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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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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Gene-trap expression screening to identify endothelial-specific genes
Masanori Hirashima,
Alan Bernstein,
William L. Stanford, and
Janet Rossant
From the Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto; and the Department of Molecular and Medical Genetics and the Institute of Biomaterials and Biomedical Engineering, University of Toronto, ON, Canada.
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, 5 gene-trapped ES cell lines showed an up-regulated expression of the gene trap lacZ reporter out of 864 ES clones screened. One of the trapped genes was endoglin, an endothelial-specific transforming growth factor- 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 early endothelial-specific genes and analyze their function in mice.

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