General Adherent Cells

General Protocol for Creation of Adherent Stable Cells Using MISSION^® Lentiviral Particles (SHVRS)

Protocol provided by our MISSION^® RNAi team

Introduction

The power of the lentiviral delivery system lies in the ability to create stable cell lines from both dividing and non-dividing cells. The lentiviral particles deliver their payload into the target cells, and then, the contents are integrated at high efficiency into the host genome. By selecting for stable integrants utilizing a selectable marker, such as puromycin resistance, a population of successfully transduced cells can be found. This general protocol is for creation of stable cells in 96-well plate format. Prior to starting the transduction experiment you must:

1. Perform a titration of lentivirus to determine the most efficient MOI to achieve high transduction efficiency.
2. Perform an Antibiotic Kill Curve Assay to determine the most efficient concentration of puromycin to achieve the complete selection of transduced cells.

Materials

1. MISSION^® lentivirus targeting your gene of interest
2. Polybrene (H9268)
3. Cells (in log growth and at 50% confluence on the day of transfection)
4. Puromycin (P9620) or G418 (A1720) for selection
5. Media
6. Tissue culture incubator—37 ° C, 5% CO₂, 100% relative humidity

Protocol

Day 0

0.1		Seed cells. Duplicate or triplicate wells for each lentiviral construct and control should be used.
0.2		Incubate 18-20 hours at 37 ° C in a humidified incubator in an atmosphere of 5-7% CO2.

Day 1

1.1		Prepare media containing polybrene such that the final concentration of polybrene is 8 µg/ml.
1.2		Add 2-15 µl of lentiviral particles to appropriate wells depending on the predetermined MOI.
1.3		Gently swirl the plate to mix.
1.4		Incubate 18-20 hours at 37 ° C in a humidified incubator in an atmosphere of 5-7% CO2.

Day 2

2.1		Remove media containing lentiviral particle and add 120 µl of fresh media to each well.
2.2		Incubate 18-20 hours at 37 ° C in a humidified incubator in an atmosphere of 5-7% CO2.

Day 3

3.1

Aspirate virus-containing medium and add fresh media containing puromycin, as determined from antibiotic optimization.

Day 4+

Replace media with fresh puromycin containing media every 3-4 days until resistant colonies can be identified

Tips

Day 1

• Seed cells and incubate overnight, but for no longer then 24 hours before transduction.
• The growth rates of cells vary greatly. Adjust the number of cells plated to accommodate a confluency of 70% upon transduction.
• Account for the length of time the cells will be growing before downstream analysis when determining the plating density.

Day 2

• It is important to remove all media from the wells. Incomplete media removal will affect the concentration of the polybrene solution. Make sure cells are not sensitive to polybrene. Include a polybrene control well in the experiment. Omit polybrene during the transduction if cells show sensitivity to the reagent. Cells will still be transduced, though at a lower efficiency.
• When transducing a lentiviral construct into a cell line for the first time, a range of MOIs should be tested including 0.5, 1, 2, or 5 to determine the optimal transduction efficiency and knockdown for that cell line.
• The amount of virus added to cells has to be determined by a titration of lentivirus. Use TurboGFP control lentiviral particles to examine the transduction efficiency of cells.
• Primary, or other difficult cell types, may require more lentiviral particles (higher MOI) or modified transduction protocols.

Day 3

• Overnight incubation with virus may present a toxicity concern. Cells may be incubated for as little as 4 hours before changing the media, though the transduction efficiency will be lower.
• Incomplete media removal will affect the concentration of the puromycin solution. Use the concentration of puromycin determined by the antibiotic kill curve.
• Some cells will show decreased viability in the presence of puromycin. Let cells recover for 24-48 hours in complete media without antibiotic and then introduce puromycin.

Day 4+

• Detection Assay Considerations.
  a. Avoid harvesting cells too early.
  b. Always use controls.