Generating Lentivirus-Infected Mouse Embryonic Stem Cells

Begin with lentiviruses carrying an antibiotic resistance gene.

Centrifuge to separate the debris. Collect the supernatant containing the viruses and pass it through a surfactant-free membrane.

This allows the lentiviruses to pass through while retaining contaminants.

Ultracentrifuge to concentrate the viruses. Remove the supernatant and add buffer to resuspend the viruses.

Then transfer the suspension to a microcentrifuge tube and vortex it.

Centrifuge to remove bubbles. Then, add the viruses to an infection medium containing mouse embryonic stem cells and mix.

Centrifuge to enhance virus-cell contact, then incubate.

The viruses deliver their genomes into the cells.

Remove the medium, add fresh medium and incubate.

The viral genome is converted to DNA and integrates into the host genome, enabling the production of mRNA that directs the synthesis of the antibiotic resistance protein.

Replace the medium with fresh medium supplemented with the selection antibiotic and incubate.

The cells expressing the antibiotic resistance protein survive, resulting in stable, virus-infected cells.

Count the cells with a hemocytometer.

Passage previously prepared mESCs into a 12 well plate format with 100,000 ce