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TRANSDUCE CELLS WITH EPISOMAL LENTIVIRAL VECTORS

The most critical parameter when transducing cells in vitro with Integrase-Deficient Lentiviral Vectors (or episomal) is to optimize the contact between cells and vector particles. To achieve this, always favor transduction in small culture volume. 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.

  •  When applicable, reducing the transduction volume allows to increase transduction efficiency without increasing the vector dose and possibly related toxicity.

The transduction volume is established according to several parameters:

  • The nature of cells, i.e. their sensitivity to transduction (see Protocol 1 and 2 for most robust cells, Protocol 3 for more sensitive cells)
  • The number of cells to transduce, i.e. the number of transduced cells needed for subsequent assay.

Find below 3 examples of protocol to help you to develop your own experimental protocols, keeping in mind the critical parameter: optimize contact between cells and lentiviral particles.

  •  The transduction is more efficient when cells are in good shape in their complete preferred culture medium so do not modify culture conditions during transduction. For instance, it's not worth removing FBS.

 

Protocol 1: Classical transduction protocol

Vectors doses for transduction

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. Note that we favored 24-well plates or 12-well plate, if you need more cells for subsequent analyses; we suggest you to distribute your transduction mix in multiple wells.
  •  After 5-6 hours of contact in minimal volume, fill with fresh medium (up to 500µL or more in 24-well plates or 1mL or more in 12-well plates or 2mL or more in 6-well plates).

 

Day 2

  •  Replace medium.

 

  Note that some sensitive cells will not tolerate simultaneous seeding and transduction and will prefer seeding, adhesion and subsequent transduction (see below).

 


 

Protocol 2: Optimized transduction protocol for high efficiency (only for robust cell lines)

Vectors doses for transduction

Day 1

  •  Collect the required number of cells.
  •  Incubate cells with Lenti-ONE particles in 0.5mL tube, in a final volume of 100µL and shake at 37°C 3 hours to prevent cell sedimentation.
  •  Place the 100µL of transduced cells in 24 wells plate with 600µL of fresh medium.

 

Day 2

  •  Replace medium.

 


 

Protocol 3: Adapted transduction protocol for sensitive cells (adherent)

Vectors doses for transduction

Day 1

  •  Seed the required number of cells 24hrs before transduction.

 

Day 2

  •  Remove medium and add Lenti-ONE™ Epi with a minimal volume of fresh medium. (200µL for 24-well plates or 400µL for 12-well plates or 800µL for 6-well plates).
  •  After 5-6 hours of transduction*, fill with fresh medium (up to 500µL or more in 24-well plates and up to 1mL or more in 12-well plates or 2mL or more in 6-well plates).

 

Day 3

  •  Replace medium.

 

  *If your cells are very sensitive, reduce transduction time (minimum contact time: 2 hours) and replace medium directly after transduction.