva false 4 Faculty of Veterinary Medicine – Skopje, Ss Cyril and Methodius University in Skopje, Lazar Pop Trajkov 5-7, 1000 Skopje, North Macedonia AUTHOR Ljupco Mickov false 5 Faculty of Veterinary Medicine – Skopje, Ss Cyril and Methodius University in Skopje, Lazar Pop Trajkov 5-7, 1000 Skopje, North Macedonia AUTHOR Ljupco Angelovski false 6 Faculty of Veterinary Medicine – Skopje, Ss Cyril and Methodius University in Skopje, Lazar Pop Trajkov 5-7, 1000 Skopje, North Macedonia AUTHOR Slavica Josifovska false 7 Laboratory for Molecular Biology, Institute of Biology, Faculty for Natural Sciences and Mathematics, Ss Cyril and Methodius University in Skopje, 1000 Skopje, North Macedonia AUTHOR Nikolaj Markov false 8 Institute of Mountain Animal Husbandry and Agriculture, Agricultural Academy-Sofia, 5600 Troyan, Bulgaria AUTHOR Nikola Adamov false 9 Faculty of Veterinary Medicine – Skopje, Ss Cyril and Methodius University in Skopje, Lazar Pop Trajkov 5-7, 1000 Skopje, North Macedonia AUTHOR Siegel, R.M., Frederiksen, J.K., Zacharias, D.A., Chan, F.K., Johnson, M., Lynch, D., Tsien, R.Y., Lenardo, M.J. 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