The cortical collecting duct regulates ion and water homeostasis in the kidney. A cell line denoted M-1 has been established from the cortical collecting duct of a transgenic mouse that exhibits many functional properties of the original tissue . While this cell line is valuable for in vitro studies, the cells are not easily transfected. Frequently, adenovirus-mediated gene transfer is used . As adenovirus production requires several additional steps, we prefer chemical methods to achieve recombinant protein expression in M-1 cells. Previous attempts to transfect this cell line were unsuccessful with far less than 10% of transfectants, although different reagents were tried. Here we compare three commercially available transfection reagents in terms of the percentage of transfected cells and the suitability for using a reporter construct in M-1 cells.
Materials and Methods
Transient EGFP transfection
One day prior to transfection, M-1 cells were seeded in 24-well plates at 60.000 cells per well for transfection reagent A and FuGENE® 6 Transfection Reagent, and at 150.000 cells for FuGENE® HD Transfection Reagent. Cells were maintained in 0.5 ml/well DMEM/F12 medium supplemented with 10% serum without dexamethasone.
For transfection reagent A, cells were transferred to DMEM/F12 without serum. 3 µl (condition 1) or 15 µl (condition 2) transfection reagent were diluted in 150 µl medium and incubated for 45 minutes. To both mixtures, 150 µl medium containing 3 µg EGFP plasmid was added. They were incubated for 15 minutes, and finally diluted with 1.2 ml medium each. To each well, 0.5 ml of the transfection complex was added and incubated for 24 hours. Then the medium was replaced with medium containing 10% serum and the cells were evaluated by fluorescence microscopy after a further 24 hours.
For FuGENE® 6 Transfection Reagent, 2 µl (condition 1) or 4 µl (condition 2) were diluted in 70 µl serum-free DMEM/F12. After 5 minutes of incubation, 1.4 µg (condition 1) or 0.7 µg (condition 2) EGFP plasmid were added and incubated for 15 minutes. To each well, 20 µl of the mixture was added. Cells were evaluated without replacing medium after 48 hours.
For FuGENE® HD Transfection Reagent, 3 µl (condition 1) or 7 µl (condition 2) were diluted in 100 µl medium containing 2 µg EGFP plasmid was added and incubated for 15 minutes. To each well, 25 µl of the mixture was added and cells were evaluated without replacing medium after 48 hours.
Transient co-transfection and reporter gene assay
Cells were seeded as described for EGFP but kept in medium containing charcoal treated serum. Transfection was performed as above; however, the total amount of DNA comprised equal proportions of a luciferase reporter construct, a constitutively expressed galactosidase plasmid, and a tagged receptor construct. 24 hours post transfection, cells were stimulated with 1 nM steroid and harvested by lysis (0.5% Triton X-100 in phosphate buffer containing protease inhibitors) 24 hours later. Luciferase and galactosidase acitivities were quantified and luciferase activity was normalized to galactosidase.
Results and Discussion
In line with our previous experiments, transfection with both transfection reagent A and FuGENE® 6 Transfection Reagent resulted in a very modest number of transfected cells (Figure 1). While there was no difference between conditions 1 (1 µl reagent per µg DNA) and 2 (5 µl reagent per µg DNA) for transfection reagent A, the higher reagent:DNA ratio (6:1 compared with 3:2) appeared to be slightly better for FuGENE® 6 Transfection Reagent. For FuGENE® HD Transfection Reagent, however, near the upper end of the recommended range (7 µl reagent per 2 µg DNA) we observed almost 50% of transfected cells. The lower reagent concentration (3 µl reagent per 2 µg DNA) was considerably less effective. We did not test more conditions, so it may be possible to obtain a higher percentage.
Reporter gene assay
In order to obtain a reliable quantification of reporter gene expression, both luciferase and the gene used for normalization, galactosidase, obviously have to be expressed at levels that are significantly higher than background values.
In our hands, transfection reagent A yielded expression levels of both galactosidase (Figure 2a) and luciferase that were very close to blank values, which led to an apparent lack of promotor induction by steroid in these experiments (Figure 2b). With the higher protein expression achieved when using FuGENE® 6 Transfection Reagent, a modest induction by steroid was visible. Much better results were obtained using FuGENE® HD Transfection Reagent. With this reagent, an almost tenfold induction was observed, which is similar to what we observed in cell lines that are transfected more easily such as HEK293.
From our results, we conclude that FuGENE® HD Transfection Reagent performs superior to other reagents we have tested in M-1 cells up to now. Although we did not extend the concentration range recommended by the manufacturer, M-1 cells are likely to require a reagent:DNA ratio at the upper end for high efficiency.
1. Stoos BA et al. (1991) Kidney Int 39:1168–1175
2. Naray-Fejes-Toth A et al. (2004) Proc Natl Acad Sci USA 101:17434–17439
This article was originally published in Biochemica 3/2007, pages 30-31. ©Springer Medizin Verlag 2007