The most critical parameter when transducing cells in vitro with Lenti-ONE™ Trans is to optimize the contact between cells and vector particles, i.e. optimize the vector concentration, rather than increasing the absolute vector dose. For this reason we've always favored transduction in small culture volumes. Indeed, transduction of a given number of cells with 1µL vector in 1 mL cell culture medium will be far less efficient than with the same dose in 200 µL cell culture medium.
Lenti-ONE™ Trans are less efficient than integrating and Integration-Deficient lentiviral vectors. The required concentration is higher, ie increase vector dose and/or reduce transduction volume to improve efficiency. These experimental conditions are more susceptible to induce toxicity and the optimal ratio Lenti-ONE™ Trans dose vs transduction volume has to be determined considering efficiency and toxicity in your particular experimental context.
Lenti-ONE™ Trans induce low level of expression despite the high number of transduced cells. Consequently transgene expression cannot be necessarily detected by regular methods such as direct fluorescence visualization for GFP (fluorescent microscopy or FACS). Transgene expression can however be detected by sensitive methods such as Western blot or ELISA.
Lenti-ONE™ Trans show particular kinetics of transgene expression. Transgene expression is detected as early as 8 hours after transduction, riches the maximum level 5-6 days after transduction, and progressively decreases for 6 days.
You will find below:
Collect the required number of cells.
Mix the determined dose of cells and vector and seed the transduction mix (cells + Lenti-ONE™ Trans) in wells.
After 3-4 hours of transduction fill with fresh medium (up to 150µl or more for 24-well plates or up to 650µl or more for 24-well plates).
You may encounter toxicity problems due to the high concentration of vectors. Toxicity can be reduced by decreasing particle concentration, reducing the vector dose and/or increasing the transduction volume.
In vitro application
CV1-5B cells conditionally express Neo or LacZ. Transduction of CV1-5B cell line with Lenti-ONE™ Trans Cre-NLS induces LoxP site recombination, excision of the Neo cassette and expression of LacZ.