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Analysis of Cell Cycle Position in Mammalian Cells
Published on: January 21, 2012
TwinGFP, a marker for cell cycle analysis in transiently transfected cells
Maarten Niemantsverdriet1, Claude Backendorf
1Department of Molecular Genetics, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, 2333 CC Leiden, The Netherlands.
Analytical Biochemistry
|February 12, 2008
Summary
Researchers developed TwinGFP, a larger fluorescent protein, to prevent leakage during cell permeabilization for cell cycle analysis. This enhanced protein allows for better detection in flow cytometry and microscopy, improving cell identification.
Area of Science:
- Cell biology
- Molecular biology
- Biotechnology
Background:
- Green fluorescent protein (GFP) is a common marker for identifying transfected cells.
- Standard GFP can be lost during cell permeabilization protocols, such as those used for cell cycle analysis with propidium iodide and ethanol.
- This loss of GFP is likely due to its small molecular size.
Purpose of the Study:
- To improve the retention of fluorescent proteins in cells during permeabilization.
- To develop a more robust fluorescent marker for use in conjunction with cell analysis techniques like flow cytometry.
- To investigate the role of protein size in cellular retention during permeabilization.
Main Methods:
- Constructing a double-sized GFP (TwinGFP) by in-frame fusion of two GFP DNA sequences.
- Transfecting HeLa cells with either standard GFP or TwinGFP constructs.
- Analyzing fluorescent signal intensity and localization in permeabilized cells using fluorescence microscopy and flow cytometry.
- Co-transfecting TwinGFP with the p53 tumor suppressor gene to demonstrate its utility in identifying specific cell populations.
Main Results:
- Cells transfected with TwinGFP exhibited significantly stronger and more uniform green fluorescence after permeabilization compared to cells with standard GFP.
- TwinGFP demonstrated improved retention within permeabilized HeLa cells.
- The study confirmed that increased protein size is a key factor in preventing fluorescence loss during permeabilization.
- TwinGFP successfully allowed for the identification and phenotypic characterization of p53-expressing cells via flow cytometry.
Conclusions:
- TwinGFP offers superior retention in permeabilized cells compared to standard GFP, making it a more reliable marker.
- The size of the fluorescent protein is a critical determinant of its retention during cell permeabilization.
- TwinGFP provides a valuable tool for advanced cell analysis, including the identification and characterization of specific transfected cell populations in flow cytometry applications.
