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TRANSDUCE CELLS WITH TRANSIENT LENTIVIRAL VECTORS

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 special vectors. The following section is aimed at highlighting their specific features that you should be aware of prior to use.

logo GEG 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.

logo GEG 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.

logo GEG 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:

  • An example of protocol to help you to develop your own experimental protocols, keeping this critical parameter in mind: optimizing the contact between cells and vector particles through the particle concentration with the optimal ratio Lenti-ONE™ Trans quantity/transduction volume to prevent toxicity and maximize efficiency.
  • Experimental results obtained with Lenti-ONE™ Trans expressing Cre-NLS recombinase
  • Details about Lenti-ONE™ Trans cycle and how the transgene is expressed.

 


 

Transduction protocol

Day 1

micropipet Collect the required number of cells.

micropipet Mix the determined dose of cells and vector and seed the transduction mix (cells + Lenti-ONE™ Trans) in wells.

micropipet 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).

 

Day 2

micropipet Replace medium.

Vectors doses for transduction

 

  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 Lenti-ONE™ Trans Cre-NLS VSV

 

In vitro application

Experimental results obtain with Lenti-ONE™ Trans Cre-NLS VSV : 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.

Transgenesis application

Experimental results obtain with Lenti-ONE™ Trans Cre-NLS VSV : transgenesis application

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

 


 

Lenti-ONE™ Trans Cycle and transgene expression

 

transient lentiviral vectors cycle and transgene expression