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. Author manuscript; available in PMC: 2015 Jan 31.
Published in final edited form as: Nature. 2014 Jun 22;511(7511):596–600. doi: 10.1038/nature13321

Extended Data Figure 7. Postnatal expression of mNaChBac and Kir2.1 using Flpo and F-FLEX switch.

Extended Data Figure 7

(a) Constitutive overexpression of mNaChBac causes a neuronal migration defect. mNaChBac or mNaChBacMut was overexpressed in a subset of pyramidal cells by in utero electroporation of pCAG-mNaChBac-T2A-tdTomato or pCAG-mNaChBacMut-T2A-tdTomato, respectively, on embryonic day 15.5 (E15.5). Representative fluorescent images of coronal sections of V1 showed that mNaChBac-expressing neurons (left panels) resided not only in layer 2/3, but also in layers 4–6 (n = 7 mice), whereas mNaChBacMut-expressing neurons (right panels) are all located in layer 2/3 (n = 5 mice). Cortical layers are indicated on the right based on the DAPI staining pattern. L: layer, WM: white matter. (b) Experimental procedures for conditional expression of mNaChBac or Kir2.1 in a subset of layer 2/3 pyramidal cells. Left: plasmids pAAV-EF1α-F-FLEX-mNaChBac-T2A-tdTomato or pAAV-EF1α-F-FLEX-Kir2.1-T2A-tdTomato together with pCAG-EGFP were in utero electroporated into V1 on E15.5. Successful transfection was indicated by the expression of EGFP. Middle: AAV-hSynapsin-Flpo was injected postnatally into V1. Right: Only those neurons that were transfected with pAAV-EF1α-F-FLEX-mNaChBac-T2A-tdTomato or pAAV-EF1α-F-FLEX-Kir2.1-T2A-tdTomato and at the same time infected by AAV-hSynapsin-Flpo will express mNaChBac-T2A-tdTomato or Kir2.1-T2A-tdTomato, respectively. (c) Representative fluorescent images of coronal sections of V1 showed that without injection of AAV-hSynapsin-Flpo, the transfected neurons did not express mNaChBac-T2A-tdTomato (left panels, n = 2 mice). The expression of mNaChBac-T2A-tdTomato in the transfected neurons was turned on by injection of AAV-hSynapsin-Flpo, and these neurons were all properly located in layer 2/3 (right panels, n = 7 mice). Cortical layers are indicated on the right based on the DAPI staining pattern. L: layer, WM: white matter. (d) Schematics of concurrent expression of mNaChBac or Kir2.1 in layer 2/3 pyramidal cells and ChR2 in PV or SOM cells. Plasmids pAAV-EF1α-F-FLEX-mNaChBac-T2A-tdTomato or pAAV-EF1α-F-FLEX-Kir2.1-T2A-tdTomato and together with pCAG-EGFP were in utero electroporated into V1 of Pv-ires-Cre or Som-ires-Cre mice on E15.5. AAV-EF1α-DIO-hChR2(H134R)-EYFP and AAV-hSynapsin-Flpo were injected into V1. ChR2 was conditionally expressed in PV or SOM cells, whereas mNaChBac or Kir2.1 was conditionally expressed in a subset of layer 2/3 pyramidal cells that were transfected by pAAV-EF1α-F-FLEX-mNaChBac-T2A-tdTomato or pAAV-EF1α-F-FLEX-Kir2.1-T2A-tdTomato, respectively.