Isolation and characterization of mammalian cells expressing the Arf promoter during eye development

Abstract

Although many researchers have successfully uncovered novel functions of the tumor suppressor p19Arf utilizing various types of cultured cancer cells and immortalized fibroblasts, these systems do not accurately reflect the endogenous environment in which Arf is developmentally expressed. We addressed this by isolating perivascular cells (PVCs) from the primary vitreous of the mouse eye. This rare cell type normally expresses the p19Arf tumor suppressor in a non-pathological, developmental context. We utilized fluorescence activated cell sorting (FACS) to purify the cells by virtue of a GFP reporter driven by the native Arf promoter and then characterized their morphology and gene expression pattern. We further examined the effects of reintroduction of Arf expression in the Arf GFP/GFP PVCs to verify expected downstream effectors of p19Arf as well as uncover novel functions of Arf as a regulator of vasculogenesis. This methodology and cell culture model should serve as a useful tool to examine p19Arf biology.

Keywords

• p19Arf
• primary vitreous
• perivascular cells
• primary cell isolation
• Arf, eye development
• hyaloid regression