Lentifectin Transfection Reagent, 1ml Lentifectin™ is a transfection reagent specially formulated with multiple cationic polymers for the
production of Lentiviral vectors in vitro. It is shown that Lentiviral vectors produced with Lentifectin™
consistently have higher titers than those produced with Calcium-phosphate transfection or with other types of lipid transfection reagents.
Protocol The following protocol allows the production of recombinant lentiviral vector up to 107 IU/ml. We
recommend including a negative control (no DNA, no transfection reagents) in your experiment
to help you evaluate your results. You will need 1.2 x 107 293FT cells for each sample.
DAY 1:
· Seed 293T cells into 10 cm dishes (add ~1.2X107 cells per 10 cm dish).
DAY 2:
· Check to make sure the cells are 80-90% confluent;
· For each 10cm dish prepare transfection complex as follows:
a. Solution A: Dilute 25ug DNA plasmids (total) in 1 ml serum-free medium;
b. Solution B: Dilute 100ul LentiFectin™ reagent in 1 ml serum-free medium;
c. Mix Solution A and Solution B together and incubate at room temperature for 20 minutes;
· Remove growth medium from the cells and add 4.5 ml serum-free medium to the cells;
· Add the transfection complex to the cells and incubate at 37oC for 4 hours.
· Add 0.65 ml FCS to each dish and incubate at 37oC overnight.
Note: Expression of the VSVG glycoprotein causes 293FT cells to fuse, resulting in the appearance of large, multinucleated cells known as syncytia. This morphological change is
normal and does not affect the production of the lentivirus.
DAY 3:
· Remove the medium from the cells and add 10 ml fresh medium to the cells.
·Incubate at 37
oC for 24 hours.
Day 4
· Collect viral supernatants* and add 10 ml fresh medium to the cells and incubate at 37oC for 24 hours.
Day 5
· Collect viral supernatants*.
*Clearing viral supernantant by centrifuging supernatants at 3000 rpm for 15 minutes at 4°C to
pellet debris.Optional: Filter the viral supernatants through a Millex-HV 0.45μm or an equivalent PVDF filter.
Caution: Remember that you are working with infectious virus after viral supernantant collection. Follow the recommended guidelines for working with BL-2 organisms (see page 4 for more information).
Determining the Lentiviral Titer with GFP Reporter Lentivirus a. The day before transduction, plate cells at 30-50% confluence in 12-well plate and incubate
cells overnight at 37°C.
b. On the day of transduction, prepare 10-fold serial dilutions of viral stocks in complete culture medium ranging from 1X to 106X.
c. Remove culture media from the cells and add 1ml of the dilutions directly to the cells.
d. If desired, add Polybrene® to each well to obtain a final concentration of 6μg/ml, and
incubate at 37°C overnight.
e. The following day, replace the media containing virus with fresh complete culture media.
f. After 2-4 days of transduction, observe the GFP expression under fluorescent microscope, and
count the GFP positive cells to estimate the titer.
Lentivirus transduction and selection a. One day before transduction, plate your target cells at 1 x 106 cells per well in a 6-well plate.
b. On the next day, mix 1.0 ml (or proper volume) of the lentiviral stock with 1.0 ml of complete
medium and add to each well.
c. Add polybrene to each well to obtain a final concentration of 6μg/ml.
d. On the next day, replace the medium containing lentiviruses with complete culture medium.
e. After 3 days of transduction, replace the medium with fresh complete medium containing
appropriate amount G418 for selection.
f. Replace medium containing G418 every 3-4 days for 3 weeks.
g. Pick up colonies for screening
Equipment 