Editing MuLV Psueduvirus (section)

=== Cell Seeding for Pseudotyped Particle Production ===

'''NOTE:'''&nbsp;Perform this step in the biosafety cabinet.

1.By standard cell culture techniques, obtain an 80–90% confluent 75 cm<sup>2</sup>&nbsp;flask of HEK-293T/17 cells passaged in complete Dulbecco's Modified Eagle's Medium (DMEM-C) containing 10% (vol/vol) fetal bovine serum (FBS), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 100 IU/mL penicillin, and 100 µg/mL streptomycin. Prepare DMEM-T medium for transfections (its composition is the same as DMEM-C but without antibiotics).

2. Wash cells with 10 mL of pre-warmed (37 °C) Dulbecco's Phosphate Buffered Saline (DPBS) twice.

'''NOTE:'''&nbsp;Handle HEK293T/17 cells with care as they easily detach.

Aspirate the supernatant and detach cells with 1 mL of 0.25% trypsin solution pre-warmed at 37 °C. Place the flask of cells at 37 °C, 5% CO<sub>2</sub>&nbsp;incubator for 3−5 min or until cells start detaching.

'''NOTE:&nbsp;'''Avoid incubating cells with trypsin for more than 5 min as this typically leads to cell clumping.

3. Deactivate trypsin by adding 4 mL of DMEM-C medium and count cells using a cell counting slide and light microscope.

'''NOTE:'''&nbsp;To avoid having to count too many cells, an additional dilution step may be required beforehand. Remember to factor in this dilution when calculating the actual cell density of trypsinized cells.

4. Dilute cells to 5 x 10<sup>5</sup>&nbsp;cells/mL with DMEM-C.

5. Seed 6-well tissue culture plate with 2 mL of cell solution per well and gently move the plate back and forth and side to side to evenly distribute cells, avoiding circular motion.

'''NOTE:'''&nbsp;This is a key step. Evenly distributed cells will ensure that cells do not clump at the center of wells. In turn, this will ensure good transfection efficiencies and pseudotyped particle production.

6. Incubate the plate overnight (16–18 h) in a 37 °C, 5% CO<sub>2</sub>&nbsp;cell culture incubator.

7. Three-plasmid Co-transfection

'''NOTE:'''&nbsp;Perform this step in the biosafety cabinet.

Observe cells under an inverted light microscope to check for the cell morphology and density.

'''NOTE:&nbsp;'''Ideally, cell density should be in the 40–60% confluency range. It is critical that cells are neither too confluent (80−90% confluent) nor too sparsely distributed (20−30% confluent) in each well. A cell density of 40−60% confluency will ensure good pseudotyped particles production.