the CPP-PEG-Ad vector with unmodified Ad vector. Each sample
is evaluated on a minimum of three duplicate wells.
1. In a 37
C water bath, warm enough DMEM medium for the
sample and control wells.
2. Carefully remove the cell culture medium (DMEM +10% CS)
from the wells (see Notes 9 and 10).
3. Gently add the transduction medium (i.e., DMEM) to each
well. The volume of transduction medium is half the normal
volume of medium used for culturing the cells (Table 1).
4. Dropwise, pipet the prepared CPP-PEG-Ad particles (Subheading 3.4, step 5), controls, or additional samples onto
each respective well.
5. Return the tissue culture plates to the incubator (humidified
atmosphere, 5% CO 2 , 37
C) and incubate for 4 h.
6. After 4 h, aspirate the serum-free medium and replace with the
appropriate volume of DMEM supplemented with 10% CS
(Table 1). Return the cell culture plates to the incubator and
store at 5% CO 2 at 37
C.
7. After 36 h, assay for reporter gene expression to assess the
effectiveness of the CPP-PEG-Ad vector.
4 Notes
1. Ad with an E1/E3 gene deletion and packaging the LacZ
reporter gene under the control of a cytomegalovirus (CMV)
promoter is purchased from Capital Biosciences (Rockville,
MD) and amplified using an HEK-293 cell line. Once the
cytopathic effect is observed, cells are collected and subjected
to three freeze/thaw cycles to lyse the cells. The virus is purified
from the cell lysate using a Vivapure Adenopack (Sartorius
Stedim, Arvada, Colorado). The virus is exchanged into a
physiological buffer, such as phosphate buffered saline (PBS),
before use in cell-based assays.
2. The virus is freshly purified on the day of the experiment and
can be stored in aliquots at 20
C for up to 1 week before use.
3. One terminal end of the PEG polymer has a thiol-reactive,
maleimide (MAL) group, and the other terminal end has an
amine-reactive, N-hydroxyl succinimidyl ester (NHS) group.
NHS and MAL enable conjugation of PEG to Ad and subsequently to the peptide, respectively. Maleimide contains a reactive C¼C double bond and is light and oxygen sensitive. Store
at 4
C in an opaque container and under a nitrogen atmosphere until use.
Surface Modification of Adenovirus Vector
363
is evaluated on a minimum of three duplicate wells.
1. In a 37
C water bath, warm enough DMEM medium for the
sample and control wells.
2. Carefully remove the cell culture medium (DMEM +10% CS)
from the wells (see Notes 9 and 10).
3. Gently add the transduction medium (i.e., DMEM) to each
well. The volume of transduction medium is half the normal
volume of medium used for culturing the cells (Table 1).
4. Dropwise, pipet the prepared CPP-PEG-Ad particles (Subheading 3.4, step 5), controls, or additional samples onto
each respective well.
5. Return the tissue culture plates to the incubator (humidified
atmosphere, 5% CO 2 , 37
C) and incubate for 4 h.
6. After 4 h, aspirate the serum-free medium and replace with the
appropriate volume of DMEM supplemented with 10% CS
(Table 1). Return the cell culture plates to the incubator and
store at 5% CO 2 at 37
C.
7. After 36 h, assay for reporter gene expression to assess the
effectiveness of the CPP-PEG-Ad vector.
4 Notes
1. Ad with an E1/E3 gene deletion and packaging the LacZ
reporter gene under the control of a cytomegalovirus (CMV)
promoter is purchased from Capital Biosciences (Rockville,
MD) and amplified using an HEK-293 cell line. Once the
cytopathic effect is observed, cells are collected and subjected
to three freeze/thaw cycles to lyse the cells. The virus is purified
from the cell lysate using a Vivapure Adenopack (Sartorius
Stedim, Arvada, Colorado). The virus is exchanged into a
physiological buffer, such as phosphate buffered saline (PBS),
before use in cell-based assays.
2. The virus is freshly purified on the day of the experiment and
can be stored in aliquots at 20
C for up to 1 week before use.
3. One terminal end of the PEG polymer has a thiol-reactive,
maleimide (MAL) group, and the other terminal end has an
amine-reactive, N-hydroxyl succinimidyl ester (NHS) group.
NHS and MAL enable conjugation of PEG to Ad and subsequently to the peptide, respectively. Maleimide contains a reactive C¼C double bond and is light and oxygen sensitive. Store
at 4
C in an opaque container and under a nitrogen atmosphere until use.
Surface Modification of Adenovirus Vector
363