TITLE: Generating Stable Cell Lines with Lentivirus
AUTHORS: Addgene the Nonprofit Plasmid Repository

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is for generating stable cell lines with lentivirus. To see the full abstract and additional resources, please visit the </span><a href="https://www.addgene.org/protocols/generating-stable-cell-lines/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Addgene protocol page</span></a><span style = ":;">.</span></div><div class = "text-block"><span style = "font-weight:bold;">Sample Data</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bkyyw.png" /></div></div>

[STEPS]
?. [Transduction]
Prepare a batch of DMEM complete + 10 µg/mL polybrene by diluting  of 10 mg/mL polybrene into  media.
20 µl
20 ml
?. [Transduction]
Rapidly thaw the lentiviral aliquot at .
37 °C
?. [Transduction]
Prepare a range of dilutions of the lentivirus in DMEM complete + 10 µg/mL polybrene.   ABC1DilutionVolume of Lentivirus (μL)Volume of DMEM complete + 10 µg/mL polybrene (µL)20050031:530020041:1015035051:503047061:1001548571:5003497
Note, this is just a sample of possible dilutions. You may want to try higher/lower dilutions depending on your downstream applications. If you’ve titered your virus beforehand, you can narrow this range according to the results of your titration.
ABC1DilutionVolume of Lentivirus (μL)Volume of DMEM complete + 10 µg/mL polybrene (µL)20050031:530020041:1015035051:503047061:1001548571:5003497
?. [Transduction]
Add  of a single viral dilution to each well.
0.5 ml
Each well gets one dilution, so a 6-well plate will hold 5 dilutions plus one 'no virus' control well.
?. [Transduction]
Perform a "reverse transduction" by seeding 50,000 cells into each well of the 6-well dish. These cells will be added to the wells that already contain 0.5 mL of virus solutions at various dilution. To seed the cells as described in 5.1 and 5.2.
Make sure to use the polybrene-containing media to make the cell solution in this step.
?. [Transduction]
Prepare a batch of cells as follows: Dilute 350,000 cells into a total volume of  of DMEM complete + 10 µg/mL polybrene. Mix well by pipetting or inverting the tube.
7 ml
?. [Transduction]
Aliquot  of cell suspension (i.e., 50,000 cells) into each well of the 6-well dish. This brings the total volume in each well up to . Since all the media in these wells was made with DMEM complete + 10 µg/mL polybrene, the final concentration of polybrene in each well should be 10 µg/mL.
1 ml
1.5 ml
*Pro-Tip* Transducing too many cells relative to the number of virus particles reduces the transduction efficiency, resulting in massive cell death upon antibiotic selection. The number of cells transduced should be enough that they can grow out in a reasonable amount of time, but not so many that they vastly outnumber the virus particles.
?. [Transduction]
Incubate the cells with the virus for  - .
?. [Selection]
Gently aspirate the media from the cells. Add  of DMEM complete containing the appropriate antibiotic.
1.5 ml
This is the beginning of the selection process, which will begin the selection of a stable cell pool.
?. [Selection]
Observe the dish every day to ensure that the cells in the untransduced well (0 µL lentivirus, above) are dying. Perform regular fluid changes the monitor the growth of the cells.
*Pro-Tips* Depending on the efficiency of your transduction, you will see different degrees of cell death upon antibiotic selection. It is important to monitor these cells regularly and replace the cell media. Cell death by some cells in the culture may adversely affect the surviving cells in the culture, so it is important to do regular fluid changes and maintain optimal growth conditions for the surviving cells. Even in the absence of cell death, the cell media should be changed every 2-3 days to maintain the dose of antibiotic, which may not be stable at  .To achieve a stable cell pool, the antibiotic selection should last at least as long as it takes the control (untransduced) cells to completely die. After that, the cells may undergo additional seleciton while the population expands. At this time, some researchers reduce the concentration of the antibiotic in culture, or remove the antibiotic entirely. If the antibiotic is reduced or removed from the culture, check the cells regularly to confirm transgene expression.
?. [Selection]
As polyclonal populations of resistant cells start coming through and the individual wells become confluent, expand into larger vessels. A confluent well of a 6-well dish can be expanded into a 10 cm dish. A confluent 10 cm dish can be expanded into two 75 cm2 flasks.
*Pro-Tip* This selection method results in a polyclonal cell population, meaning that the transgene has integrated in different locations in the various cells in the culture. This is because lentiviral integration is random. Given the MOI of the lentivirus used, cells may also exhibit varying numbers of integration events. In other words, if an MOI > 1 was used, some cells may have 1 copy of the transgene, while others have >1 copy of the transgene in their genome. This can result in varying expression levels of the transgene from different cells in the population.
?. [Selection]
Once the polyclonal populations are growing well and have been sufficiently expanded, prepare cell stocks and/or harvest to test for protein expression.
Typically cells transduced with lower dilutions of the virus will have higher levels of expression. Consider expanding populations transduced with a variety of dilutions and pick the population that has the most desirable level of expression.
?. [Selection]
Over time, transgene expression in a polyclonal population may drop. This is because cells that express high levels of the transgene may have reduced growth rates, especially if that transgene is toxic. Eventually, the rapidly growing low-level transgene expressors may take over the culture. To overcome this, consider generating monoclonal lines from the early polyclonal populations.