Isolation and propagation of yolk-sac-derived endothelial cells from a hypervascular transgenic mouse expressing a gain-of-function fps/fes proto-oncogene.
We report on the isolation and propagation of endothelial cells from the mouse embryonic yolk sac, the earliest site of blood vessel development, and on the advantages of a hypervascular transgenic mouse source of these cells. These transgenic mice express multiple copies of an activated allele of the human fps/fes proto-oncogene and display hypervascularity progressing to multifocal hemangiomas. This phenotype suggested a role of the fps/fes proto-oncogene in vasculogenesis and angiogenesis and led us to investigate the growth characteristics of yolk-sac-derived endothelial cells from transgenic fps/fes embryos. We have established eight independent cell clones from a mixture of transgenic and control yolk sacs from Day 12 embryos. Southern blot hybridization analysis showed all eight clones to be derived from transgenic cells, suggesting a growth advantage of cells carrying the activated fps/fes gene. A cell line, Clone 166 (C166), established from one of these clones, was more fully characterized. C166 exhibits normal endothelial characteristics, such as rearrangement into tubelike structures when placed on Matrigel, expression of angiotensin converting enzyme, retention of cobblestone morphology at confluence, and the presence of cell surface receptors for acetylated low density lipoprotein. The cells constitutively express murine endothelial cell adhesion molecule VCAM-1 and the vascular addressin identified by antibody MECA-99. As expected, the cell line expresses high levels of the cytoplasmic protein-tyrosine kinase encoded by the fps/fes proto-oncogene. The clone we have described as well as other endothelial cell lines that we have established from the mouse embryonic yolk sac should prove useful for the study of endothelial cell differentiation and for the determination of the mechanisms underlying the establishment of organ-specific endothelial cell heterogeneity.
Wang
S J
SJ
Center for Developmental Biology, University of Wisconsin, Madison 53706, USA.
Greer
P
P
Auerbach
R
R
D000483:Alleles; D000818:Animals; D015139:Blotting, Southern; D015153:Blotting, Western; D003470:Culture Media; D004730:Endothelium, Vascular / Q000166:cytology*; D005260:Female; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D018919:Neovascularization, Physiologic / Q000235:genetics; D011247:Pregnancy; D011505:Protein-Tyrosine Kinases; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D011519:Proto-Oncogenes; D019010:Vascular Cell Adhesion Molecule-1 / Q000235:genetics; D015017:Yolk Sac / Q000166:cytology*
D003470:Culture Media; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D019010:Vascular Cell Adhesion Molecule-1; C052602:proto-oncogene protein c-fes-fps; D011505:Protein-Tyrosine Kinases