Gene Delivery and Gene Therapy for Alzheimer’s Disease - Gene Delivery and Therapy for Neurological Disorders - page 103

Table 3
Reagents

Reagent Volume (ml) Component Final concentration

2.1 Transfection into AAV-293 cells
Complete DMEM
(cDMEM) (1 l)

880 DMEM (Invitrogen,

12491-015)

100 Fetal bovine serum 10 %

10 Penicillin/

streptomycin

100 U/ml and
100 µg/ml

10 L -glutamine 2 mM

2× HEPES-buffered
saline (HBS)

NaCl 280 mM

HEPES 50 mM

Na 2 HPO 4 1.5 mM

pH 7.12

2.2 Crude AAV particle extraction
Cell suspension buffer

Tris-HCl, pH 8.5 50 mM

NaCl 150 mM

dH 2 O to volume

2.3 Iodixanol gradient purifi cation
PBS-MK (1 l)

100 10 × PBS 1×

2.0 0.5 M MgCl 2 1 mM

5.0 1 M KCl 5 mM

893 ddH 2 O
2.4 HiTrap Q HP column chromatography and concentration
Buffer W (wash buffer) Tris-HCl, pH 8.5 20 mM

NaCl 15 mM

ddH 2 O to volume

Buffer E (elution buffer) Tris-HCl, pH 8.5 20 mM

NaCl 500 mM

ddH 2 O to volume

3.2 Generation
and Purifi cation
of Recombinant AAV

rAAV particles are produced by transfection of AAV-293 cells with
three plasmids ( see Fig. 1 ). Two days post transfection, the cells are
harvested and crude rAAV particles are extracted by freeze-thaw
cycle. The particles are purifi ed with an iodixanol gradient, followed
by Sepharose column chromatography ( see Note 1 ).

1. The following components are needed:
(a) cis -plasmid pAAV2-MCS-WPRE-[cDNA of interest] from
Sect. 1
(b) p5E18RXC1: AAV1 trans plasmid with the AAV1 Rep and
Cap genes (Xiao et al. [ 37 ])
(c) pAd

3.2.1 Transfection into
AAV-293 Cells to Generate
Hybrid rAAV

∆ F6: adenoviral helper plasmid