Inducible gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in mice.
The retina is a powerful experimental system for the analysis of angiogenic blood vessel growth in the postnatal organisms. The three-dimensional architecture of the vessel network and processes as diverse as endothelial cell (EC) proliferation, sprouting, perivascular cell recruitment, vessel remodeling or maturation can be investigated at high resolution. The characterization of physiological and pathological angiogenic processes in mice has been greatly facilitated by inducible and cell type-specific loss-of-function and gain-of-function genetics. In this paper, we provide a detailed protocol for tamoxifen-inducible gene deletion in neonatal mice, as well as for retina dissection, whole-mount immunostaining and the quantitation of EC sprouting and proliferation. These methods have been optimized by our laboratory and yield reliable results. The entire protocol takes approximately 10 d to complete.
Pitulescu
Mara E
ME
Department of Tissue Morphogenesis, Faculty of Medicine, Max Planck Institute for Molecular Biomedicine, University of Münster, Münster, Germany. mara.pitulescu@mpi-muenster.mpg.de
Schmidt
Inga
I
Benedito
Rui
R
Adams
Ralf H
RH
D000818:Animals; D049109:Cell Proliferation; D004210:Dissection / Q000379:methods; D042783:Endothelial Cells / Q000166:cytology / Q000502:physiology; D017353:Gene Deletion; D018390:Gene Targeting / Q000379:methods*; D007150:Immunohistochemistry / Q000379:methods; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence / Q000379:methods; D018919:Neovascularization, Physiologic; D037121:Plant Lectins / Q000032:analysis; D012160:Retina / Q000254:growth & development; D012171:Retinal Vessels / Q000254:growth & development*; D013629:Tamoxifen / Q000494:pharmacology*; D001616:beta-Galactosidase / Q000032:analysis
C045475:Griffonia simplicifolia lectins; D037121:Plant Lectins; D013629:Tamoxifen; D001616:beta-Galactosidase