Production of Pseudovirus Using Plasmid Transfection in Human Cells

Begin with a culture flask containing human embryonic kidney cells in a nutrient-rich medium.

Replace the medium with fresh medium containing chloroquine to enhance plasmid delivery into the cells.

Take a mixture of plasmids encoding influenza surface proteins, lentiviral core proteins, and the lentiviral genome, along with calcium phosphate. Incubate to form calcium phosphate–DNA complexes.

Add the mixture to the cells and mix gently. Incubate to allow the complex to enter the cells through endocytosis. Chloroquine promotes the endosomal escape of plasmid DNA.

Inside the cell, the plasmid DNA drives the expression of both influenza and lentiviral components.

Replace the medium to remove excess complexes.

Incubate to allow the viral components to assemble and bud off as pseudoviruses, which are released into the culture medium.

Harvest the medium containing the pseudoviruses and centrifuge to pellet cellular debris.

Collect the pseudovirus-containing supernatant and store it at low temperatures for downstream assays.

To begin, make a single cell suspension of HEK 293FT cells in a complete DMEM medium. Add ten milliliters of the single cell suspension to a T75 flask, followed by incubation at thirty-seven degrees Celsius.

Two hours before transfection, replace the old medium with ten milliliters of fresh complete medium with chloroquine. Mix the reagents and plasmid DNA as described in the manuscript. Then transfer this mixture into the medium above the cells and rock gently.

After incubation, replace the medium with fifteen milliliters of fresh complete DMEM Medium. After sixty-five hours, harvest the supernatant by centrifugation. Collect the supernatant and aliquot it.