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

1.将VEZF1cDNA-IRES-EGFP片段插入ES细胞稳定表达载体PHPCAG中，构建VEZF1正、反义表达载体(VEZF1-S和VEZF1-AS)，并将其导入ES细胞系MG1.19。我们发现VEZF1在明胶上培养的MG1.19细胞中有VEZF1的表达。虽然我们最初在pHPCAG中使用了IRES-EGFP作为模拟载体，但在载体中没有检测到EGFP的表达。因此，我们在PHPCAG中使用了绿色荧光蛋白作为模型。FCM检测到绿色荧光蛋白在Mock、VEZF1-S和VEZF1-AS中均有表达。RT-PCR和Western印迹检测到VEZF1在三种转染体中的表达水平相同。此外，在没有LIF的OP9细胞中，内皮祖细胞的标志物VEGFR2(Flk-1)在分化系统中的表达是相同的。我们发现VEZF1在体外和体内对血管内皮细胞的生长、迁移和网络的形成是必不可少的(我们未发表的数据)。然而，由于VEZF1-AS7不能抑制VEZF1的表达，我们还不能明确VEZF1在内皮细胞分化和血管生成中的作用。我们想尝试使用siRNA。

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 We found VEZF1 was essential for endothetial growth, migration, and network formation in Vitro and in Vivo (our unpublished data).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.