Material:

• Packaging Cells, e.g. Phoenix cells (an adenovirus Ad5-transformed human embryonic kidney cell line 293T, transfected with two MoMLV packaging gene constructs: CMV-Env-PolyA, and RSV-Gag/Pol-Tyt2-PolyA.) See the Nolan lab website for further details and for obtaining and MTA. Use passage number < 20.

• Trypsin/EDTA (Gibco)

• Retroviral plasmid DNA (usually based on MMLV based with LTRs, the packaging signal sequence and containing the gene of interest, a promotor and a selectable marker like antibiotica resistance, GFP or CD8) and control vector without gene of interest. Ecotropic vectors infect rodent cells, amphotropic or xenotropic are able to infect human cells

• 2M CaCl2 (5.88 g in 20 mL H2O 0.2 µm filtered, store aliquots at -20°C)

• 2x HBS (Hepes buffered saline)

• 100 mM chloroquine (0.516g chloroquine diphosphate, 10 mL H2O, 0.2 µm filtered, store aliquots at -20°C)

• DMEM with 10% FBS and 1x penicillin-streptomycin

• Polybrene 1000x (4 mg/ mL) in PBS, stock sterile filtered

• Lipopolysaccharide (LPS) from Salmonella typhimurium, 1000x stock [50 µg/ µL]

• B cell medium

• PBS

• Gentamycin 500x (50 mg/ mL)

• Puromycin 200x (300 µg/ mL)

• Hygromycin B 500x (50mg/ mL)

• G418 = neomycin

Method:

1. Detach the Packaging Cells with 1mL Trypsin (1min/ 37°C), resuspend in 10 mL DMEM with 10% FBS, count an aliquot and seed 4.5 - 6 x 106 cells per 10 cm plate. Allow to grow 18-24 hr prior to transfection.

2. Prepare in a sterile 5 mL tube 20 µg plasmid DNA, 62.5 µl 2M CaCl2 and H2O (sterile) ad 500 µl. Agitate constantly by an automated pipet (air bubbles) and add dropwise 500 µl 2x HBS. Precipitation will occur within 5 min at room temperature.

3. Remove media from the packaging cells, add carefully 10 mL DMEM medium containing 25¨µM chloroquine (2.5 µl 100mM stock). Add precipitate dropwise. Incubate 9-10 hr. Remove medium and gently replace with 5mL DMEM to collect virus SN for 24-36 hr.

4. 12 hr after Packaging Cell transfection (when supernatant collection has been started), 7.5 x 105 MEFs should be seeded (at subconfluent density) per 10 cm plate.

5. Remove the medium from the MEFs. Combine5 µL polybrene (final concentration 4mg/ mL; infection enhancer) to the virus-containing SN of the Packaging Cell plate and filter through a 0.45 µm filter on the MEF plate. Add another 5 ml DMEM to the Packaging Cells for ongoing supernatant collection.

6. Superinfect the same plate of MEFs by repeating the infection (as described in the previous step) at 6 and 18 hours after the initial infection.

7. Allow the cells to grow and express the infected genes for 24 hr after the (last) infection. GFP (green fluorescent protein) encoding vectors can be easily selected by flow cytometric sorting. Collect sorted cells in fresh medium supplemented with 200 µl gentamycin 500x. Alternatively, antibiotic selection (corresponding to the vector encoded resistance gene) can be carried out (usually after splitting the cells) in 1.5-2.5 µg/mL puromycin for 2 days or in 100-200 µg/mL hygromycin B for 5 days. Individual drug concentration and duration of selection may vary and should result in 100% killing of uninfected cells as control.