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Blood, 1 August 2004, Vol. 104, No. 3, pp. 711-718.
Prepublished online as a Blood First Edition Paper on April 15, 2004; DOI 10.1182/blood-2004-01-0254.
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Submitted January 22, 2004
Accepted March 23, 2004
Gene trap expression screening to identify endothelial-specific genes
Masanori Hirashima, Alan Bernstein, William L Stanford, and Janet Rossant*
Samuel Lunenfeld Research Institute, Toronto, Ontario, Canada
Samuel Lunenfeld Research Institute, Toronto, Ontario, Canada; Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada
Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada
* Corresponding author; email: rossant{at}mshri.on.ca.
The endothelial cell is a key cellular component for blood vessel formation. Many signaling receptors expressed in endothelial cells play critical roles in vascular development during embryogenesis. However, downstream response genes required for vascular differentiation are still not clearly identified. Here we describe the development of a protocol for gene trap expression screening in embryonic stem (ES) cells for endothelial-specific genes. ES cells were differentiated into endothelial cells on an OP9 feeder cell layer in 96-well plates. In a pilot screen, five gene trapped ES cell lines showed an upregulated expression of the gene trap lacZ reporter out of 864 ES clones screened. One of the trapped genes was Endoglin, an endothelial-specific TGF- type III receptor, and another was ASPP1, a p53-binding protein. In vivo expression analysis of the lacZ reporter confirmed that both genes are specifically expressed in endothelial cells during early mouse embryogenesis. Gene trap expression screening can thus be used to identify endothelial-specific genes and analyze their function in mice.
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