Biology Reference
In-Depth Information
4.
Vector purity assessment
. 40 % acrylamide solution (AppliChem;
#A4989); 10 % ammonium persulfate in H
2
O (APS;
AppliChem; #A2941); tetramethylethylenediamine (TEMED;
Sigma-Aldrich, St-Louis, MO, USA; #T9281); gel cassette
and electrophoresis system (Mini-PROTEAN Tetra
Electrophoresis System, Bio-Rad, Hercules, CA, USA);
SimplyBlue SafeStain (Life Technologies, #LC6060)
4× loading buffer: mix 6 g sodium dodecylsulfate (SDS), 3 ml
β
-mercaptoethanol, 15 g glycerol and 25 mg Coomassie G250
(Bio-Rad; #161-0406). Complete the volume to 50 ml with
TrisHCl 150
μ
m pH 7.0.
1.
Injection
. 10
l syringe (Microliter™; Hamilton, Reno, NV,
USA); polyethylene tubing (A-M Systems, Sequim, WA,
USA); 30G insulin syringe (BD Micro-Fine™; Becton
Dickinson, Franklin Lakes, NJ, USA); Fast Green FCF (Sigma-
Aldrich, St-Louis, MO, USA; #F7258)
2.
Animal anesthesia
. Medetomidine 1 mg/ml (Dorbene
®
; Dr.
Graeub AG, Bern, Switzerland); Midazolam 5 mg/ml
(Dormicum
®
, Roche, Basel, Switzerland); Atipamezol 1 mg/
ml (Alzane
®
; Dr. Graeub AG) (
see
Note 5
)
3.
Tissue analysis
. Pentobarbitalum natricum 150 mg/kg
(Esconarkon
®
, Streuli Pharma, Uznach, Switzerland); 5UI/
ml Heparinum natricum (Liquemin
®
, Drossapharm, Basel,
Switzerland); 4 % Paraformaldehyde (PFA) (Sigma-Aldrich,
St-Louis, MO, USA; #P6148); 30 % sucrose solution (in PBS)
to prevent freezing artifacts; Cryomatrix (Thermo Scientifi c,
Waltham, MA, USA); PBS-azide solution; Bovine serum albu-
min (BSA) (PAA Laboratories, Pasching, Austria; #K41-001);
Triton
®
X-100 (Sigma-Aldrich; #T8787); Mouse anti-GFP
antibody mAb3E6 (Molecular Probes
®
, Life Technologies;
#A11120); Alexa Fluor 488 goat anti-mouse antibody
(Molecular Probes
®
; #A11029); Mowiol (Calbiochem, San
Diego, CA, USA)
μ
2.3 AAV6 Vector
Injection and Analysis
of Transgene
Expression
3
Methods
3.1 AAV6 Vector
Production
1.
Packaging cells
. 4
E
7 HEK 293-AAV cells are plated in 500 cm
2
Petri dishes and grown in DMEM supplemented with 10 %
FBS and 1 % penicillin/streptomycin. Typically, fi ve cell facto-
ries are needed for one AAV6 vector production for in vivo
applications.
2.
Transfection
. One day post-plating, prepare the transfection
mix for each cell factory as follows: mix 2.5 ml CaCl
2
0.5 M,
200
μ
g helper plasmid (pDP6), and 100
μ
g shuttle plasmid
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