shRNA-mediated knockdown of Ngn2 and time course of axonal projection using ex vivo electroporation coupled with organotypic slice culture. (A) The bicistronic pSCV2 plasmid was engineered to allow for the visualization of cells expressing the shRNA. This plasmid contains a nonspecific control shRNA under the control of the pol-III U6 promoter and the YFP variant Venus under control of the pol-II CAG promoter. (B,C) The control vector with the nonspecific shRNA does not reduce expression of Myc-tagged Ngn2 in P19 cells. (B) Myc-tagged Ngn2 expression is drastically reduced in the presence of shRNA targeting Ngn2 (Ngn2KD). This shRNA effectively knocks down Ngn2 expression even at a 1:10 ratio of shRNA to Myc-tagged Ngn2 plasmids. (C) In order to rescue shRNA-mediated knockdown of Ngn2, we generated a Ngn2 rescue mutant (Ngn2Rescue) containing a single noncoding point mutation that reduces the ability of the shRNA to target Ngn2. A 1:1 ratio of shRNA to myc-tagged Ngn2Rescue allows for weak expression of Ngn2, and a 1:4 ratio of shRNA to myc-tagged Ngn2Rescue allows for expression of Ngn2 equal to control shRNA. (D) Diagrams depicting the areas imaged in (E-H): left, low magnification images reveal the area of the cortex electroporated; middle, high magnification of the medial cortex to visualize medial projecting axons; right, high magnification of the lateral cortex to visualize lateral projecting axons. (E-H) For the axonal projection time course, embryonic cortices were electroporated with a plasmid encoding the YFP variant Venus under the control of the CAG promoter at time points varying from E13.5 to E16.5. After 5 days in vitro (DIV), the electroporated neurons were immunostained using anti-GFP to assess the axonal projections of cortical neurons labeled at each time point. (E) At E13.5, a large proportion of the electroporated neurons have laterally projecting axons. Starting at E14.5, the number of laterally projecting axons steadily decreases (F), and by E15.5 there are very few laterally projecting axons (G). (H) Virtually no laterally projecting axons were seen following electroporation at E16.5.