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Sabrina V. Klein, Yong Liang, Norbert Kociok, Elizabeth Abari, Eva-Maria Hammer, Kerstin Winter, Stefan Weger, Regine Heilbronn, Antonia Joussen; Expression And shRNA Mediated Inhibition Of Netrin-4 Using rAAV-Vectors. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4837.
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Netrins are a laminin-related family of matrix-binding secreted proteins, known to play a role in establishment of the nervous system and in normal and pathological neuroangiogenesis. They were originally identified as axonal guidance molecules that interact with the Unc5 (uncoordinated-5) receptor family, but also with neogenin and DCC (deleted in colorectal carcinoma).Previous studies have shown that angiogenesis is inhibited through binding of netrin-4 to its receptors, but the exact role of netrin-4 in neuronal degeneration and neoangiogenesis is still not understood. This study aims to examine the function of netrin-4 using rAAV vectors for repression and overexpression in the mouse eye.
Full length netrin-4 and Unc5B cDNAs were synthesizied and cloned into a CMV-driven expression vector. In addition, 8 selected shRNA sequences directed against netrin-4 were cloned into a H1-PolIII based RNA interference vector. Verification of overexpression and repression was performed in cell culture.The plasmids were packed into rAAV vectors for in vivo injection into the mouse eye.Furthermore gfp expressing rAAV vectors were vitreally injected into the mouse eye to distinguish the target localization of the virus in vivo.
We were able to show the at least one of the predicted shRNAs can repress the expression of netrin-4 more than 80%, while others have only a small effect on the protein level. Netrin-4 and Unc5B can be strongly overexpressed in cell culture.
We set up a tool for overexpression as well as (partial) repression of netrin-4, which can be used in different animal models. The morphological and functional characterization of netrin-4 will be useful for the understanding of normal and pathological neovascularisations.
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