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Lentivirus Transduction Protocol


Lentivirus is a modified HIV virus and although unable to replicate in a host, it must be handled with caution.

Lentivirus Transduction Protocol

Concerns:

Lentivirus is a modified HIV virus and although unable to replicate in a host, it must be handled with caution. When working with these viruses, work only in a BL2+ designated hood or viral vector room. All handling, storage and disposal of biohazard waste must be carefully treated.

Procedures:

Day1:

1. Split your interested cell line at proper density in a 6-well tissue culture plate in 2ml of DMEM media containing 10% FBS and 1% Pen-Strep.

Note: ensure two or three replicates each sample and be sure to include the proper controls.

Day2:

1. Remove medium from cells

2. Add proper amount of virus for infection in a fresh microtube

3. Bring up the volume to 1ml (Polybrene could be added to help lentivirus integration)

4. Add the virus medium into cells, and gently mix to ensure virus cover cells well

Day3:

1. Remove the virus media and replace with normal DMEM media supplemented with 10% FBS and 1% PenStrep.

Day5:

1. Replace the media on the cells with 2ml of DMEM media supplemented with 10% FBS, 1% PenStrep and the proper concentration of Puromycin.

Note: the concentration of puromycin depends on cell types.

Day7-20:

1. Watch the cells carefully over the next 1-3 weeks, passaging the cells and changing the media as necessary.

Note: during this period, puromycin should be added constantly.

2. Puromycin challenge could be stopped until the uninfected control cells are completely dead.

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