TRANSDUCE CELLS WITH TRANSIENT LENTIVIRAL VECTORS
The most critical parameter when transducing cells in vitro with Reverse-Transcriptase Deficient Lentiviral Vectors (transient) is to optimize the contact between cells and vector particles. To achieve this, always favor 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.
- RTDLV are special vectors. The following section is aimed at highlighting their specific features that you should be aware of prior to use.
RTDLV 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 vector dose vs transduction volume has to be determined considering efficiency and toxicity in your particular experimental context.
RTDLV induce low level of expression despite the high number of transduced cells. Consequently transgene expression cannot necessarily be 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.
RTDLV show particular kinetics of transgene expression. Transgene expression is detected as early as 8 hours after transduction, reaches the maximum level 5-6 days after transduction, and progressively decreases for 6 days.
You will find below:
- An example of protocol to help you develop your own experimental protocols, keeping this critical parameter in mind: optimizing the contact between cells and vector particles with the optimal ratio vector quantity/transduction volume to prevent toxicity and maximize efficiency.
- Experimental results obtained with RTDLV expressing Cre-NLS recombinase
- Details about RTDLV cycle and how the transgene is expressed.
Transduction (infection) protocol
- Collect the required number of cells.
- Mix the determined dose of cells and vector and seed the transduction mix (cells + Lenti-ONE) in wells.
- After 3-4 hours of transduction fill with fresh medium (up to 150µl for 96-well plates, or up to 650µl 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.
Experimental results obtained with RTDLV Cre-NLS VSV
In vitro application
CV1-5B cells conditionally express Neo or LacZ. Transduction of CV1-5B cell line with Lenti-ONE™ Cre-NLS induces LoxP site recombination, excision of the Neo cassette and expression of LacZ.
Reverse-Transcriptase Deficient Lentiviral Vectors: cycle and transgene expression