Lentiviruses represent a powerful tool in research applications to transduce a wide range of cell types. During virus production, low titers and toxicity can be challenging. This easy-to-use protocol uses X-tremeGENE HP Transfection Reagent for fast and robust lentiviral preparation, with high virus titers being suitable to successfully transduct target cells.
Figure 1.Transfection efficiency after 48 hours. Delivery of high amounts of viral plasmids into packaging cells using X-tremeGENE HP Transfection Reagent. (A) bright field microscopy, (B) fluorescence microscopy.
Note: Plasmid preparations should be of high quality and endotoxin free.
B) Dilute DNA into 0.2 mL transfection medium (serumfree Opti-MEM® medium).
C) Add 6 μL of X-tremeGENE HP Reagent, briefly vortex and incubate 15 minutes at room temperature.
3. Carefully add the transfection mixture to the cells and agitate gently to achieve a homogeneous distribution of compounds.
Replace medium with 2 mL of growth medium. Note: the discarded media contains small amounts of virus particles and should be handled as waste according to biosafety level 2.
Seed 1 x 104 cells (here also TLA-HEK293T) per well of a poly-lysine coated 96-well plate in a volume of 200 μL of growth medium.
To determine whether this protocol produced sufficiently pure and concentrated virus suitable for cell culture applications, cells were transfected according to the procedure described above. Microscopy analysis clearly revealed high levels of GFP expression (<90%) without toxicity (Figure 1).
Furthermore, the microscopy analyses following transduction using the produced virus-containing supernatant revealed yields of GFP-positive cells at 1.2 x 106 pfu/mL. Thus, the protocol established here allows the production of lentiviral viruses in suitable amounts. To avoid multiple insertions of virus DNA into the host genome, it is recommended to infect the cells at a multiplicity of infection (MOI) below 0.1. This protocol provides virus amounts derived from one well of a standard 6-well plate for the transduction of at least 107 cells.
For life science research only. Not for use in diagnostic procedures.