Protocol of gene-trap screen for endothelial-specific genes. IRESβgalNeo(-pA) gene-trap vector consists of Engrailed-2 splice acceptor (SA), IRES, β-gal, polyA signal, human β-actin promoter, neo, and Pax2 splice donor (SD).22 After electroporation of ES cells with IRESβgalNeo(-pA) gene-trap vector, individual G418-resistant ES cell colonies were tested for lacZ expression before and after differentiation of ES cells on the OP9 feeder cell layer for 5 days in 96-well plates. ES clones with up-regulated lacZ reporter expression on the OP9 feeder cell layer were further tested for lacZ expression on embryonic fibroblasts, in embryoid bodies, and in differentiated ES cells on the OP9 feeder cell layer for 1 to 6 days. Trapped genes were identified by RACE, followed by the identification of the vector integration site. Mouse lines were generated from gene-trapped ES cells, and in vivo expression and phenotypic analysis were carried out.
