Related Experiment Video
Updated: Aug 15, 2026
12:19
In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells
Published on: May 2, 2008
Effects of epigenetic modulation on reporter gene expression: implications for stem cell imaging
Manickam Krishnan1, Jinha M Park, Feng Cao
1The Molecular Imaging Program at Stanford (MIPS), The Department of Radiology and Bio-X Program, Stanford University, Stanford, CA 94305-5344, USA.
Summary
Reporter gene silencing in stem cells is reversed by epigenetic modulation. 5-azacytidine treatment restored firefly luciferase expression, improving in vivo imaging of transplanted cells.
Area of Science:
- Stem cell biology and regenerative medicine.
- Epigenetics and gene expression regulation.
- Biotechnology and bioimaging.
Background:
- Tracking transplanted stem cells requires stable reporter gene expression.
- Reporter gene silencing is a common challenge in long-term stem cell studies.
- Epigenetic mechanisms, such as DNA methylation, can silence reporter genes.
Purpose of the Study:
- To investigate the long-term stability of reporter gene expression in embryonic rat cardiomyoblasts.
- To determine the role of epigenetic modulation in reversing reporter gene silencing.
- To assess the efficacy of 5-azacytidine in restoring reporter gene expression for in vivo imaging.
Main Methods:
- Stable transfection of embryonic rat cardiomyoblasts with CMV-Fluc reporter construct.
- Long-term serial passaging of transfected cells (3-8 months).
- Treatment with epigenetic modulators (5-azacytidine, trichostatin A, retinoic acid).
- Quantification of firefly luciferase activity, mRNA, and protein levels.
- DNA methylation analysis of the CMV promoter.
- In vivo bioluminescence imaging (BLI) of transplanted cells.
Main Results:
- Progressive decrease in firefly luciferase activity observed over 60 passages.
- 5-azacytidine treatment maximally rescued reporter gene activity compared to other agents.
- Increased firefly luciferase mRNA and protein levels following 5-azacytidine treatment.
- Inverse correlation between 5-azacytidine dosage, promoter methylation, and reporter gene expression.
- Extended in vivo monitoring duration for 5-azacytidine treated cells via BLI.
Conclusions:
- Reporter gene expression in stem cells is prone to epigenetic silencing over time.
- DNA methyltransferase inhibition with 5-azacytidine effectively reverses reporter gene silencing.
- Epigenetic modulation can enhance the utility of reporter genes for long-term stem cell tracking.
- These findings have significant implications for improving stem cell transplantation imaging.
Related Concept Videos
02:11Reporter Genes
Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
Commonly used reporter...
Commonly used reporter...
01:32Chromatin Modification in iPS Cells
Chromatin modification alters gene expression; therefore, scientists can add histone-modifying enzymes, histone variants, and chromatin remodeling complexes to somatic cells to aid reprogramming into pluripotent stem (iPS) cells.
Compact chromatin makes reprogramming difficult. Enzymes, such as histone demethylases and acetyltransferases, are often added during reprogramming to loosen the chromatin, making the DNA more accessible to transcription factors. Molecules that inhibit histone...
Compact chromatin makes reprogramming difficult. Enzymes, such as histone demethylases and acetyltransferases, are often added during reprogramming to loosen the chromatin, making the DNA more accessible to transcription factors. Molecules that inhibit histone...
01:29Somatic to iPS Cell Reprogramming
Reprogramming alters the gene expression in somatic cells, transforming them into induced pluripotent stem (iPS) cells over several generations. Scientists can reprogram cells by introducing genes for four transcription factors—Oct4, Sox2, Klf4, and c-Myc (OSKM) by viral or non-viral methods. These factors are also known as Yamanaka factors after Shinya Yamanaka, who first generated iPS cells using mouse skin cells. Yamanaka was awarded the Nobel Prize in Physiology or Medicine in 2012 for this...
01:37Epigenetic Regulation
Epigenetic changes alter the physical structure of the DNA without changing the genetic sequence and often regulate whether genes are turned on or off. This regulation ensures that each cell produces only proteins necessary for its function. For example, proteins that promote bone growth are not produced in muscle cells. Epigenetic mechanisms play an essential role in healthy development. Conversely, precisely regulated epigenetic mechanisms are disrupted in diseases like cancer.
X-chromosome...
X-chromosome...
02:30Genomic Imprinting and Inheritance
Diploid organisms inherit genetic material through chromosomes from both parents. Copies of the same gene are known as alleles. In most cases, both alleles are simultaneously expressed and allow various cellular processes to function optimally. If one of the alleles is missing or mutated, the expression of the other allele can compensate; however, this is not true for all genes.
The expression of some genes depends on which parent passed the gene to the offspring, through a phenomenon known as...
The expression of some genes depends on which parent passed the gene to the offspring, through a phenomenon known as...