Abstract
The human tyrosinase gene TYR is a multifunctional reporter gene with potential use in photoacoustic imaging (PAI), positron emission tomography (PET), and magnetic resonance imaging (MRI). We sought to establish and evaluate a reporter gene system using TYR under the control of the Tet-on gene expression system (gene expression induced by doxycycline [Dox]) as a multimodality imaging agent. We transfected TYR into human breast cancer cells (MDA-MB-231), naming the resulting cell line 231-TYR. Using non-transfected MDA-MB-231 cells as a control, we verified successful expression of TYR by 231-TYR after incubation with Dox using western blot, cellular tyrosinase activity, Masson-Fontana silver staining, and a cell immunofluorescence study, while the control cells and 231-TYR cells without Dox exposure revealed no TYR expression. Detected by its absorbance at 405 nm, increasing concentrations of melanin correlated positively with Dox concentration and incubation time. TYR expression by Dox-induced transfected cells shortened MRI T1 and T2 relaxation times. Photoacoustic signals were easily detected in these cells. 18F-5-fluoro-N-(2-[diethylamino]ethyl)picolinamide (18F-5-FPN), which targets melanin, quickly accumulated in Dox-induced 231-TYR cells. These show that TYR induction of melanin production is regulated by the Tet-on system, and TYR-containing indicator cells may have utility in multimodality imaging.
Highlights
On reporter genes, using a probe that binds to the gene product
In gene therapy and related gene studies[26], it has been demonstrated that the timing and degree of gene expression with an activator substance is much better than the sustained expression of a gene product, as the sustained expression of exogenous genes or proteins may result in some unexpected adverse effects[27]
Melanin began to decrease 4 h after the withdrawal of Dox and returned to normal levels at about 48 h (Fig. 4C). This suggests that Dox should be withdrawn in advance if we want to stop the effect of the reporter gene
Summary
On reporter genes, using a probe that binds to the gene product. Based on the same principle, some PET probes, such as (N-[2-(diethylamino) ethy1]-6-18F-fluoropicolinamide) (18F-MEL050), have demonstrated high and specific binding to melanin both in vitro and in vivo[22]. Previous studies have demonstrated that TYR can be used as a multifunctional reporter gene for PAI/ MRI or PAI/MRI/PET imaging both in vitro and in vivo[24,25]. TYR, to act as a reporter gene, needs to be transfected and integrated into cells, and the Tet-on tetracycline gene induction system is widely used for inducible expression, as it can effectively control gene expression in vivo and in vitro using doxycycline (Dox) as the activator[27,32]. The system was evaluated in vitro under the control of Dox for providing the feasibility of multimodality imaging
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