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First published online September 21, 2006
Stem Cells Vol. 25 No. 1 January 2007, pp. 39 -45
doi:10.1634/stemcells.2006-0173; www.StemCells.com
© 2007 AlphaMed Press

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EMBRYONIC STEM CELLS: CHARACTERIZATION SERIES

Production of Green Fluorescent Protein Transgenic Embryonic Stem Cells Using the GENSAT Bacterial Artificial Chromosome Library

Mark J. Tomishimaa,b, Anna-Katerina Hadjantonakisa, Shiaoching Gongc, Lorenz Studera,b

aDevelopmental Biology Program and
bDivision of Neurosurgery, Sloan-Kettering Institute,
cRockefeller University, New York, New York, USA

Key Words. Neural differentiation • Embryonic stem cell • Bacterial artificial chromosome transgenesis • Fluorescent reporter

Correspondence: Mark J. Tomishima, Ph.D., Developmental Biology & Neurosurgery, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Box 256, New York, New York 10021, USA. Telephone: 212-639-3913; Fax: 646-422-2062; e-mail: tomishim{at}mskcc.org

Received March 23, 2006; accepted for publication September 14, 2006.
First published online in STEM CELLS EXPRESS   September 21, 2006.



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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