Adenoviral Gene Transfer into Insulinoma Cell Lines

In order to optimize the infection of insulinoma cell lines, it is important to test the efficiency of each constructed recombinant adenovirus individually. Efficiency of gene transfer can vary based on the stability and toxicity of the expressed gene product itself in insulinoma cells. The adenoviral gene transfer

Insulinoma Cell Line

Fig 1. Analysis of the infection efficiency using different titers of the GFP adenovirus. (A): MIN6 cells infected with various titers of the GFP adenovirus. The adenoviral titer is given next to each panel. After the addition of the various concentrations of the adenovirus, plates were incubated for 1 h at 37°C with gentle agitation and then placed in a 37°C humidified incubator with 10% CO2 for an additional 17 h. (B): INS-1 cells infected with various titers of the GFP adenovirus. The infection procedure is the same as described in (A).

method described in this chapter is optimized for use with the pAdEasy adenovirus system (8).

Infection of a confluent mouse insulinoma (MIN6) cell line (approx 1 X 106 cells) with different titers of a recombinant adenovirus that only expresses GFP [obtained after recombination of pAdTrackCMV and pAdEasy-1 vectors in bacteria (8)] resulted in infection of 85% of cells using an adenoviral titer of 1 X 108 plaque-forming unit (pfu)/mL (see Fig. 1A). This corresponds to a MOI (multiplicity of infection) of 100. Concentrations of virus greater than or equal to 1 X 109 resulted in very high GFP expression but appeared to be toxic to MIN6 cells with more than 20% of the cells detaching from the plate. The infection efficiency for the rat insulinoma-1 (INS-1) cells (approx 2 X 105 cells) with the GFP adenovirus was close 100% using an adenoviral titer of 1 X 107 pfu/mL, corresponding to a MOI of 50 (Fig. 1B). Increasing the adenoviral titer does not necessarily result in linear increases in protein expression levels, as shown in Fig. 2. The infection efficiency can be increased not only by using higher adenoviral titers but also by prolonging the length of infection. When using an adenoviral titer of 1 X 107 pfu/mL, MIN6 cells display maximal infection efficiency when they are exposed to the GFP adenovirus for 18 h. INS-1 cells also give

Fig. 2. Western blot analysis of GFP protein levels in MIN6 cell extracts infected with the GFP adenovirus using a monoclonal anti-GFP antibody (Clontech). The adenoviral titer used for infection of the MIN6 cells is given above each lane.

maximal infection efficiency with a titer of 1 X 106 pfU/mL when exposed to it for 18 h. Exposure of either cell line to the given concentrations of virus for 24 h did not result in increased infection, but in both cases, it leads to the detachment of more than 20% of the cells. These experiments show that although MIN-6 and INS-1 cells are both insulinoma cell lines, they display different infection efficiencies. Maximal infection of INS-1 less requires about 10-fold less virus than MIN-6 cells

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