1
D10 medium (Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, 100 U of penicillin per ml, 100 mg of streptomycin per ml, and 13 amphotericin B)
Maintenance of Cos and HeLa cell monolayer cultures
9261407
2
AAV4 stocks (American Type Culture Collection, no. VR-646)
Source of AAV4 for virus propagation
3
Helper plasmid (modified to remove homologous sequence)
Minimization of wild-type AAV particle formation during recombinant virus production
4
DNA sequence (ABI 373A automated sequencer, FS dye terminator chemistry)
DNA sequencing
5
PCR (Deep Vent Polymerase, New England Biolabs)
Cloning of ITR
6
XbaI and EcoRI
Restriction digestion post-PCR
7
Agarose gel electrophoresis
Purification of the amplified DNA band
8
Bluescript SKII+ (Stratagene)
Cloning vector for recovered DNA fragment
9
Competent Sure strain bacteria (Stratagene)
Transformation for plasmid propagation
10
Pfu polymerase (Stratagene)
PCR for producing helper plasmid
11
Simian virus 40 origin of replication containing plasmid
Helper plasmid construction for recombinant virus production
12
B-galactosidase reporter vector
Reporter gene for transduction efficiency analysis
13
Human erythrocytes (RBC) (type O)
Hemagglutination (HA) assay
14
EDTA-buffered saline
Dilution buffer for virus in HA assay
15
5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) (ICN Biomedicals)
Staining for β-galactosidase activity
16
Trypsin–0.02% EDTA solution (Biofluids)
Trypsinization of cells before transduction
