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Fast generation of stable cell lines expressing fluorescent marker molecules to study pathogen induced processes
Jens Bernhard Bosse1, Jessica Ragues, Harald Wodrich
1Department of Molecular Biology, Princeton University, Princeton, NJ, USA.
Methods in Molecular Biology (Clifton, N.J.)
|September 3, 2013
Summary
This study presents a rapid method to create stable cell lines with fluorescent protein-tagged proteins for studying host-pathogen interactions in real-time using advanced imaging. This technique enhances virology research by providing essential genetic tools for live-cell analysis.
Area of Science:
- Virology
- Cell Biology
- Microscopy
Background:
- Fluorescent proteins (FPs) are crucial for visualizing viral and cellular components in live-cell imaging.
- Studying host-pathogen interactions in real-time requires high-quality genetic tools for advanced imaging technologies.
- Existing methods for generating stable cell lines can be time-consuming.
Purpose of the Study:
- To describe a universal and reliable method for rapidly generating stable cell lines expressing FP-tagged proteins.
- To provide a protocol applicable to various transgenes and FPs for host-pathogen interaction studies.
- To exemplify the method using autophagosomes and the actin cytoskeleton.
Main Methods:
- Utilizes the commercial Flp-In™ and Gateway™ systems.
- Employs Gateway™ technology for rapid generation of FP-tagged transgenes.
- Involves recombination into a cell line with a single, transcriptionally active genomic locus.
Main Results:
- Successfully generated stable cell lines expressing FP-tagged proteins.
- Demonstrated the protocol's applicability to autophagosomes and actin cytoskeleton.
- Established a rapid and reliable method for creating essential genetic tools.
Conclusions:
- The described method offers a universal and efficient approach to generate FP-tagged cell lines for host-pathogen interaction studies.
- This protocol facilitates the advancement of live-cell imaging in virology and cell biology.
- The technique provides researchers with valuable tools for real-time analysis of complex cellular processes.
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