Biomedical Engineering Reference
In-Depth Information
(c) Add 2.5
μ
l of purified rAAV virus of pTR-UF
3
into A1 and mix well by pipetting.
(d) Make serial dilutions (from 10
−
2
to10
−
11
) by transferring 25
μ
l of well-mixed
solution beginning from A1 to the next well (A2) and continually from A9 to A10,
changing pipette tip after each transfer.
2 In a new tube, add 25
μ
lofAd5
y
into 975
μ
l of fresh media; mix well.
3 Take day 1 seed 96-well plate from incubator; decant old media.
4 Transfer 100
μ
l of serial dilutions (normally select from 10
−
8
to 10
−
11
)toperwell
(duplicate or triple).
5 Pipette appropriate volume
z
of Ad5 (MOI 20) to each well.
6 Pipette 100
μ
l of fresh media to per well cells (duplicate or triple) and pipette the same
volume as in step 5 of Ad5 (MOI 20) to each well.
aa
.
7 Pipette 100
μ
l of fresh media to per well (duplicate or triple) without adding Ad5.
bb
8 Incubate at 37
◦
C, 5% CO
2
for 48 h.
cc
9 Score the cells infected with rAAV-UF3 visually using a fluorescence microscope.
10 Calculate the IP/ml according the score and dilution.
dd
Notes
v
C-12 cell line contains integrated wild-type AAV rep and cap genes for both the ICA and
fluorescent cell assay.
w
Transfer 1.5ml of the cell suspension into a new T-75 flask containing 15ml of fresh media;
incubate at 37
◦
C, 5% CO2 for further use.
x
The accured volume should be put in 247.5
μ
l fresh media.
y
Ad5 is co-infected C-12 along with rAAV. It is titrated using the same C-12 cell line in a serial
dilution cytopathic effect (CPE).
z
The volume (
μ
l) depends on the titer of Ad5 and the confluency of C-12 cells for MOI 20.
aa
A negative control (Ad5).
bb
A negative control (medium).
cc
The amount of Ad producing well-developed CPE in 48 h on C-12 was used to provide a helper
function for both the ICA and FCA.
dd
The PP/ml and the IP/ml differ by a factor 2 or less in most cases.
12.3 Troubleshooting
•
The quality of plasmids
: Impurities in the plasmid preparation can be deleterious to
transfection efficiency. The plasmids (rAAV plasmids and helper plasmid of pDG), which
are purified by CsCl-ethidium bromide equilibrium centrifugation, are good quality to
be transfected. The quality of the plasmids, which are purified by QIAGEN Plasmid Midi
and Maxi Kits, are acceptable.
•
Low passages (
<
50) of HEK 293 cells
: The higher titer of AAV would be produced in
low passage of HEK 293 cells. It has recently been found [35] that the tumorigenicity
