title: 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 p19(Arf) 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 p19(Arf) 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 p19(Arf) 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 p19(Arf) biology.
authors.affiliation: Department of Pediatrics, Division of Hematology/Oncology, University of Texas Southwestern Medical Center, Dallas, TX.
authors.forename: Nida S
authors.initials: NS
authors.lastname: Iqbal
authors.affiliation: Department of Pediatrics, Division of Hematology/Oncology, University of Texas Southwestern Medical Center, Dallas, TX.
authors.forename: Lin
authors.initials: L
authors.lastname: Xu
authors.affiliation: Department of Pediatrics, Division of Hematology/Oncology, University of Texas Southwestern Medical Center, Dallas, TX.
authors.forename: Caitlin C
authors.initials: CC
authors.lastname: Devitt
authors.affiliation: Department of Pediatrics, Division of Hematology/Oncology, University of Texas Southwestern Medical Center, Dallas, TX; Center for Cancer and Blood Disorders, Children's Medical Center, Dallas, TX.
authors.forename: Stephen X
authors.initials: SX
authors.lastname: Skapek
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics* / Q000378:metabolism; D005123:Eye / Q000254:growth & development*; D005434:Flow Cytometry / Q000379:methods*; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins / Q000235:genetics; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D011401:Promoter Regions, Genetic; D014822:Vitreous Body / Q000166:cytology* / Q000254:growth & development
chemical_list: C417743:Cdkn2a protein, mouse; D019941:Cyclin-Dependent Kinase Inhibitor p16; D049452:Green Fluorescent Proteins
keywords: Arf, eye development; hyaloid regression; p19Arf; perivascular cells; primary cell isolation; primary vitreous