Preliminary Study On The Receptor For Axon Guidance Factor Netrin-4

Objective Netrins are secreted proteins that elicit both attractive and repulsive responses in migrating cells in the central and peripheral nervous systems. These bifunctional effects of guidance cues could result from activation of different types of receptors for attraction (DCC) and repulsion (UNC5H). Netrin-4 found by our group is a new member of Netrin gene family. The full-length of its cDNA is 3179bp, coding a 591-amino acid protein. It has 31% homology to Netrin-1 gene. Some studies indicate that Netrin-4 has the outgrowth-promoting activity of Netrin-1 on axons. However, the mechanism of interaction between the second messengers in determining the polarity of the guidance responses is largely unknown. Here, we establish a primary culture model of olfactory bulb and cortical neurons of newborn rat and investigate the localization of Netrin-4 receptor on the neurons. And study whether the known Netrin-1 receptors, DCC and UNC5H1, are coreceptors for Netrin-4. Methods We amplified Netrin-4 and Netrin-1 gene (1.8Kb), then sub-cloned into plasmid APTag4. The vectors were then introduced into E. coli MC1061 cells. SDS-PAGE and Western Blot analyzed expressed proteins. We improved Koh method to cultivate the primary neurons of newborn rat.