Adeno-Associated Viral Gene Therapy for Retinal Disorders - Gene Delivery and Therapy for Neurological Disorders - page 216

Table 1
Calculating ratio of plasmids for virus production

Plasmids Size (kb) Amount per transfection ( μ g)

pHelper 18.7 18.7 × R

pRep-cap 7 7 × R

pTransgene t t × R

Total K kb 500 ʼ g

Ratio ( R ) mass/kb 500/ K

between all layers of the HYPERFlask. Then, turn the HYPERFlask
to an upright position and fi ll completely with media (an additional
300-350 ml) until the fl ask is full to the lowest thread on the neck.
Gently tap the fl ask to dislodge any trapped air bubbles.
After 72 h, transfect the cells in each HYPERFlask with a total
of 500 g DNA. The ratio of helper plasmid to rep/cap plasmid

to transgene plasmid is dependent on the size of the plasmids.
Add the size of each plasmid to provide a total in kb. Divide 500
(the amount of total DNA required) by that total to give the ratio
value ( R ). Multiply each plasmid size by the R to give the amount
in

ʼ

g of each plasmid to be used (Table 1 ). Then, determine the
volume in

ʼ

l required of each plasmid that provides the respective
amount of DNA.
Measure the three plasmids into a 50-ml Falcon tube, and make
up to 10 ml with 150-mM NaCl. To make a 150-mM sodium chlo-
ride solution, dilute 4.38 g NaCl in 500 ml dH 2 O. In a separate
Falcon tube, measure out 1.125 ml 10 mM polyethylenimine (PEI)
transfection reagent (linear polyethylenimine, MW 25,000,
Polysciences, 23966-2), and make up to 10 ml with 150 mM NaCl.
Add the PEI drop by drop into the Falcon tube containing the
DNA, gently swirling the mixture continuously while doing so. If
the PEI is added too quickly, precipitation will occur and the con-
tents of the tube will go cloudy. Incubate the mixture at room tem-
perature for 20 min. Add this to a 500 ml bottle of DMEM
containing 4.5 g/l glucose, 2 % fetal bovine serum, 2 mM L -
glutamine, 100 U/ml penicillin, and 100

ʼ

g/ml streptomycin.
Slowly drain the old media from the HYPERFlask and fi ll with
media containing the DNA and transfection agent as prepared
above. Top up the HYPERFlask with additional media if required,
ensuring again there are no air bubbles. Incubate at 37 °C for 72 h.

ʼ

4.4 Harvesting
and Lysing Cells

Collect the cells from each HYPERFlask into two Corning 500-ml
centrifuge tubes. Remove 100 ml of media before dividing approx-
imately 150 ml media from the HYPERFlask into the two centri-
fuge tubes, and then shake the HYPERFlask vigorously to loosen