Biology Reference
In-Depth Information
specificity [
6
,
7
]. Combination of other components may also
allow to evade the host immune response [
8
] or to induce different
gene expression or gene regulation profiles.
As chimeric adenoviruses maintain most of their original
genes, their replication and assembling mechanisms should be
maintained. Therefore, chimeric HAdV-5-derived vectors must to
be produced in permissible HEK-293 cells. However, since chi-
merism can alter infectivity, efficiency, virion intracellular traffick-
ing, viral genome replication, and viral assembly, the standard
production protocols have to be adjusted for every new chimeric
vector. Indeed, as other adenoviral-derived vectors, chimeric
adenoviruses are genetically modified organisms (GMO) and have
to be produced on a Biosafety Level 2 laboratory.
2
Materials
1. Dulbecco's Modified Eagle's Medium (DMEM) supplemented
with 10 % or 2 % Fetal Bovine Serum (FBS).
2. HEK-293 cells.
3.
Pac
I restriction enzyme.
4. 150 mM NaCl.
5. Polyethylenimine (PEI).
2.1 Generation
of Viral Pre-stock
2.2 Purification
of Viral Pre-stock
1. Ultracentrifuge: Beckman Coulter Optima L90K o L100XP
with rotors SW32 and SW40Ti.
2. Polyallomer centrifuge tubes for SW32 and SW40 rotor.
3. CsCl solutions: 1.4 g/mL, 1.34 g/mL, and 1.25 g/mL in
1× PBS.
4. 18G needles, 2 mL syringes, pipette-aid, 10 mL pipettes.
5. PD-10 columns Sephadex G-25.
6. 1× PBS Ca
2+
/Mg
2+
.
7. Glycerol, anhydrid.
1. A viral pre-stock (fully characterized). It is recommended to
use vectors carrying a reporter gene.
2. HEK-293 cells.
3. Growing medium: DMEM supplemented with 10 % FBS and
1 % Penicillin-Streptomycin.
4. Infection medium: DMEM supplemented with 2 % FBS and
1 % Penicillin-Streptomycin.
5. 24-well and 96-well plates.
2.3 Tittering and
Analysis of Adenoviral
Replicative Cycle
