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. 2016 Mar 31;9:22. doi: 10.3389/fnmol.2016.00022

FIGURE 2.

FIGURE 2

Glycine receptor (GlyRs) knock-down NSCs. (A) Experimental protocol to purify NSCs from (i) uninjected, (ii) Ctrl MO-injected, and (iii) glra4a MO-injected. gfap:GFP embryos were injected at the one-cell stage and let develop until 20hpf. NSCs were sorted by FACS and harvested for total RNA extraction. (B) glra4a morpholino targets 24 nucleotides (in red) downstream to the transcription starting site. A 100 bp fragment encompassing the end of the 5UTR and the 5 part of the first exon was cloned in frame with eGFP coding sequence downstream to the CMV promoter. (C) Embryos co-injected with the glra4a-eGFP construct and Ctrl morpholino depict broad GFP fluorescence at 20 hpf (upper right). However, co-injection with glra4a morpholino abolishes fluorescence (lower right) thus validating the use of both Ctrl and glra4a morpholinos.