Abstract
Reporter gene imaging allows for non-invasive monitoring of molecular processes in living cells, providing insights on the mechanisms underlying pathology and therapy. A lysine-rich protein (LRP) chemical exchange saturation transfer (CEST) MRI reporter gene has previously been developed and used to image tumor cells, cardiac viral gene transfer, and oncolytic virotherapy. However, the highly repetitive nature of the LRP reporter gene sequence leads to DNA recombination events and the expression of a range of truncated LRP protein fragments, thereby greatly limiting the CEST sensitivity. Here we report the use of a redesigned LRP reporter (rdLRP), aimed to provide excellent stability and CEST sensitivity. The rdLRP contains no DNA repeats or GC rich regions and 30% less positively charged amino-acids. RT-PCR of cell lysates transfected with rdLRP demonstrated a stable reporter gene with a single distinct band corresponding to full-length DNA. A distinct increase in CEST-MRI contrast was obtained in cell lysates of rdLRP transfected cells and in in vivo LRP expressing mouse brain tumors (p=0.0275, n = 10).
Highlights
Magnetic resonance (MR) reporter genes can non-invasively monitor transgene expression in vivo at high resolution with unlimited tissue p enetration[1]
The lysine-rich protein (LRP) reporter gene in its current form is unstable, as the highly repetitive DNA sequence encoding the reporter leads to DNA recombination events and the expression of a range of truncated protein fragments with fewer lysine amide protons, resulting in decreased and limited chemical exchange saturation transfer (CEST) s ensitivity[5,32]
We report the redesign of an improved, stable, and robust LRP-based CEST-MRI reporter gene, using an RHGP amino acid motif that consisted of arginine, histidine, glycine, and proline amino acids separated by different numbers of lysines
Summary
Magnetic resonance (MR) reporter genes can non-invasively monitor transgene expression in vivo at high resolution with unlimited tissue p enetration[1]. Turnbull and their colleagues have taken a creative approach to express a multi-component reporter in endothelial c ells[10] Another strategy relied on overexpression of the organic anion transporting protein (Oatp1a1). An alternative approach was to use hyperpolarized xenon as a substrate for enhancing the MRI contrast of a genetically encoded r eporter[19]. All these innovative approaches relied on administration of a substrate or a secondary compound that is not accessible to all tissues and requires repetitive injections for longitudinal imaging. The reporter stability and contrast enhancement ability were evaluated in cell lysates from human embryonic kidney (HEK293T) and murine glioma (GL261N4) cell lines as well as from in vivo LRP expressing brain tumors in mice
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