Lentiviral Transduction of Chondrocytes

Dominik Haudenschild, December 2009 UCD

(1) The day before transduction ( day 1), trypsinize and count the cells, plating them in a 6-well plate such that they will be 30-50% confluent at the time of transduction, you can either culture them overnight or wait 4-8 hours to transduce the cells.

Note: seed approximately 200,000 cells per 6-well dish

Note: prefer shorter plating time with chondrocytes, ~4 hours, passage on morning of transduction day.

(2) On the day of transduction ( day 2), thaw your lentivirus stock and prepare 10 fold serial dilution to a final volume of 1 ml. Remove the culture medium from the cells. Mix each dilution gently by inversion and add to one well of cells

Note: for chondrocytes use 2-fold concentrated viral supernatant, 1 to 1.5ml/well

(3)  Right after adding virus, add Polybrene to each well to a final concentration of 6 ug/ml, swirl the plate gently to mix (Polybrene is hexadimethrine bromide 6 mg/ml stock in water) Incubate with virus and polybrene at 37 C overnight.

Note: spin cells after adding virus/polybrene for approximately 30 minutes at R.T. (500g), this seems to help increase transfection efficiency

(4) The following day ( day 3), remove the media containing virus and replace with 2 ml of complete culture medium. If selection is to be done, start 2 - 4 days after the end of viral transduction

(5) Replace medium with fresh medium containing antibiotic every 2-3 days.

(6) After 10-12 days of selection ( day 14-16),  you should see no live cells in the mock well and discrete antibiotic-resistant colonies in one or more of the dilution wells.