Journal: Nature Communications
Article Title: Non-invasive imaging using reporter genes altering cellular water permeability
Figure Lengend Snippet: AQP1 enables the imaging of gene expression in intracranial tumour xenografts. ( a ) Experimental approach to establishing bilateral tumours in the striatum, inducing transgene expression, and performing diffusion-weighted MRI. ( b ) Representative diffusion-weighted image of a horizontal brain slice with bilateral tumour xenografts, 48 h after doxycycline injection. Inset shows a diffusion-weighted image of the same mouse acquired before doxycycline injection. Images were acquired at Δ eff =98 ms and b -value=1,000 s mm −2 . Dashed lines indicate the tumour ROI(s). Scale bar, 2 mm. ( c ) Average diffusion-weighted image intensity of AQP1-expressing tumours relative to contralateral GFP-expressing tumours before and after doxycycline induction. n =5 biological replicates. Error bars±s.e.m. ( d ) Confocal fluorescence image of a representative 100 μm section of a mouse brain implanted with GFP and AQP1 tumours. The AQP1 tumour appears dimmer due to diminished GFP translation from the IRES sequence. Cell nuclei are counterstained using TO-PRO iodide (red). Scale bar, 2 mm. ( e ) Low ( × 10) and ( f ) high ( × 30) magnification images of 5 μm haematoxylin–eosin-stained sections of intracranial tumour xenografts expressing AQP1 and GFP. Scale bars, 30 and 10 μm, respectively. ( g ) Longitudinal measurements of tumour growth in bilateral subcutaneous xenografts induced using doxycycline to express AQP1 or GFP 11 days following tumour inoculation. ( h ) Mean end-point tumour mass and ( i ) images of AQP1- and GFP-expressing subcutaneous tumours harvested 9 days after doxycycline induction of gene expression. n =4 biological replicates. Scale bar, 1 cm. Error bars±s.e.m.
Article Snippet: Construction of aquaporin and GFP-expressing cell lines Human AQP1 (NM_198098.1) and AQP4 (NM_001650.4) complementary DNAs were ordered from OriGene (Rockville, MD) and subcloned into a lentiviral vector downstream of a constitutive CMV or doxycycline-regulated CMV promoter (Clontech, Mountain View, CA) and an N-terminal FLAG tag.
Techniques: Imaging, Expressing, Diffusion-based Assay, Magnetic Resonance Imaging, Slice Preparation, Injection, Mass Spectrometry, Fluorescence, Sequencing, Staining