Retroviral Mediated Gene Transduction: A Gene Transfer Technique to Deliver a Transgene in Cultured Cells Using Engineered Retroviral Vectors
Transcript
To transduce host cells with RNA containing retroviral vectors, take a tube containing packaged vectors in suitable media. Each vector contains a viral capsid, containing the transgene's RNA complement to be integrated into the host genome and important viral enzymes. A lipid bilayer surrounds the capsid to facilitate entry into the host cell.
Add the vectors in the presence of a suitable transfection reagent to the well of a culture plate containing the host cell suspension. Cover the plate. Centrifuge at low speed to allow spin transduction. This brings the viral vector closer to the host cell surface, increasing transduction efficiency.
Simultaneously, the polycations in the transfection reagent neutralize negative charges on the virus and the host cell, promoting viral adsorption onto target cells.
Consequently, receptors on the virus surface facilitate its fusion with the host membrane. The viral capsid enters the cell and ruptures, releasing transgenic RNA and viral enzymes into its cytoplasm.
Inside the cytoplasm, the viral reverse transcriptase converts the RNA into a single-stranded complementary DNA, later changing it into double-stranded proviral DNA. The resultant DNA enters the nucleus. Viral integrase enzyme mediates the integration of proviral DNA into the host genome, resulting in transduced cells.
