Background and aims: Tumour endothelial marker-8 (TEM-8) has been found to be selectively up regulated in tumour-associated endothelial cells, it is implicated in tumour specific angiogenesis, but its mechanism in angiogenesis is not defined. Methods: A ribozyme transgene (TEM-8) was cloned into a suitable mammalian expression vector (pc DNA 3.1-GFP-NT) and transfected into HECV cells. Various domains of TEM-8 were designed and cloned into pEF6/V5-His TOPO TA vector and transfected into Chinese Hamster ovarian cells (CHO), which do not form tubules and do not express TEM-8 in general (CHOvW, CHOTM, CHOvW/TM, CHOAE, CHOAC, CHOIC, and CHOFL domains, respectively). The effect of TEM-8 knocked out HECV cells was tested (by angiogenesis and migration assays), and the effect of each cleavage domain of TEM-8 was tested by microtubule formation assay. Results: TEM-8 stable transfectants (HECVDelta TEM8a) manifested a complete loss of TEM-8 gene expression at mRNA and protein levels. In contrast, control GFP plastrid (HECVpControl) and wild-type HECV cells (HECVWT) had similar levels of TEM-8 expression. TEM-8 transfected cell (HECVDelta TEM8a) significantly decreased the micro-vessels formation compared with controls (HECVpControl) (mean +/- SE, 20.3 +/- 4.03 pm; p = 0.0086 vs. control 39.5 +/- 10.1 mu m), and migration (38.52 +/-2.17; p < 0.05 vs. control 80.23 +/- 3.19), and micro-vessel formation of HECVDelta TEM8a cell was also reduced compared with wild-type (HECVWT) (mean SE, 20.3 + 4.03 mu m; p = 0.0078 vs. wild-type 42.5 +/-9.1 pin) and migration (38.52 2.17 pm; p < 0.05 vs. wild-type 82.4 +/-4.45 mu m). vW together with transmembrane domains of TEM-8 (CHOvW/TM) and full-length CHOFL showed formation of tubule-like structure in CHO cells, whereas the other domains showed no effect. Conclusion: Targeting the TEM-8 gene by way of a hammerhead ribozyme knocks out TEM-8 cells, and is an effective way to reduce the micro-vessel formation or migration potential in turnour-associated endothelial cell through its vW domain. These results suggest that the vW domain together with the transmembrane domain of TEM-8 may play an important biological role in TEM-8 related tubule formation. (c) 2005 Elsevier Inc. All rights reserved.