In vivo imaging of mlc2v-luciferase, a cardiac-specific reporter gene expression in mice1

Abstract

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

Imaging Stem Cells Implanted in Infarcted Myocardium

“…Cardiac specific GFP reporter (or its variants) would be extremely useful for screening or selection of stem cells undergoing cardiac differentiation by fluorescent microscopy or flowcytometry (161). To monitor stem cells undergoing cardiac differentiation in vivo, luminescent (e.g., Fluc) or NIS reporter under cardiac specific promoters would be suitable; in vivo luminescent imaging of MLC2v-Fluc expression in the mouse heart has been achieved (47), suggesting that this approach is feasible for monitoring cardiac differentiation of stem cells in small animals. Gamma camera and micro-PET imaging of cardiac alpha myosin heavy chain-NIS expression in the heart in a transgenic mouse model has been demonstrated recently (139); this reporter gene is more relevant to clinical application than Fluc because of the nonimmunogenic nature of NIS and the availability of clinical counterparts of the imaging modalities.…”

“…Reporter genes would be extremely useful in assessing survival status of the implanted cells because the reporter will be expressed as long as the cells are alive and will be passed to daughter cells upon cell division; this approach has been used to monitor the uncontrolled growth of embryonic stem cells into a tumor (teratoma) when a large number of cells are implanted, as shown in Figure 1D. If a cardiac specific promoter is used to control the expression of the reporter (46,47), then the cardiac differentiation of stem cells in their physiological environment also can be monitored. The reporter gene approach would allow selection for optimal cell type and engraftment sites that promote survival or differentiation of implanted cells.…”

In vivo bioluminescence for tracking cell fate and function

“…This provides exciting opportunities for transcriptional targeting, double reporter labeling for BLI monitoring, as well as gene therapy. For example, by linking the expression of luciferase to the cardiac-specific promoter myosin light chain 2v, it is possible to monitor cells undergoing cardiac differentiation over time via the detection of reporter gene expression by BLI (27,35).…”

Emerging Approaches for Cardiovascular Stem Cell Imaging

“…In addition to cell survival and proliferation, BLI can be also used to track stem cell differentiation by inserting the luciferase reporter gene downstream of tissue-specific promoters [44].…”