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Updated: Jul 19, 2026
Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli
Published on: January 6, 2015
Production of green fluorescent protein transgenic embryonic stem cells using the GENSAT bacterial artificial
Mark J Tomishima1, Anna-Katerina Hadjantonakis, Shiaoching Gong
1Developmental Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA. tomishim@mskcc.org
Abstract:
Transgenic green fluorescent protein (GFP) reporter embryonic stem (ES) cells are powerful tools for studying gene regulation and lineage choice during development. Here we present a rapid method for the generation of ES cells expressing GFP under the control of selected genes. Bacterial artificial chromosomes (BACs) from a previously constructed GFP transcriptional fusion library (Gene Expression Nervous System Atlas [GENSAT]) were modified for use in ES cells, and multiple BAC transgenic ES cell lines were generated. Specific GFP expression in transgenic cell lines was confirmed during neural differentiation marking neural stem cells, neuronal precursors, and glial progeny by Hes5, Dll1, and GFAP, respectively. GFP was dynamically regulated in ES cell progeny in response to soluble factors that inhibit Notch signaling and a factor that directs astroglial fate choice. Our protocols provide a simple and efficient strategy to utilize the whole GENSAT BAC library to create hundreds of novel fluorescent cell lines for use in ES cell biology.
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