Lentivirus/Retrovirus Transduction Protocol for Adherent Cell Types

Purpose This protocol may be used for the Transduction of adherent cell lines with recombinant Retroviral vectors (Lenti- and MMLV-)*. *Please note that this process is variable and expression efficiency may vary based on
cell type. Optimization of conditions may be needed to maximize expression.
Materials 1 6-well Tissue Culture Plate at ~5.0x105 cells per well and appropriate media 1 Disposable 9in Glass Pasteur Pipette 0.15ml Retroviral supernatant per well (1x concentration)** 2.5l linker molecule*** per well (optional) 1 6 1 1 1 1 1 5ml Pipette 30l Pipette Tips Pipette Aid 20l Pipette-Man Tissue Culture Hood 37C Incubator 32oC Incubator (optional)

Eppendorf 5810R Centrifuge w/ multi-well plate swinging bucket (optional)

**Final virus concentration of up to 5x has been shown to increase transduction efficiency when necessary. ***Polybrene or Protamine Sulfate stock solution made at 4mg/ml dissolved in Milli-Q water and 0.22m Filter Sterilized prior to use. See note #1 under optimization section for further information.

Procedure 24 hours prior to transduction: - Split target cells into a 6-well tissue culture plate at 2.03.0x105 per well. The cells should reach the desired confluency of 40-60% the following day for optimal transduction. Transduction: - Observe cells under microscope to verify that they look healthy and are at the right confluence. - In a Tissue Culture Hood, aspirate off old cell media and replace with 1.35ml fresh cell media in each well. - Add 0.15mls viral supernatant per well for 1x concentration (see Table #1 in optimization section). - Add linker molecule (polybrene) if desired directly to media at a final concentration of 8g/ml (see note #1 in optimization section). - Gently rock the plates to ensure even distribution of virus and linkers. - Incubate cells in incubator at 37oC for 8-24 hours. Optional spin inoculation can be performed to enhance percent efficiency see optimization step #2.

Aspirate off any viral supernatant from the plate, replace with cell specific media in each well, and return to incubator. Analyze trans-gene expression 72 hours post infection. In some instances, trans-gene expression may take up to 7 days post Infection.

Optimization If needed, the following steps may be taken to increase transduction efficiency with retrovirus in some cell types. 1. Linker molecules have been shown to greatly improve transduction efficiency in many (but not all) cell types. Polybrene is the most commonly used polylinker, and seems to be the most effective in a wide range of cell types. However, polybrene can be toxic to some cell lines. Alternately, Protamine Sulfate can also be used effectively, and it is less toxic to cells. The concentration of Polybrene or Protamine can also be raised or lowered if needed. It is important to note that due to the variability of cell lines, linker molecules can actually make transduction efficiency lower in a few cell lines; therefore, it may be necessary to optimize transduction efficiencies in some cell lines. Table 1. Suggested set-up for optimizing cell transduction efficiencies. Shown is amount for 1x final virus concentration. Up to 5x final virus concentration can be used. The media plus linker molecule wells serve as controls to monitor any cell death, and the use of both linker molecules is optional.
1.35ml Media 0.15ml 10x viral sup 1.35ml Media 0.15ml 10x viral sup 2.5l 4mg/ml Polybrene 1.5ml Media 2.5l 4mg/ml Polybrene (optional) 1.35ml Media 0.15ml 10x viral sup 2.5l 4mg/ml Protamine Sulfate (optional) 1.35ml Media 2.5l 4mg/ml Protamine Sulfate

1.5ml Media

2. Spin-inoculate the cells by placing the plate in a multi-well plate swinging bucket rotor and place in Eppendorf 5810R Centrifuge. Spin the plate at 2500rpm for 30 minutes at 30C. Note: spin inoculation can improve transduction efficiencies by 10-20%. 3. Incubate the cells at 32oC with 5%CO2 for 16-48h.

Extra Notes - Antibiotics may be added to the media or supernatant if contamination is present, ONLY AFTER the transduction media has been changed. - The starting number of cells may be varied to account for total cell availability. Successful transductions have been carried with as little as 1.0 x 105 cells per well. - The duration that the viral supernatant sits on the cells can range from 4 hours to 2 days. We usually let cells go overnight (16-24 hrs). We have seen that a longer exposure can produce higher transduction efficiency; however, this is also proportional to increasing cell death, as the VSVG viral envelope and the localization molecules can be toxic. - Multiple infections may be preformed on the same cells to increase the transduction efficiency. To do this allow the 1 infection to stay on the cells overnight, replace the supernatant with cell-specific media and allow the cells to recover for 24h and then infect again overnight. This cycle may be repeated 3 times.