Figure 6. Inducible lentiviral shRNA mediates effective knockdown of Connexin 36.

(a) Schematic illustration of the shRNA-expressing lentiviral constructs. The Ctrl-shRNA and the Cx36-shRNA inserts contained 21-nt sense and antisense strands. Sense and antisense strands were linked through a standard 5′- CTCAAGAGA -3′ loop structure specific to mammalian cells. (b) Western blot of protein samples isolated from cultured E18 mouse primary cortical neurons infected with Ctrl-shRNA and Cx36-shRNA lentiviruses or WT cells (uninfected cells) and probed with anti-Cx36 and anti-β-tubulin antiserum. β-Tubulin served as a loading control (n=4 pregnant mice). (c,d) Quantitative analysis of western blot (c) and real-time PCR (d) showed that both the protein (∼50%) and mRNA levels (∼68%) of Cx36 were significantly reduced in Cx36-shRNA-transfected cells as compared with WT cells and Ctrl-shRNA-transfected cells; no significant difference was noted between WT and Ctrl-shRNA groups (P>0.05). Cx36 mRNA expression levels normalized for GAPDH. (e) Schema showing the method of virus injection, using a tiny self-made glass injector to inject the virus into the gap between the dura and cortical layer 1 at P1 with CD-1 mice (also see Methods). (f) Image of virus injection into neocortical layer 1 at P1. The fast green injected along with virus helped in estimating the extent of diffusion of the virus in neocortical layer 1. (g) Representative distribution of lentivirus-labelled GFP-positive (GFP⁺) cells. GFP⁺ cells densely packed in neocortical layer 1, but sparsely distributed in deep layers, were detected at P15. Scale bar, 50 μm. *P<0.05, **P<0.01, ***P<0.001, two-tailed paired t-test. Error bars represent mean±s.e.m.