Journal: Journal of Veterinary Pharmacology and Therapeutics

Article Title: Functional Characterization of the Cat and Dog Wild‐Type and Mutant MDR1 Carrier Proteins and Frequency of the MDR1 Gene Mutation in 800 Cats From Germany

doi: 10.1111/jvp.70041

Figure Lengend Snippet: Transwell transport experiments with Rh123 and [ 3 H]digoxin in MDCKII cells. MDCKII cells lacking endogenous canine MDR1 (MDCKII MDR1ko ) were stably transfected with the canine MDR1 wild‐type (dog MDR1 wt), canine MDR1 knockout (dog MDR1 mut), feline MDR1 wild‐type (cat MDR1 wt), and feline MDR1 knockout (cat MDR1 mut) constructs, respectively. Non‐transfected MDCKII MDR1ko cells and the parent MDCKII cell lines were additionally analyzed for control. Transport experiments were performed in both apical‐basolateral (AB) and basolateral‐apical (BA) directions in the presence (blue bars) and absence (red bars) of the MDR1 inhibitor tariquidar (TQD). Samples from the donor chamber and the receiver chamber were taken at different time points. Fluorescence of Rh123 was quantified using a fluorescence plate reader. The amount of radiolabeled [ 3 H]digoxin in the samples was measured by liquid scintillation counting. Efflux ratios (ER) were calculated as described in the Material and Methods section. Data are means ± SD from three independent experiments each with triplicate determinations ( n = 9, Rh123) or from two independent experiments each with triplicate determinations ( n = 6, [ 3 H]digoxin). *Significantly higher in the absence of TQD compared to the presence of TQD according to two‐way ANOVA with p < 0.05.

Article Snippet: For transwell transport experiments in the MDCKII cell lines, 3 × 10 5 cells were seeded per 12‐mm/0.4 μm transwell membrane inserts (Sarstedt, Numbrecht, Germany).

Techniques: Stable Transfection, Transfection, Knock-Out, Construct, Control, Fluorescence