DEV02354 Supplementary Material

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• Supplemental Figure 1 -

  Fig. S1. Knock down of NCAD by RNAi causes a defect in the migration of LRN/ECN neurons in vivo. (A) A transverse section of E14.5 mouse caudal hindbrain hybridized with antisense digoxigenin-labeled riboprobes for Ncad. An arrow indicates the marginal stream of LRN/ECN neurons. A mouse Ncad cDNA fragment was obtained by PCR using cDNAs from embryonic mouse brains as a template and cloned into the pGEM-T Easy vector (Promega) for generation of in situ hybridization probes. The used primers are (forward) 5'-agcacccacctcagtcgaca-3' and (reverse) 5'-gctctttatcccgccgtttcatcca-3'. (B-E) E18.5 mouse hindbrains electroporated with a control short hairpin RNA (shRNA) vector (B and D) or an Ncad shRNA vector (C and E) together with an EGFP vector at E12.5. Dorsal (B and C) and ventral (D and E) views. Most of neurons expressing Ncad shRNAs remain on the ipsilateral side, whereas control neurons have already reached contralateral destinations. Rostral is upwards. Asterisks represent the labeled side. VM, ventral midline; AEMS, anterior extramural migratory stream; PEMS, posterior extramural migratory stream. For construction of an Ncad shRNA vector (pSuper-Ncad), a sense oligonucleotide (5'-GATCCCCGATGTTTACAGCGCAGTCTTTCAAGAGAAGACTGCGCTGTAAACATCTTTTTGGAAA-3') and an antisense oligonucleotide (5'-AGCTTTTCCAAAAAGATGTTTACAGCGCAGTCTCTTGAAAGACTGCGCTGTAAACATCGGG-3') were annealed and ligated into a linealized pSuper vector (OligoEngine: catalog VEC-PBS-0011). This target sequence corresponds to that of RNAi against rat Ncad reported previously (Fairless et al., 2005). As a negative control, an shRNA vector consisting of scrambled target sequence of Ncad (pSuper-Ncad-scrambled) was designed, and a sense oligonucleotide (5'-GATCCCCGCTATCGCTACGTGTAAGTTTCAAGAGAACTTACACGTAGCGATAGCTTTTTGGAAA-3') and an antisense oligonucleotide (5'-AGCTTTTCCAAAAAGCTATCGCTACGTGTAAGTTCTCTTGAAACTTACACGTAGCGATAGCGGG-3') were used for construction. For ex utero electroporation, pCAGGS-EGFP (0.5 mg/ml) and shRNA constructs (1 mg/ml) were mixed and used. Scale bars: 200 μm in A; 1 mm in B-E.

  Reference

  Fairless, R., and Barnett, S. C. (2005). N-cadherin differentially determines Schwann cell and olfactory ensheathing cell adhesion and migration responses upon contact with astrocytes. Mol. Cell. Neurosci. 28, 253-263.