JoVE Encyclopedia of Experiments
Microbiology
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Begin with mouse intestinal organoids that have been partially dissociated to facilitate lentiviral transduction.
Take the lentiviral vectors in a transduction medium.
The lentivirus carries a gene of interest for stable integration into the host genome.
Add the lentivirus to the well.
Incubate the mixture to allow viral entry.
The viral RNA gets reverse transcribed into DNA and integrates into the host genome.
Add an organoid medium and transfer the mixture to a tube.
Centrifuge to remove any unbound viruses.
Mix the pellet in a basement membrane matrix.
Place the organoids in a well and allow them to solidify.
The matrix forms a network that entraps organoids and provides structural support.
Add organoid medium supplemented with growth promoters to support organoid proliferation.
This results in budding organoids containing the transduced gene, which can be used for further functional analysis.
Transfer the organoids in this last small amount of medium into a well on a 48-well plate.
Add the previously prepared high titer lentivirus in transduction medium to the organoids and resuspend. Incubate the organoid-virus mixture for one hour at 37 degrees Celsius in a culture incubator to allow transduction. After one hour, add one milliliter of organoid culture, medium to the organoid virus mixture, resuspend, and transfer the mixture to a micro centrifuge tube centrifuge at 850 g for five minutes.
To pellet the organoids, remove the supernatant and resuspend the pellet in 20 microliters of ice-cold Matrigel. Put the droplet in the middle of a well. In a 48 well plate, incubate at 37 degrees Celsius for 15 minutes, for the droplet to solidify. After 15 minutes, carefully add 250 microliters of organoid culture medium supplemented with nicotinamide and kinase inhibitors.