Generating Neurod5kb and RET Fusion Constructs

Neurod5kb:jip3-mCherry was previously described (Drerup and Nechiporuk, 2013 (link)). Neurod5kb:mCherry plasmid was generated by recombining the neurod5kb promoter (Mo and Nicolson, 2011 (link)) p5e entry vector into the pDEST394 vector in the Gateway system by previously described methods (Kwan et al., 2007 (link)). Human RET9-mCherry and RET51-mCherry constructs in the EGFP-N1 vector were obtained from the Mulligan lab (Crupi et al., 2015 (link)) and digested with HindIII, NotI, and SphI to release the whole tagged construct and digest the EGFP-N1 vector backbone. Released RET fusion construct cassettes were ligated into HindIII/NotI-digested Gateway pME-MCS vector and subsequently recombined with the neurod5kb or cmv/sp6 promoter p5e entry vectors to yield neurod5kb:RET9/51-mCherry or cmv/sp6:RET9/51-mCherry in the pDEST394 destination vector (Kwan et al., 2007 (link)). Jip3 deletion constructs Δp150 and ΔJNK were derived from pME-Jip3 (Drerup and Nechiporuk, 2013 (link)) using the Quickchange II kit (Agilent) (primers in Table 1). mRNA was synthesized from Jip3 constructs or cmv/sp6:RET9/51-mCherry using SP6 mMessage Machine (Life Technologies) and microinjected at 500 pg/embryo for Jip3 constructs and 50 pg/embryo for RET9/51-mCherry constructs.