Biology Reference
In-Depth Information
plasmid as template and the oligonucleotides designed to
generate the S100E substitution (see Note 2).
2. Confirm the mutagenesis by sequencing the
pAAV2
.
1
-
CMV
-
S100E
plasmid.
3. Make a large plasmid preparation of the
pAAV2
.
1
-
CMV
-
S100E
using endotoxin-free solutions and materials.
The proper expression of the EPO and the S100E proteins from
the pAAV2.1 plasmids can be assessed in vitro prior to the genera-
tion of AAV vectors.
3.2 Expression
Analysis of the EPO
and the S100E
Proteins In Vitro
1. Maintain COS7 cells in DMEM containing 10% FBS and
2 mM l-Glutamine and 1× antibiotic-antimycotic solution.
2. Plate the cells in 6-well-plates at a density of 1 × 10
5
cell/well.
3. Twenty-four hours later, transfect the cells with 2
3.2.1 COS7 Cells
Transfection
g of either
pAAV2.1-CMV-EPO, pAAV2.1-CMV-S100E or pAAV2.1-
CMV-EGFP as negative control, using the PolyFect
Transfection Reagent (follow the protocol supplied by the
provider).
4. Thirty-six hours later incubate the cells in serum-free
DMEM.
5. Harvest the media and the cells 12 h later.
6. Collect the media and spin at 4000 ´
g
for 10 min at 4°C in a
microfuge to remove any cellular debris.
7. Store the media at 4°C (or −80°C for long storage).
8. Scrape the adherent cells in 1 mL of 1× PBS, spin the cells at
4000 ´
g
for 10 min at 4°C in a microfuge.
9. Lyse the cells in RIPA buffer (50
μ
L) in the presence of 1×
protease inhibitors and 1 mM PMSF for 30 min at 4°C.
10. Spin the cell lysates at 16,000 ´
g
for 15 min at 4°C in a
microfuge to remove the cellular debris and membranes.
11. Quantify both the cell lysates and the media using standard
procedures.
12. Separate the medium and lysate samples by 12% SDS-PAGE
and blot on a membrane following standard procedures (see
Note 11).
13. Block the membrane in 5% milk/TBS-T for 1 h on a shaker.
14. Incubate the membrane with the anti-EPO antibody diluted
1:400 in 5% milk/TBS-T for 2 h at room temperature on a
shaker.
15. Wash the membrane three times in TBS-T for 5 min each time
at room temperature on a shaker.
μ
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