[Construction of a retroviral vector for RNA interference targeting human telomerase reverse transcriptase]

Area of Science:

• Molecular Biology
• Gene Therapy
• Cancer Research

Context:

• Human telomerase reverse transcriptase (hTERT) is crucial for telomere maintenance and is upregulated in most human cancers.
• RNA interference (RNAi) offers a potential strategy for targeted gene silencing.
• Developing effective delivery vectors for RNAi is essential for therapeutic applications.

Purpose:

• To construct a recombinant retroviral vector capable of delivering small interfering RNA (siRNA) targeting hTERT.
• To validate the vector's functionality in vitro through gene expression and cell viability assays.

Summary:

• A retroviral shuttle plasmid, pLXSN, was modified to create pLXSN-EGFP-U6-siTERT, incorporating sequences for enhanced green fluorescence protein (EGFP), a U6 promoter, and an hTERT-targeting siRNA.
• Successful construction was confirmed by restriction enzyme digestion and sequencing.
• Transfection of NIH3T3 cells showed 24.1% EGFP expression via flow cytometry, while Hela cells exhibited a 53.2% cell death rate 72 hours post-transfection, indicating potent RNAi-mediated growth inhibition.

Impact:

• The successful development of this recombinant retroviral plasmid demonstrates the potential of RNA interference for suppressing hTERT expression in mammalian tumor cells.
• This vector system could serve as a valuable tool for further research into hTERT-targeted cancer therapies.
• The findings highlight the therapeutic promise of RNAi in oncological applications.