Generation of Recombinant Lentivirus in Mammalian Cells

Begin with a culture of mammalian cells in a low-serum medium.

Add the DNA-lipid complex to the cells.

The complex contains lentiviral packaging plasmids and an expression plasmid encoding the gene of interest.

Incubate to allow the cellular entry of the complex.

Replace the medium to remove residual transfection reagent and incubate.

Inside the cell, the plasmid DNAs enter the nucleus and direct the synthesis of viral RNA carrying the gene of interest and viral components, including capsid and envelope proteins, reverse transcriptase, and integrase.

These components assemble into recombinant lentiviral particles that bud from the host membrane.

Collect the medium containing the released viral particles.

Centrifuge to pellet cell debris and filter the viral supernatant to remove residual contaminants.

Centrifuge at high speed to pellet intact viral particles.

Discard the supernatant and resuspend the viral pellet in a small volume of fresh medium.

Store the concentrated recombinant lentivirus at a low temperature for downstream applications.

To begin the production of the concentrated lentivirus, plate 1.5 times 10 to the 6 HEK-293T cells into a 60-millimeter dish, and culture the cells with n