Notes

1. The HEK293 cell line used in this study was obtained from Quantum Biotechnologies Inc. and was grown on DMEM media with 5% FBS. For 6-cm, 10-cm, and 15-cm culture dishes, the amount of media used was 5 mL, 10 mL, and 25 mL, respectively.

2. The pAdEasy adenoviral expression system used in this study is described in detail at http://www.coloncancer.org/adeasy/protocol.htm and in ref. 8. The adenoviral system described here lacks the E1 gene that is required for replication. Therefore, the obtained recombinant adenoviruses need to be amplified in HEK293 cells, which express the E1 gene and facilitate the amplification of the virus.

3. Lipofectamine transfection was carried out according to the manufacturer's (Invit-rogen) protocol and a description of this transfection technique can be found in Chapter 5 of this volume.

4. Although we used for infection of b-cell lines the GFP adenovirus here, the same conditions apply also for infection with other recombinant adenoviruses containing b-cell-specific genes such as the pancreas-specific transcription factor PDX-1.

5. This step should yield about 106-107 pfu/mL. At this point, the presence of the recombinant adenovirus can be checked either by polymerase chain reaction (PCR) or by immunoblotting with specific antibodies.

6. We have obtained 4 mL of viral supernatant with a titer of about 1 X 109 pfu/mL from six 15 cm of 80% confluent HEK293 cells infected with the GFP adenovirus. The viral titer is determined by infecting confluent HEK293 cells with a 1: 10,000 dilution of the concentrated GFP adenovirus for 18-24 h, as described previously (9). After this incubation period, the number of GFP-positive cells per field is counted three times using 100 X magnification. The average number of GFP-positive cells is multiplied by 107 and the obtained number is defined as plaque-forming units per milliliter. This number should be proportional to the number of infective viral particles in the amplified adenoviral preparation.

7. For decontamination of recombinant adenoviruses, dishes, pipets, and tubes were rinsed with 10% bleach and autoclaved.

8. The high level of protein in the DMEM + 10% FBS and RPMI + 10% FCS can interfere with the binding of the adenovirus particles to their cellular receptors. For this reason, washing with 1X PBS is critical to the infection process as well as the use of a serum-free media during the infection.

9. The amount of virus needed will be dependent on the cell type. For p-cell lines, a MOI of 30-100 (30-100 virus particles per cell) is a good starting point.

10. The length of infection time will be dependent on the cell type as well as the gene that is being introduced.

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