Department of Radiology, Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital and Harvard Medical School, 149 13th Street, Suite 2301, Charlestown, MA, 02129, USA.
Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.
Sci Rep. 2020 Nov 26;10(1):20664. doi: 10.1038/s41598-020-77576-z.
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 ([Formula: see text], n = 10).
报告基因成像是一种非侵入性的监测活细胞内分子过程的方法，可以深入了解病理学和治疗学的机制。之前已经开发并使用富含赖氨酸的蛋白质（LRP）化学交换饱和传递（CEST）MRI 报告基因来对肿瘤细胞、心脏病毒基因转移和溶瘤病毒治疗进行成像。然而，LRP 报告基因序列的高度重复性质会导致 DNA 重组事件和一系列截断的 LRP 蛋白片段的表达，从而大大限制了 CEST 的敏感性。在这里，我们报告了使用重新设计的 LRP 报告基因（rdLRP），旨在提供出色的稳定性和 CEST 敏感性。rdLRP 不含 DNA 重复序列或 GC 丰富区，带正电荷的氨基酸减少了 30%。转染 rdLRP 的细胞裂解物的 RT-PCR 显示出稳定的报告基因，只有一条与全长 DNA 相对应的独特条带。转染 rdLRP 的细胞裂解物和体内表达 LRP 的小鼠脑肿瘤中的 CEST-MRI 对比明显增加（[Formula: see text]，n = 10）。
