RESEARCH Generation and Characterization of Yeast Two-Hybrid cDNA Libraries Derived From Two Distinct Mouse Pluripotent Cell Types

The Author(s) 2012. This article is published with open access at Springerlink.com Abstract Pluripotent stem cells have the therapeutic potential in future regenerative medicine applications. Therefore, it is highly important to understand the molec-ular mechanisms governing the pluripotency and differen-tiation potential of these cells. Our current knowledge of pluripotent cells is largely limited owing to the candidate gene/protein approach rather than studying the complex interactions of the proteins. Experimentally, yeast two-hybrid system (Y2H) is by far the most useful and widely used method to detect the protein–protein interactions in high-throughput screenings. Unfortunately, currently there is no GAL4-based pluripotent stem cell-specific cDNA library available for screening the interaction proteins impeding the large-scale studies. In this study, we report the construction of Y2H cDNA libraries derived from mouse pluripotent embryonic stem cells (ESCs) and mul-tipotent adult germ-line stem cells (maGSCs) in GAL4-based Y2H vector system with very high transformation efficiency. Furthermore, we have constructed two different baits and screened for interaction partners in an effort to characterize the libraries and also as a part of our ongoing studies. Consequently, many putative interaction proteins were identified in both cases and their interaction was further validated by direct-Y2H. The observed interactions between bait proteins and their respective analyzed puta-tive interaction proteins were further confirmed using two independent approaches in mammalian cells, thus high-lighting the biological significance of the identified inter-actor (s). Finally, we would like to make these cDNA libraries as a resource that can be distributed to the research community