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Lentivirus Transduction Protocol

Lentiviral vector is an efficient gene delivery tool in both discovery research and cell and gene therapies. After transduction, the gene of interest cargo in lentiviral vectors is integrated into the host genome in a random integration fashion. 

 

The following lentiviral vector transduction protocol can be applied to adherent cells (HEK193T is used in this protocol) in a 24-well plate scale. You may scale up or down depending on the size of the cell culture plate. 

Tissue Culture Vessel Growth area, cm2/well
96-well plate 0.35
24-well plate 2
12-well plate 4
6-well plate 9.5
35 mm plate 8

 

Day 1 Seed cells

Seed 2x104 HEK 293T cells in each well of 24-well plate. The number of cells seeded can be adjusted depending on the size of your target cells, in order to reach around 50% of confluency at the time of transduction the next day. Incubate cells overnight at 37°C, 5% CO2.

 

Day 2 Transduction

1.    Calculate the amount of lentiviral vector needed per cell according to the desired multiplicity of infection (MOI).

2.    Thaw the lentiviral GFP vectors or your GOI lentiviral vector on ice. Gently spin down before opening to collect the vector to the bottom of the tube. Keep them on ice. Mix gently before use.

3.    Remove the spent media from wells and add appropriate amount of lentiviral GFP viral vectors, culture media and polybrene (final concentration is 8 ug/mL) to a total volume of 0.5 mL. Gently swirl the plate to mix. Polybrene usually increases the transduction efficiency of lentiviral vectors. If polybrene toxicity to your cells is observed, protamine sulfate can be used instead to assist lentiviral vector transduction. 

4.      Incubate cells overnight at 37°C, 5% CO2.

Note: When transducing a cell line for the first time, a range of MOI should be tested. You can use Forecyte Bio’s premade  lentiviral GFP vectors  to conduction the MOI optimization studies.  

 

  Multiplicity of Infection (MOI):

Multiplicity of Infection (MOI) is the number of transducing lentiviral vector particles per cell.

Total volume of lentiviral vector (mL) to add to each well = (Total number of cells per well x Desired MOI) / Lentiviral vector titer (TU/ml)  

 

Day 3 Change Media

Remove media containing the lentiviral vector and replace with fresh growth media. If cell toxicity is observed after overnight lentiviral vector incubation, cells can be incubated with the lentiviral vector for a shorter period of 4 to 8 hours instead.

 

Day 5 Evaluation Of Gene Expression

Transduction efficiency can be evaluated under the fluorescence microscope if lentiviral GFP vector is used for transduction. For gene specific expression, you can harvest cells for Flow or Western Blot with gene specific antibody, or any other assays. 

If stable cell selection is desired, the cells can be cultured with puromycin containing media. 

Note: Perform a cell kill curve experiment to select the correct dose of puromycin for cell line selection.

 

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