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08:51
Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling
Published on: June 25, 2015
Inducible gene expression using an autoregulatory, tetracycline-controlled system.
Penny Shockett1, David Schatz2
1Southeastern Louisiana University, Hammond, Louisiana.
Current Protocols in Cell Biology
|January 30, 2008
Summary
This study details protocols for transfecting cells to express specific genes, enabling inducible transactivator and target gene protein expression for research applications.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Stable transfection of adherent cells is crucial for creating cell lines with inducible gene expression.
- The tetracycline-inducible system offers precise control over gene expression.
- Developing robust protocols for generating and validating these cell lines is essential for reproducible research.
Purpose of the Study:
- To describe detailed protocols for the stable transfection of adherent cells.
- To establish methods for generating cell lines with inducible transactivator and target gene expression.
- To provide procedures for testing and validating inducible gene expression in transfected cells.
Main Methods:
- Cotransfection of fibroblast cell lines with plasmids encoding a transactivator (pTet-tTAk) and selectable markers.
- Sequential stable cotransfection to introduce target genes under inducible control.
- Methods for testing stably transfected cell lines for inducible gene expression and gene detection.
Main Results:
- Establishment of stably transfected fibroblast cell lines exhibiting inducible transactivator expression.
- Successful generation of cell lines with inducible target gene expression.
- Validated protocols for testing inducible gene expression and detecting the transactivator gene.
Conclusions:
- The described protocols enable the creation of versatile cell lines for studying inducible gene expression.
- These methods facilitate the development of precise genetic tools for molecular and cell biology research.
- The protocols are applicable to various research settings, including cell-based assays and transgenic models.
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