Inducible gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in mice

• Published in: Nature protocols Vol. 5; no. 9; pp. 1518 - 1534
• Main Authors: Pitulescu, Mara E, Schmidt, Inga, Benedito, Rui, Adams, Ralf H
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.09.2010; Nature Publishing Group
• Subjects: 631/136/16; 631/1647/1513/1967; 631/1647/664; 631/378/2613/1786; Analytical Chemistry; Angiogenesis; Animals; beta-Galactosidase - analysis; Biological Techniques; Blood vessels; Cell culture; Cell Proliferation; Computational Biology/Bioinformatics; Dissection; Dissection - methods; Endothelial cells; Endothelial Cells - cytology; Endothelial Cells - physiology; Gene Deletion; Gene targeting; Gene Targeting - methods; Genetic aspects; Genetics; Immunohistochemistry - methods; Life Sciences; Methods; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microarrays; Microscopy, Fluorescence - methods; Neonates; Neovascularization; Neovascularization, Physiologic; Organic Chemistry; Physiological aspects; Physiology; Plant Lectins - analysis; Protocol; Quantitation; Retina; Retina - growth & development; Retinal Vessels - growth & development; Stem cells; Tamoxifen - pharmacology; Germany
• ISSN: 1754-2189; 1750-2799
• DOI: 10.1038/nprot.2010.113

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 ~10 d to complete.