3. Dilute 40 μl of the PEG stock solution with 10 μl of 50 mM
HEPES buffer (pH 8.0).
4. Dropwise, add 50 μl of the diluted PEG solution to the side of
the microcentrifuge tube containing the virus while gently
vortexing so that the final concentration of PEG is 4 μg/
10
6 vp.
5. Incubate for 45 min at room temperature while pulse vortexing
every 7–10 min.
6. See Note 7 for details on how to determine the extent of the
reaction between PEG and Ad.
3.4 Formation of
CPP-PEG-Adenovirus
1. Prepare an 800 μl stock solution of 2 mg CPP/ml in 20 mM
HEPES buffer (pH 7.0) (see Note 5).
2. Dilute 31.25 μl of the CPP stock solution into 18.75 μl of
20 mM HEPES buffer (pH 7.0).
3. Dropwise, add 50 μl of the CPP solution (from step 2) onto
the side of the microcentrifuge tube containing the PEGylated
virus while gently vortexing so that the final concentration of
CPP is 6.25 μg/10
6 vp.
4. Incubate for 45 min while pulse vortexing every 7–10 min.
5. Purify the CPP-PEG-Ad particles by removing unreacted peptide and polymer using a Pierce protein concentrator with
10 kDa MWCO, as follow:
(a) Place the sample into the concentrator sample chamber.
(b) Place the cap and the sample chamber and insert the
sample chamber into a collection tube.
(c) Place the concentrator assembly into the microcentrifuge
with a proper counterbalance and centrifuge at 500 Â g
until the sample remaining in the sample chamber is half
the original volume.
(d) Add approximately 100 μl of 20 mM HEPES buffer
(pH 7.0) to the sample chamber to bring the volume
back to 200 μl. Repeat steps 5b–d twice to further purify
the sample.
(e) After purification with the protein concentrator, return
the volume to 200 μl with 20 mM HEPES buffer
(pH 7.0).
6. See Note 8 for details on how to determine the extent of the
reaction between the PEGylated virus and the CPP.
3.5 Transduction
of NIH/3T3 Cells
For our purposes, we expose cells to a PBS control that allows us to
measure background gene expression levels. Also, additional Ad
virus is needed if one is interested in comparing the efficiency of
362
Yasmine Gabal and Joshua D. Ramsey
HEPES buffer (pH 8.0).
4. Dropwise, add 50 μl of the diluted PEG solution to the side of
the microcentrifuge tube containing the virus while gently
vortexing so that the final concentration of PEG is 4 μg/
10
6 vp.
5. Incubate for 45 min at room temperature while pulse vortexing
every 7–10 min.
6. See Note 7 for details on how to determine the extent of the
reaction between PEG and Ad.
3.4 Formation of
CPP-PEG-Adenovirus
1. Prepare an 800 μl stock solution of 2 mg CPP/ml in 20 mM
HEPES buffer (pH 7.0) (see Note 5).
2. Dilute 31.25 μl of the CPP stock solution into 18.75 μl of
20 mM HEPES buffer (pH 7.0).
3. Dropwise, add 50 μl of the CPP solution (from step 2) onto
the side of the microcentrifuge tube containing the PEGylated
virus while gently vortexing so that the final concentration of
CPP is 6.25 μg/10
6 vp.
4. Incubate for 45 min while pulse vortexing every 7–10 min.
5. Purify the CPP-PEG-Ad particles by removing unreacted peptide and polymer using a Pierce protein concentrator with
10 kDa MWCO, as follow:
(a) Place the sample into the concentrator sample chamber.
(b) Place the cap and the sample chamber and insert the
sample chamber into a collection tube.
(c) Place the concentrator assembly into the microcentrifuge
with a proper counterbalance and centrifuge at 500 Â g
until the sample remaining in the sample chamber is half
the original volume.
(d) Add approximately 100 μl of 20 mM HEPES buffer
(pH 7.0) to the sample chamber to bring the volume
back to 200 μl. Repeat steps 5b–d twice to further purify
the sample.
(e) After purification with the protein concentrator, return
the volume to 200 μl with 20 mM HEPES buffer
(pH 7.0).
6. See Note 8 for details on how to determine the extent of the
reaction between the PEGylated virus and the CPP.
3.5 Transduction
of NIH/3T3 Cells
For our purposes, we expose cells to a PBS control that allows us to
measure background gene expression levels. Also, additional Ad
virus is needed if one is interested in comparing the efficiency of
362
Yasmine Gabal and Joshua D. Ramsey