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.

listRTDLV 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.

listRTDLV 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.

listRTDLV 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

Cells / well (HEK293T)
Vector Quantity (TU)
Transduction volume
Growing volume
8.104 – 1.105
1.104 – 5.104
50 µl
150 µl
1.5 105 – 2.5 105
5.105 – 1.106
150 µl
500 µl

Day 1

  •  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).

Day 2

  •  Replace medium.

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

Experimental results: transgenesis 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.

Transgenesis application

Experimental results: in vitro application

Rosa26 mice conditionally express Neo or LacZ. Injection of Lenti-ONE™ Cre-NLS in ROSA26 zygote induces LoxP site recombination, excision of the Neo cassette and expression of LacZ.

Reverse-Transcriptase Deficient Lentiviral Vectors: cycle and transgene expression

transient lentiviral vectors cycle