Adeno-Associated Viral Gene Therapy for Retinal Disorders - Gene Delivery and Therapy for Neurological Disorders - page 215

4.1.1 Transformation
Protocol

This is based on the manufacturer's guidelines. Thaw 75 ʼ l

XL10-Gold bacteria on ice and add 3 -mercaptoethanol, and

gently mix every 2 min for 10 min. Incubate the cells with 5

ʼ l ʲ

fl of
the ligation mixture on ice for 30 min. Heat shock at 42 °C for
30 s and then incubate for 2 min on ice. Add 925

ʼ

l of SOC
Medium (Invitrogen) and incubate at 37 °C in a shaker rotating at
225 rpm for 1 h. Spread 200

ʼ

ʼ l of the transformation mix on LB

agar plates containing 100 g/ml ampicillin, and incubate at 37 °C

for 16 h. Pick single colonies, culture in 4-ml LB/ampicillin for
16 h, and isolate the plasmid using a Miniprep Kit (Sigma-Aldrich).
To generate the high concentrations of plasmid required for virus
preparation, culture an isolated plasmid (fully sequenced for con-
fi rmation of the transgene insertion) in 2 l of LB/ampicillin and
isolate using a Megaprep Kit (QIAGEN).

ʼ

Due to the nature of the secondary structure formed by AAV ITRs,
we have found that confi rmation of their presence following clon-
ing is diffi cult. Direct sequencing and isolation via polymerase
chain reaction (PCR) both give variable and often incomplete
results. Therefore, we have found that the most accurate method
of confi rming the presence of ITRs is via a restriction digest.

4.2 Checking ITRs

Two Xma I restriction sites are present within the ITR sequence
(Fig. 3 ); check that this enzyme does not cut anywhere else in the
expression cassette inserted. If the distance between the two ITRs
is approximately half the plasmid size, a second restriction enzyme
with only one recognition site between the ITRs will also be
required.
Incubate 500 ng plasmid with 1

4.2.1 Method

ʼ l Xma I , 2.5 ʼ l buffer, and

2.5 l with sterile water for 2 h at

37 °C. Separate the products using agarose gel electrophoresis,
and the size of bands seen will indicate whether the clones are posi-
tive for both ITRs and the inserted transgene.

ʼ l BSA and make up to 25 ʼ

4.3 Transfecting
Cells for Virus
Production

Transfecting the large number of cells needed for virus production
is most easily done in a HYPERFlask (Corning). This is a multilay-
ered fl ask with a total growth area of 1,720 cm 2 .
Seed a 75 cm 2 tissue culture fl ask with 17 × 10 6 HEK 293T
cells in Dulbecco's Modifi ed Eagle Medium (DMEM) with 4.5 g/l
glucose, 2-mM L -glutamine, 10 % fetal bovine serum, 100 U/ml
penicillin, and 100

g/ml streptomycin. Each fl ask will then seed
one HYPERFlask. We recommend using two HYPERFlasks and
therefore seeding two fl asks at the above concentration per virus to
be made.
After approximately 72 h, or when the cells are 80 % confl uent,
loosen the cells and resuspend in 200 ml media. Gently transfer the
cells from one 75 cm 2 fl ask into a HYPERFlask. This is most easily
done with the HYPERFlask tilted to about 60°, so the cells are divided

ʼ