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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.

Aquaporins

Aquaporins

Aquaporins or AQPs are a family of integral membrane proteins whose primary function is to transport water, while some called aquaglyceroporins also transport glycerol. In addition, aquaporins have also been suspected to be involved in transporting volatile substances, such as carbon dioxide and ammonia, across membranes. Such AQPs that act as gas channels are often highly expressed in cells involved in the gaseous exchange, such as red blood cells, epithelial cells, and pulmonary capillaries.

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Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

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GFP Reporter System Reveals Cell-to-Cell Variability in Aquaporin-2 Expression.

Lihe Chen¹, Adrian Murillo-de-Ozores¹, Euijung Park¹

¹Epithelial Systems Biology Laboratory, Systems Biology Center, National Heart, Lung, and Blood Institute, NIH, Bethesda, Maryland.

Biorxiv : the Preprint Server for Biology

|December 25, 2025

Summary

This summary is machine-generated.

Vasopressin (AVP) regulates aquaporin-2 (Aqp2) gene transcription. A new reporter cell line shows Aqp2 expression heterogeneity linked to cell cycle state, offering a tool for studying AVP regulation.

Keywords:

Aqp2 GFP reporter RNA-seq cell cycle

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Area of Science:

Molecular biology
Cell biology
Renal physiology

Background:

Vasopressin (AVP) is a key regulator of water reabsorption in the kidneys.
Aquaporin-2 (Aqp2) water channel gene transcription is a primary target of AVP action in collecting duct principal cells.
Understanding Aqp2 gene regulation is crucial for comprehending kidney water balance.

Purpose of the Study:

To develop a reporter cell line for studying Aqp2 gene transcription regulation.
To investigate the mechanisms underlying heterogeneity in Aqp2 expression.
To explore the relationship between cell cycle and Aqp2 expression.

Main Methods:

Engineered an Aqp2 reporter cell line using CRISPR/Cas9 in mpkCCD cells, inserting a GFP cassette at the endogenous Aqp2 locus.
Stimulated cells with dDAVP (vasopressin analog) and forskolin, assessing GFP and Aqp2 expression via flow cytometry.
Performed cell sorting of GFP-positive and GFP-negative populations, followed by restimulation, cell cycle analysis (flow cytometry), and RNA-sequencing.

Main Results:

The Aqp2-GFP reporter cell line faithfully tracked endogenous Aqp2 expression upon dDAVP stimulation and withdrawal.
Significant heterogeneity in Aqp2 expression was observed, with fewer than 50% of cells expressing GFP/Aqp2 even after stimulation.
GFP-negative and GFP-positive cells, when regrown, produced both populations, indicating a dynamic regulation.
Cell cycle analysis revealed that GFP-positive cells were predominantly in G0/G1, while GFP-negative cells showed more cells in G2/S.
RNA-seq identified increased cell-cycle related transcripts in GFP-negative cells.

Conclusions:

Heterogeneity in Aqp2 gene expression is intrinsically linked to the cell cycle state of collecting duct principal cells.
The developed Aqp2-GFP reporter cell line is a valuable tool for dissecting the transcriptional regulation of Aqp2.
This finding provides new insights into the dynamic regulation of water channel expression in the kidney.