Testing Functionality

Having designed and built a transgenic reporter gene, it is extremely prudent to test whether it is capable of acting as a functional expression unit prior to microinjection. This is easily achieved by transfection of the intact plasmid into cultured cells. Most cell lines are suitable for this purpose, although there may be instances where your particular reporter gene is not expressed in a certain cell line (see Note 9). A standard, inexpensive, transfection method using calcium phosphate is described (51), although other methods, such as those utilizing liposome formulations, are equally suitable.

1. Harvest cells by trypsinization, and reseed at a density of 1-3 x 105/cm2 in 85mm diameter tissue-culture dishes. Incubate for 4-16 h at 37°C in a CO2 incubator to allow the cells to adhere to the dish.

2. Prepare DNA for each point in a sterile microfuge tube containing; 10 pg of DNA, 95 pL of 2 M CaCl2, and sterile distilled water up to a total volume of 750 pL (see Note 10).

3. Slowly add each DNA sample dropwise to a tube (e.g., Falcon 2059) containing 750 pL of 2X HBS, with continuous mixing by gentle vortexing. Incubate the samples at room temperature for 30 min to allow a DNA-calcium phosphate coprecipitate to form.

4. Add fresh cell-culture medium, and then overlay the precipitate onto the cells, swirling gently to ensure even distribution. Incubate cells for 20 h at 37°C in a CO2 incubator.

5. Remove the medium, and wash the cells twice with warm (37°C) PBS. Add fresh medium, and reincubate the cells for 24 h.

6. Rinse the cell monolayer twice with wash solution, and incubate at room temperature for 5 min with 8 mL of cell fixative. Wash three times for 5 min at room temperature with wash solution.

7. For lacZreporters, add 8 mL of X-gal stain solution, and incubate at 37°C in the dark for 1 h to overnight (see Note 11). Wash cells to remove stain solution, and store at 4°C in 50% (v/v) ethanol in PBS.

8. For alkaline phosphatase reporters, heat-treat the cells for 45 min at 65°C in PBS to reduce preferentially the activity of endogenous alkaline phosphatases. Add 8 mL of AP stain solution, and incubate at room temperature in the dark for 1 h to overnight. Wash cells with PBS containing 20 mM EDTA to stop the staining reaction, and store at 4°C in 50% (v/v) ethanol in PBS.

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