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A gene trap vector system for identifying transcriptionally responsive genes.
E Medico1, G Gambarotta, A Gentile
1Institute for Cancer Research and Treatment, University of Torino School of Medicine, 10060 Candiolo, Italy. emedico@ircc.unito.it
Nature Biotechnology
|June 1, 2001
Summary
We developed a novel gene trapping method using a green fluorescent protein-nitroreductase (GFNR) fusion protein. This technique efficiently identifies genes regulated by external signals through positive and negative selection.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Identifying genes regulated by external stimuli is crucial for understanding cellular responses.
- Existing methods for gene discovery can be time-consuming and lack efficiency.
Purpose of the Study:
- To develop a fast and efficient method for trapping genes regulated by exogenous stimuli.
- To enable the identification and isolation of specific responsive gene populations.
Main Methods:
- Construction of a promoterless retroviral vector encoding a green fluorescent protein-nitroreductase (GFNR) fusion protein.
- Utilizing flow cytometry for cell sorting based on GFNR expression.
- Employing positive and negative pharmacological selection strategies using nitroreductase (NTR).
- Application in liver cells stimulated with hepatocyte growth factor (HGF).
Main Results:
- Successful isolation of cell populations with gene traps in induced or suppressed genes.
- Identification of several distinct responsive clones with confirmed regulated gene expression at the RNA level.
- Demonstration that selection can be calibrated for varying basal expression and regulation levels.
Conclusions:
- The GFNR-based gene trap screening procedure is flexible and efficient.
- This method is suitable for large-scale surveys of transcriptionally regulated genes.
- The technique facilitates the discovery of genes responsive to external signals.