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A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo
Published on: January 4, 2012
Gene expression and cell fusion analyzed by lacZ complementation in mammalian cells
1Department of Molecular Pharmacology, Stanford University Medical Center, CA 94305-5322, USA.
Summary
We adapted the Escherichia coli lacZ gene for mammalian cells, creating a reporter system. This system detects when two proteins are expressed simultaneously, aiding cell biology research.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Reporter genes indicate protein expression levels within cells.
- Intracistronic complementation relies on the interaction of protein fragments to restore function.
Purpose of the Study:
- To adapt the Escherichia coli lacZ gene for intracistronic complementation in mammalian cells.
- To develop a reporter system for detecting coincident protein expression and cell fusion.
Main Methods:
- Adapted the lacZ gene for intracistronic complementation in mammalian cells.
- Utilized a novel fluorescent substrate (Fluor-X-Gal) for beta-galactosidase detection.
- Employed quantitative biochemical assays, flow cytometry, and microscopy for analysis.
Main Results:
- Enzymatic activity was detected upon convergent expression of mutant lacZ peptides in single cells or fused cells.
- The system allows for simultaneous microscopic visualization of other fluorescent markers using Fluor-X-Gal.
- Successful adaptation of lacZ complementation for mammalian systems was demonstrated.
Conclusions:
- Enzymatic complementation of lacZ provides a sensitive reporter for coincident protein expression in mammalian cells.
- This method facilitates studies on cell fusion, cell lineage, and signal transduction.
- The developed system offers a versatile tool for various cell biology applications.
