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Reporter Genes

Reporter 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.

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Tet-Regulated Expression and Optical Clearing for In Vivo Visualization of Genetically Encoded Chimeric

Liliya Maloshenok^1,2, Gerel Abushinova^1,2, Natalia Kazachkina¹

¹Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, 33, Bldg. 2 Leninsky Ave., 119071 Moscow, Russia.

Materials (Basel, Switzerland)

|February 11, 2023

Summary

CRISPR-Cas9 gene editing tools, specifically catalytically inactive Cas9 (dCas9), were tested for visualizing cellular structures in live cells and tumor xenografts. Doxycycline induction enabled rapid visualization of dCas9-fluorescent protein fusions in tumors within 24 hours.

Keywords:

FLIM MRI dCas9 orthologs doxycycline inducible Tet-On expression fluorescence imaging fluorescent proteins gadobutrol multimodal imaging optical clearing subcutaneous tumor xenograft

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Area of Science:

Molecular Biology
Biotechnology
Cell Biology

Background:

Catalytically inactive Cas9 (dCas9) offers biomedical applications, including transcriptional regulation and gene mapping.
dCas9 is emerging as a tool for visualizing cellular components in live cells and tissues.

Purpose of the Study:

To evaluate dCas9-fluorescent protein constructs for visualizing nuclear compartments in live human cells.
To assess the time-dependent expression and visualization of dCas9-fluorescent protein fusions in live cells and tumor xenografts.
To investigate the utility of optical clearing and contrast-assisted MRI for in vivo imaging of dCas9 expression in tumors.

Main Methods:

Investigated doxycycline-inducible (Tet-On) expression of dCas9 orthologs (St, Nm) fused with EGFP and mCherry in A549 cells.
Monitored time-dependent dCas9-fluorescent protein expression in live cells and tumor xenografts.
Employed fluorescence imaging, optical clearing (OC) with gadobutrol, and in vivo contrast-assisted magnetic resonance imaging (MRI) for tumor assessment.

Main Results:

Doxycycline induction enabled intracellular distribution studies of dCas9-EGFP and dCas9-mCherry in human cells.
In vivo expression of dCas9-mCherry in tumor xenografts was visualized as early as 24 hours post-induction.
Optical clearing enhanced contrast for fluorescence imaging of dCas9-FP expression in tumors, showing a 1.1-1.2-fold increase in fluorescence intensity.

Conclusions:

dCas9-fluorescent protein systems are effective for visualizing nuclear compartments and tracking gene expression dynamics in live cells.
Rapid in vivo visualization of dCas9 expression in tumor xenografts is achievable, facilitating real-time monitoring.
Optical clearing combined with contrast agents improves the sensitivity and resolution of in vivo fluorescence imaging for dCas9-based applications.