Biology Reference
In-Depth Information
3. MEM eagle, Joklik modifi cation (MEM, Life Technologies/
Gibco, Darmstadt, Germany) for cultivating 116 cells in sus-
pension, supplemented with 5 % FCS, 100 U/mL penicillin-
streptomycin, 2 mM L-glutamine, and 0.1 mg/mL hygromycin B.
4. Fetal calf serum (FCS).
5. Trypsin-EDTA.
6. Penicillin-Streptomycin-Glutamine.
7. PBS w/o Ca
2+
, Mg
2+
.
8. 60-mm and 150-mm cell culture dishes for adherent cells.
9. Spinner fl asks, preferably 250-mL and 3 L. Magnetic stirrer.
1. JetPEI (Polyplus, Illkrich, France).
2. HC-Ad vector plasmid.
3. Optional, but recommended: pHC-AdEGFP as control for
transfection/amplifi cation.
4. Helper virus.
5. PBS w/o Ca
2+
and Mg
2+
.
2.2 HC-Ad
Vector Rescue
2.3 HC-Ad Vector
Serial Amplifi cations
1. Optional but recommended: fl uorescence microscope with
appropriate laser/fi lter set to excite EGFP (488 nm excitation,
512 nm emission).
2. Helper virus and the corresponding plasmid.
3. QIAamp DNA Mini Kit or equivalent.
4. Glycerol 100 %.
5. Liquid nitrogen and an appropriate container for freeze-thaw.
2.4 HC-Ad Vector
Purifi cation
1. Ultracentrifuge, e.g., Beckman XPN-90 with SW41Ti rotor.
2. Ultraclear centrifuge tubes 12.8 mL.
3. HEPES 50 mM pH 8.0.
4. PD-10 disposable desalting columns.
5. Glycerol 100 %.
6. 2 mL syringes and 18-G needles.
3
Methods
Careful routine cultivation of 116 cells is mandatory for successful
rescue and amplifi cation of HC-Ad vectors. Only healthy cells with
a high level of
Cre
expression allow for high-titer HC-Ad vector
preparations with low helper virus contamination. Furthermore, a
well-organized cultivation schedule guarantees availability of
healthy cells when needed for the different amplifi cation steps and
3.1 Cultivation
of 116 Cells
