The role of a transcription factor, VEZF1 in endothelial differentiation and vasculogenesis

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1. We made VEZF1 sense and antisense expression vectors (VEZF1-S and VEZF1-AS) by inserting the VEZF1 cDNA-IRES-EGFP fragment into pHPCAG, a stable expression vector for ES cells to monitor the transfection efficiency and introduced the vector intoMG1.19, an ES cell line by a supertransfection method.2. We found that VEZF1 was expressed in MG1.19 cultured on gelatin in the presence of LIF : in the undiflerentiation system.3. Although we used IRES-EGFP in pHPCAG as a Mock at first, EGFP expression was not detected in the vector. Therefore, we use EGFP in pHPCAG as a Mock.4. EGFP was detected in Mock, VEZF1-S, and VEZF1-AS by FCM. However the expression level of VEZF1 measured by RT-PCR and Western blot was the same among these three transfectants.5. Furthermore, the expression of VEGFR2 (Flk-1), a marker of endothelial progenitor cells was the same in these three transfectants on OP9 cells without LIF: in the differentiation system.6. We found VEZF1 was essential for endothetial growth, migration, and network formation in Vitro and in Vivo (our unpublished data). However, we could not clarify the involvement of VEZF1 in endothelial differentiation as well as in angiogenesis because of failure in inhibiting VEZF1 expression by VEZF1-AS.7. We would like to try using siRNA.