Development of a New Reporter Gene System—dsRed/Xanthine Phosphoribosyltransferase-Xanthine for Molecular Imaging of Processes Behind the Intact Blood-Brain Barrier

Abstract

We report the development of a novel dual-modality fusion reporter gene system consisting of Escherichia coli xanthine phosphoribosyltransferase (XPRT) for nuclear imaging with radiolabeled xanthine and Discosoma red fluorescent protein for optical fluorescent imaging applications. The dsRed/XPRT fusion gene was successfully created and stably transduced into RG2 glioma cells, and both reporters were shown to be functional. The level of dsRed fluorescence directly correlated with XPRT enzymatic activity as measured by ribophosphorylation of [14C]-xanthine was in vitro (Ki = 0.124 +/- 0.008 vs. 0.00031 +/- 0.00005 mL/min/g in parental cell line), and [*]-xanthine octanol/water partition coefficient was 0.20 at pH = 7.4 (logP = -0.69), meeting requirements for the blood-brain barrier (BBB) penetrating tracer. In the in vivo experiment, the concentration of [14C]-xanthine in the normal brain varied from 0.20 to 0.16 + 0.05% dose/g under 0.87 + 0.24% dose/g plasma radiotracer concentration. The accumulation in vivo in the transfected flank tumor was to 2.4 +/- 0.3% dose/g, compared to 0.78 +/- 0.02% dose/g and 0.64 +/- 0.05% dose/g in the control flank tumors and intact muscle, respectively. [14C]-Xanthine appeared to be capable of specific accumulation in the transfected infiltrative brain tumor (RG2-dsRed/XPRT), which corresponded to the 585 nm fluorescent signal obtained from the adjacent cryosections. The images of endogenous gene expression with the "sensory system" have to be normalized for the transfection efficiency based on the "beacon system" image data. Such an approach requires two different "reporter genes" and two different "reporter substrates." Therefore, the novel dsRed/XPRT fusion gene can be used as a multimodality reporter system in the biological applications requiring two independent reporter genes, including the cells located behind the BBB.

Tracking Stem Cells for Cellular Therapy in Stroke

“…In addition, grafted cells that express HSV1-tk are vulnerable to anti-herpes drugs, such as acyclovir and gancyclovir [75,76], thus it will be important to identify or develop alternative enzyme-substrate systems that are compatible with PET. One example of an alternative system is the xanthine phosphoribosyl transferase reporter enzyme, which has the added advantage that xanthine reporter probes can cross the BBB [77]. Further characterization of this and other probe systems will help increase the utility of PET for monitoring transplanted cells.…”

Noninvasive Molecular Neuroimaging Using Reporter Genes: Part II, Experimental, Current, and Future Applications

“…Another study that exemplifies a search for new reporter systems with capabilities of imaging processes behind an intact BBB is that of Doubrovin et al, 83 who investigated Escherichia coli xanthine phosphoribosyltransferase for nuclear imaging with radiolabeled xanthine. Again, by using autoradiography, they found that 14 C xanthine was capable of specific accumulation in transfected infiltrative brain tumors.…”

Imaging Virus-Associated Cancer

“…A few examples include use of the herpes simplex virus type 1 TK (HSV1-TK) [17], Escherichia coli (E. coli) and yeast cytosine deaminases (CD and yCD) [18], and E. coli xanthine phosphoribosyltransferases (XPRT) [19]. Among these, the wild type and mutant HSV1-tk genes are the most frequently used PET reporter genes.…”