Rationale and Objectives-A reporter or marker gene that is detectable by in vivo imaging permits longitudinal monitoring of certain fundamental biological processes (eg, differentiation) within the context of physiologically authentic environments. Tissue-specific expression of a reporter gene can be achieved when it is under the transcriptional control of a tissue-specific promoter. The objective of this study was to construct a plasmid vector containing firefly luciferase (Fluc) marker gene downstream of the promoter sequence of rat ventricular myosin light chain 2 (MLC2v); to detect the in vivo expression of this cardiac-specific reporter (MLC2v-Fluc) in the mouse heart by bioluminescent imaging; and to correlate the bioluminescent signal with postmortem luminometer assay.Materials and Methods-MLC2v-Fluc plasmid was generated by molecular cloning of 3 kb promoter sequence into a pGL3-Basic vector containing the Fluc reporter. Twenty μg of MLC2v-Fluc plasmid DNA in phosphate-buffered saline was directly injected into mouse myocardium through a midline sternotomy.Results-At 1 week after injection, MLC2v-Fluc expression was detected by in vivo bioluminescent imaging in 60% of injected animals; the average in vivo signal intensity was (1.5 ± 0.6) × 10 4 radiance (p/sec/cm 2 /sr); in vivo signal was well above the detection threshold over 3 weeks after injection. In vivo bioluminescent signal is correlated (r 2 = 0.8) with the luminometer assay results from homogenized heart samples. Conclusion-The capability of noninvasive imaging of the MLC2v-Fluc in the heart will encourage applications that aim at monitoring and tracking the marker gene expression over time in cells undergoing cardiac differentiation. KeywordsCardiac ventricular myosin light chain 2 (MLC2v); bioluminescence; luciferase; cardiac; reporter gene Reporter (or marker) genes whose expression can be detected in vivo by noninvasive imaging modalities hold great promise for longitudinal monitoring of certain fundamental biological processes in a live animal. Reporter genes for various in vivo imaging modalities have been
We reported previously the in vivo detection of ectopic and transient expression of creatine kinase gene (ck) in the liver by phosphorus-31 magnetic resonance spectroscopy ((31)P MRS). Here we demonstrate the feasibility of using ck as a reporter gene to monitor the transfer of low-density lipoprotein receptor (LDLr) gene to LDLr(/) mice, a preclinical model for familial hypercholesterolemia. A recombinant adenovirus was generated that carries the creatine kinase gene (ck) and human LDL receptor gene (hLDLr) linked by an internal ribosomal entry site sequence. Intravenous injection of the adenovirus into LDLr(/)mice (1 x 10(11) viral particles/mouse) resulted in transduction of more than 90% of hepatocytes in the liver. Simultaneous expression of ck and LDLr was confirmed by Western analysis of the transduced livers. Through precise regulation of transgene expression in hepatocytes in vitro, an excellent correlation (R(2) = 0.96) between LDLr and ck expression was demonstrated over a wide range of viral dose. In vivo 31P MRS was employed to detect the metabolic product (i.e., phosphocreatine) of the creatine kinase protein (CK) reaction. CK activity, which is a true measure of ck gene expression, was quantified in vivo by magnetization transfer. Because ck is expressed abundantly in human muscle and brain but is absent from the liver, ck is useful to monitor any liver directed gene transfer. Use of the ck reporter would facilitate the clinical translation of gene therapy by providing a nondestructive readout of the level and duration of therapeutic gene expression.
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