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Regulatable in vivo biotinylation expression system in mouse embryonic stem cells
Qin Wang1, Ryan T Wagner, Austin J Cooney
1Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, United States of America.
Plos One
|May 14, 2013
Summary
We developed a novel biotinylation system to study gene regulation in mouse embryonic stem (ES) cells. This system allows for inducible control of gene expression and protein interactions, aiding in understanding ES cell pluripotency.
Area of Science:
- Stem cell biology
- Molecular biology
- Gene regulation
Background:
- Embryonic stem (ES) cells possess unique self-renewal and differentiation capabilities.
- Understanding the molecular mechanisms regulating these properties is crucial for stem cell research.
Purpose of the Study:
- To develop and validate a regulatable in vivo biotinylation system for studying gene function in mouse ES cells.
- To investigate the role of hKlf4 in ES cell regulation using this novel system.
Main Methods:
- Constructed a doxycycline-inducible expression system using E. coli biotin ligase (BirA) and the AviTag peptide.
- Utilized homologous recombination and Cre recombinase for gene insertion into the ROSA26 locus.
- Performed Western blot and chromatin immunoprecipitation (ChIP) assays to confirm protein interactions and target enrichment.
Main Results:
- Successfully established a system where doxycycline induces both BirA and AviTag-tagged hKlf4 expression.
- Demonstrated that induced hKlf4 represses mouse ES cell growth, independent of LIF.
- Showed that AviTag-tagged hKlf4 can be enriched at the Nanog enhancer via streptavidin pulldown.
Conclusions:
- The developed regulatable biotinylation system is effective for studying gene function in mouse ES cells.
- This system facilitates the investigation of protein-DNA interactions and gene regulatory networks in stem cells.
- The findings highlight the utility of inducible biotinylation for dissecting complex biological processes in stem cell biology.
