Lentiviral Transduction Troubleshooting
Lentiviral transduction is the method of choice whenever generating stable cell lines from both dividing and non-dividing cells is required. Thereby, viral vectors are used as a vehicle for integrating DNA into the host genome, at high efficiency. One of the most critical factors for successful lentiviral transduction is the viral titer. Also, other factors can influence your transduction efficiency such as the viability of target cells and the quality of transfer vector DNA.
If this is the case for you, have no fear! This ZAGENO troubleshooting guide is here to help.
Low Transduction Efficiency
- Target cell line is difficult to transduce: Use a chemical transduction enhancer which neutralizes charge repulsions between virus and cells
- Cells are of poor quality Optimize the growth conditions: Check growth medium, mycoplasma contaminations and the cell density (cells should have about 50-80% confluency at transfection stage)
- Viral titer is not infectious titer: Measuring the titer using RT-PCR tends to lead to overestimation because of non-effective particles. Check the infectious titer by transducing cells with serial dilutions of your protein expressing lentiviral vector
- Volume of infecting supernatant is too high Concentrate your virus by using ultracentrifugation to achieve absorption of viral particles to the cells
- Multiplicity of infection (MOI) is too low: Increase the amount of lentivirus
- Truncated viral RNA transcript: The 3’-LTRs itself acts as a polyA signal, any foreign polyadenylation between the LTR elements can reduce the amount of transductionally active genomic RNA and decrease the viral titer
- Low DNA quality: Ensure that your transfer vector DNA is of “transfection-grade”. Use either Plasmid Purification Kits or phenol/chloroform extraction followed by a cesium chloride (CsCl) gradient
- Cells are sensitive to transduction enhancer: Use another transduction enhancer
- Cells are sensitive to lentiviral treatment: Use a lower amount of lentivirus and change the growth media 4 hours after transduction
- No expression of the expression construct: The maximal expression is expected 72 hours after infection. Some cell line show delayed expression. Measure again after 96 hours
If you plan on repeating this experiment, follow these tips to make things easier
- Use healthy cells that are well maintained, passaged regularly and free of contaminations
- Use lentiviral expression constructs of high quality
- Make sure you have a high titer (>10^8 IFU/ml)
- Always use a positive control
Hopefully by following these tips your problems will soon become a thing of the past. If you're still having issues - or have extra tips - let us or the community know in the comments!