Biomedical Engineering Reference
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Fig. 1. The vector plasmid (p) HSV-PrpUC, which serves as the viral backbone for
all of our viral-mediated gene transfer studies.
Lim et al.
(3)
showed that the transfection of the vector DNA at the start of the
packaging procedure was signifi cantly more effi cient using lipofection than
using calcium phosphate, and thereby achieved a favorable ratio (>1) of vector
to helper. Since then, using a total of three passages (infections/harvests) in 2-2
cells, we have achieved vector/helper ratios greater than 100
1. Surprisingly,
this ratio becomes less favorable, rather than more favorable, when more
than three passages are used. An additional improvement to the packaging
procedure, the banding of the virus on a sucrose step gradient, followed
by a high-speed centrifugation to pellet the virus, has reduced further the
cytotoxicity of the virus preparations. This step simultaneously removes toxic
factors present in the crude cell lysates and enables concentration of the vector
to titers exceeding 10
8
infectious units (iu)/mL.
3.1.1. Transfect 2-2 Cells
1. Maintain 2-2 cells at 37°C in a humidifi ed, 10% CO
2
incubator in DMEM +
10% FBS. When a fresh aliquot of cells is thawed, it should be passaged at least
two times before the cells are plated for the transfection.
10
5
per 60-mm dish in
2. Two days before transfection, plate 2-2 cells at 2.5
×
5 mL of DMEM.
3. Dilute 2
µ
g of DNA (purifi ed by QIAGEN column) with 250
µ
L of OptiMEM in a
sterile 1.5-mL microcentrifuge tube. Dilute 12
µ
L of LipofectAMINE with 250
µ
L
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