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In Vitro MR Imaging of Regulated Gene Expression¹

¹ From the Department of Radiology, Philipps University, Baldingerstrasse, 35043 Marburg, Germany. Received December 7, 2001; revision requested February 18, 2002; revision received October 28; accepted December 10. Address correspondence to H.A. (e-mail: alfke@mailer.uni-marburg.de).

View larger version (39K): [in a new window]   Figure 1. Schematic representation of the tetracycline (tet)-regulated tyrosinase expression system. A, The pUHD15.1 vector constituently expresses the tetracycline transactivator (tTA, triangle). B, The expression of the human tyrosinase cDNA is under control of the tetracycline response element (TRE). Expression of the tyrosinase gene is induced only after the tetracycline-controlled transactivator binds to the tetracycline response element. C, Binding of doxycycline (Dox) to the transactivator inhibits binding to the tetracycline response element and gene expression. PCMV = cytomegalovirus promotor.

View larger version (86K): [in a new window]   Figure 2. Polyacrylamide gel electrophoresis: detection of tyrosinase expression in stable transfected MCF-7 cells under the control of the tetracycline-off regulated system by means of reverse transcription polymerase chain reaction, followed by the amplification of a tyrosinase-specific DNA fragment. Cell clones were grown with (+) or without (-) doxycycline in the supernatant. The arrow shows the tyrosinase-specific SI with the expected value of 156 base pairs (bp). Kb ladder = kilobase DNA ladder.

View larger version (171K): [in a new window]   Figure 3a. Photomicrographs show tyrosinase immunohistochemical and melanin staining. (a) Image obtained at immunohistochemical examination of tyrosinase-expressing cells (right) in comparison to control cells (left) shows the presence of the tyrosinase protein only in the former. (b) Melanin staining of tyrosinase-expressing cells shows brownish granular in the cytoplasm (arrows), which is identical to melanin (see text for details) (original magnification, x100).

View larger version (176K): [in a new window]   Figure 3b. Photomicrographs show tyrosinase immunohistochemical and melanin staining. (a) Image obtained at immunohistochemical examination of tyrosinase-expressing cells (right) in comparison to control cells (left) shows the presence of the tyrosinase protein only in the former. (b) Melanin staining of tyrosinase-expressing cells shows brownish granular in the cytoplasm (arrows), which is identical to melanin (see text for details) (original magnification, x100).

View larger version (122K): [in a new window]   Figure 4. T1-weighted three-dimensional fast low-angle shot gradient-echo (50/10.3; acquisition time for the entire experiment, 1 hour 8 minutes 18 seconds) MR image with cell clones that express or do not express human tyrosinase. Image of the two cell clones (clone 2, left column; clone 3, right column) cultured with conditions that allowed tyrosinase expression (top row) or did not allow gene expression (bottom row). For imaging, 107 cells were pelleted by means of centrifugation in the bottom of 1.5-mL Eppendorf tubes. All the tubes were imaged together in the experiments. Cultured with iron- and holotransferrin-enriched medium, cells that were tyrosinase positive showed higher SI on T1-weighted MR images because of the presence of metallomelanin (see text for details).