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Gene-trap expression screening to identify endothelial-specific genes.

Masanori Hirashima¹, Alan Bernstein, William L Stanford

¹Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada.

|April 20, 2004

Summary

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This study introduces a new method for identifying genes crucial for blood vessel formation. The gene-trap screening successfully identified endoglin and ASPP1 as key endothelial-specific genes during early development.

Area of Science:

Developmental biology
Cell biology
Genetics

Background:

Endothelial cells are vital for blood vessel formation, with many surface receptors influencing vascular development.
Identifying downstream response genes essential for endothelial differentiation remains a challenge.

Purpose of the Study:

To develop and validate a gene-trap expression screening protocol for identifying endothelial-specific genes in embryonic stem cells.
To uncover novel genes involved in vascular differentiation during embryogenesis.

Main Methods:

Embryonic stem (ES) cells were differentiated into endothelial cells using OP9 feeder layers in 96-well plates.
A pilot screen utilized gene-trap expression screening to identify upregulated lacZ reporter expression in ES cell clones.
In vivo analysis confirmed the expression patterns of identified genes in mouse embryos.

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Main Results:

The protocol successfully screened 864 ES clones, identifying 5 gene-trapped cell lines with upregulated lacZ reporter expression.
Endoglin (a TGF-β type III receptor) and ASPP1 (a p53-binding protein) were identified as endothelial-specific genes.
In vivo studies confirmed specific expression of endoglin and ASPP1 in endothelial cells during early mouse embryogenesis.

Conclusions:

Gene-trap expression screening is an effective method for discovering early endothelial-specific genes.
This approach facilitates the functional analysis of newly identified genes in mouse models of vascular development.