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Figure 1 | Stem Cell Research & Therapy

Figure 1

From: Neurotransplantation of stem cells genetically modified to express human dopamine transporter reduces alcohol consumption

Figure 1

Human DAT construct used in the stable transfection of C17.2 neural stem cells and characterization of its functional expression in selected clones. (a) Expression vector used for transfection. The construct contained the cytomegalovirus (CMV) enhancer element, chicken β-actin promoter, the coding sequence of hDAT, an intron (IVS), an internal ribosome entry site (IRES), and the coding sequence of the puromycin resistance gene. (b) Clones that survived selection with puromycin were screened for specific [3H]DA uptake (±1 mM cocaine). Several clones were chosen for expansion and further analysis based on their uptake of DA (arrows). Clones b7 (C17.hDAT) and f2 (C17.mock), identified by black arrows, were chosen for the transplantation and alcohol-consumption experiments. (c) Kinetic analysis of specific [3H]DA uptake in a C17.hDAT clone. Three independent experiments revealed average values for Km of 5 ± 1 μM and Vmax of 180 ± 5 pmol/min/mg protein. (d) In similar [3H]DA-uptake kinetic experiments, rat striatal synaptosomes had values for Km of 0.10 ± 0.02 μM and Vmax of 108 ± 27 pmol/min/mg protein (n = 3). (e) Selectivity experiments in the C17.hDAT clone revealed that [3H]DA uptake was blocked only by the DAT inhibitor (GBR 12909, 1 μM), but not by selective inhibitors of norepinephrine or serotonin transporters, desipramine, or fluoxetine (1 μM), respectively. *P < 0.01 compared with control, Tukey test (three independent experiments). (f) Western immunoblot for hDAT showing both mature, glycosylated (DAT) and immature, nonglycosylated forms of hDAT (ngDAT) in the C17.hDAT clone. Beta-actin was detected as a control for protein loading.

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