Journal: Neuron

Article Title: Axo-axonic Innervation of Neocortical Pyramidal Neurons by GABAergic Chandelier Cells Requires AnkyrinG-associated L1CAM

doi: 10.1016/j.neuron.2019.02.009

Figure Lengend Snippet: (A1-A3) Experimental strategy to target/manipulate PyN gene expression and label ChCs in the same neocortical layer. (A1) Schematic drawing of E15.5 in utero electroporation (IUE) targeting nascent layer II/III (LII/III) PyNs in embryos from Swiss Webster (SW) females that were bred with Nkx2.1-CreER+/−;Rosa26-loxpSTOPloxp-tdTomato (Ai9)+/+ males. The position of the positive (+) and negative (−) electrodes used to target neocortical progenitors in the ventricular zone (VZ) is depicted. (A2) Tamoxifen (TMX) administration at E18.5 induces Cre activity and excision of a STOP cassette resulting in tdTomato red fluorescent protein (RFP) expression in ChC progenitors. (A3) Representative 200 μm × 200 μm confocal image of a single RFP+ ChC and neighboring electroporated GFP+ PyNs in LII of somatosensory cortex. Scale bar, 20 μm. Enlarged view of the boxed area showing a GFP+ PyN innervated at its AIS by an RFP+ ChC cartridge (arrow) is depicted on the right. Scale bar, 5 μm. AISs are visualized by immunostaining for ankyrin-G (AnkG) (blue).

Article Snippet: Plasmid: pCAG-ER T2 CreER T2 , Gift from C. Cepko , Addgene plasmid #13777( Matsuda and Cepko, 2007 ).

Techniques: Gene Expression, In Utero, Electroporation, Activity Assay, Expressing, Immunostaining

Journal: Neuron

Article Title: Axo-axonic Innervation of Neocortical Pyramidal Neurons by GABAergic Chandelier Cells Requires AnkyrinG-associated L1CAM

doi: 10.1016/j.neuron.2019.02.009

Figure Lengend Snippet: (A) Temporal profile of PyN AIS innervation by ChCs in LII of somatosensory cortex from Nkx2.1-CreER;Ai9 mice. Representative images of RFP+ ChC cartridges, AISs of neighboring PyNs, and gephyrin puncta at time points spanning P8 to P28. Scale bar, 5 μm.

Article Snippet: Plasmid: pCAG-ER T2 CreER T2 , Gift from C. Cepko , Addgene plasmid #13777( Matsuda and Cepko, 2007 ).

Techniques:

Journal: Neuron

Article Title: Axo-axonic Innervation of Neocortical Pyramidal Neurons by GABAergic Chandelier Cells Requires AnkyrinG-associated L1CAM

doi: 10.1016/j.neuron.2019.02.009

Figure Lengend Snippet: (A) Representative images of PyNs innervated by ChC cartridges in LII of somatosensory cortex from Nkx2.1-CreER;Ai9 mice electroporated at E15.5 with plasmids expressing EGFP and shCtrl, shL1CAM#1, shL1CAM#2, or shL1CAM#1 + human L1CAM (hL1CAM) and sacrificed at P28. Scale bar, 10 μm.

Article Snippet: Plasmid: pCAG-ER T2 CreER T2 , Gift from C. Cepko , Addgene plasmid #13777( Matsuda and Cepko, 2007 ).

Techniques: Expressing

Journal: Neuron

Article Title: Axo-axonic Innervation of Neocortical Pyramidal Neurons by GABAergic Chandelier Cells Requires AnkyrinG-associated L1CAM

doi: 10.1016/j.neuron.2019.02.009

Figure Lengend Snippet: (A) Representative images of PyNs innervated by ChC cartridges in LII of somatosensory cortex from Nkx2.1-CreER;Ai9 mice electroporated at E15.5 with plasmids expressing EGFP and shCtrl, shL1CAM#1, shL 1 CAM# 1 +hL 1CAM-WT, or shL1CAM#1+hL1CAM-Y1229H and sacrificed at P28. Scale bar, 10 μm.

Article Snippet: Plasmid: pCAG-ER T2 CreER T2 , Gift from C. Cepko , Addgene plasmid #13777( Matsuda and Cepko, 2007 ).

Techniques: Expressing

Journal: Neuron

Article Title: Axo-axonic Innervation of Neocortical Pyramidal Neurons by GABAergic Chandelier Cells Requires AnkyrinG-associated L1CAM

doi: 10.1016/j.neuron.2019.02.009

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Plasmid: pCAG-ER T2 CreER T2 , Gift from C. Cepko , Addgene plasmid #13777( Matsuda and Cepko, 2007 ).

Techniques: Virus, Control, Recombinant, Protease Inhibitor, DNA Ligation, Bicinchoninic Acid Protein Assay, Western Blot, Sequencing, shRNA, Plasmid Preparation, Software, Imaging

Journal: PLoS ONE

Article Title: miR-132 Enhances Dendritic Morphogenesis, Spine Density, Synaptic Integration, and Survival of Newborn Olfactory Bulb Neurons

doi: 10.1371/journal.pone.0038174

Figure Lengend Snippet: ( A ) qRT-PCR of miR-132 fold-change normalized to control RNA U6 from the ipsilateral bulbs containing pSico 132 -expressing neurons (red) and from the contralateral bulbs (black) at 5 weeks post-tamoxifen (wpt) injections given 2 wpe (N = 5 mice each). ( B and C ) Plots of the summed dendrite length (B) and bar graphs of the total dendritic length (C) of pSico SCR (n = 47) and pSico 132 -containing neurons (n = 51) at 5 wpt injections given at 7 dpe (i.e. 6 wpe). ( D ) Bar graphs of the frequency of GABAergic PSCs in pSico SCR - and pSico 132 -containing neurons at 5 wpt (n = 13 black and 15 red, respectively). ( G ) Sample images illustrating the density of pSico SCR and pSico 132 neurons in OB coronal sections. ( H and I ) Bar graphs of absolute (H) and normalized (I) RFP + ( i.e. pSico SCR , black and pSico 132 , red) neuron density in the GCL (N = 8 and 9 mice, 3–4 images per mouse, respectively). Scale bar: 100 µm.

Article Snippet: The pSico plasmids were co-injected with the tamoxifen-inducible Cre-recombinase expression vector pCAG- ERT2 Cre ERT2 (Addgene, C. Cepko) as well as a pCAG-tdTomato (noted RFP) vector that was constructed using the pCMV-tdTomato vector from Clontech.

Techniques: Quantitative RT-PCR, Expressing

Journal: Nature Communications

Article Title: Convulsive seizures from experimental focal cortical dysplasia occur independently of cell misplacement

doi: 10.1038/ncomms11753

Figure Lengend Snippet: ( a ) Diagram illustrating the vectors and strategy used. ( b , c , e , f ) Images of cGFP+tdTomato CAG or cRheb CA +GFP CAG -electroporated neurons co-stained for pS6 (red and B&W for pS6 only, ( e , f )). Scale: 100 μm. Neurons were co-electroporated with an inducible Cre plasmid and injected with tamoxifen at P6. ( d , g ) Bar graphs of the soma size of cGFP + ( N =4) neurons and cRheb CA+ ( N =3) neurons ( d ) and pS6 immunoreactivity per cell ( g ). * P <0.05 (Student's t -test). ( h ) Representative examples of EEG recordings from mice electroporated with plasmid encoding cGFP (left) and cRheb CA (right). Scale bar, 10 s, 210 μV. ( i ) Bar graphs show the number (#) of seizures/day and seizure duration in seizing cRheb CA animals ( N =5). When compared to control (cGFP) littermates ( N =6; * P <0.05; ** P <0.01; Student's t -test). Error bars, s.e.m.

Article Snippet: To generate epilepsy-associated FCD without heterotopia and dyslamination, a solution of loxP-containing vector pCALNL-Rheb S16H (cRheb CA , 1.5 μg μl −1 )+a Tamoxifen-inducible pCAG-ER T2 CreER T2 (3 μg μl −1 , Addgene)+pCAG-GFP (1.5 μg μl −1 ) was injected in half of the fetuses while the remaining half received a solution containing pCALNL-GFP (cGFP, 1.5 μg μl −1 , Addgene)+pCAG-ER T2 CreER T2 (3 μg/μl)+pCAG tdTomato (1.5 μg μl −1 ) as controls.

Techniques: Staining, Plasmid Preparation, Injection

Journal: Stem Cell Reports

Article Title: Genetic Engineering of Human Embryonic Stem Cells for Precise Cell Fate Tracing during Human Lineage Development

doi: 10.1016/j.stemcr.2018.09.014

Figure Lengend Snippet: Tight Control and Efficient Cleavage of PAX6-P2A-Cre or FOXA2-P2A-Cre Fusion Proteins (A) Schematic diagram for constructing PAX6-P2A-Cre and FOXA2-P2A-Cre hPSCs lines through a gRNA-guided CRISPR/Cas9 system. Genotyping PCR primer sets are labeled with arrows. (B) Genomic DNA PCR results identify five monoallelic HR colonies after genetic engineering. (C) Immunolabeling of FOXA2 and Cre in FOXA2-P2A-Cre lines differentiated toward a cortical NP or FP fate for 6 or 12 days. Nuclei are counter stained with Hoechst. Scale bar, 100 μm. (D) Western blot analysis of FOXA2-P2A-Cre lines differentiated toward a cortical NP or FP fate for 6, 12, or 20 days. (E) Genomic DNA PCR results identify two monoallelically recombined PAX6-P2A-Cre colonies after genetic engineering. (F) Immunolabeling of PAX6 and Cre in PAX6-P2A-Cre lines at hESC stage, day 10 NE, day 17 cortical NPs, or day 17 ventral NPs. Nuclei are counter stained with Hoechst. Scale bar, 100 μm. (G) Western blot analysis of PAX6-P2A-Cre lines along with neural differentiation. See also Figure S1 .

Article Snippet: PAX6-P2A-Cre, FOXA2-P2A-Cre, PAX6-Cre ERT2 , and FOXA2-Cre ERT2 donor plasmids were constructed by ligating the left and right homologous arms with Cre or Cre ERT2 cassettes PCR amplified from pCAG-Cre-ERT2 (Addgene, no. 14797) ( ) into Oct4-2a-eGFP-PGK-puro vector (Addgene, no. 31938) ( ) with BamHI and EcoRI.

Techniques: CRISPR, Labeling, Immunolabeling, Staining, Western Blot

Journal: Stem Cell Reports

Article Title: Genetic Engineering of Human Embryonic Stem Cells for Precise Cell Fate Tracing during Human Lineage Development

doi: 10.1016/j.stemcr.2018.09.014

Figure Lengend Snippet: Tracing PAX6 Expressing NE In Vitro and In Vivo (A) AAVS1 left and right TALENs and AAVS1-LSL-GFP donor plasmids are electroporated into PAX6-P2A-Cre H9 hESCs. Genomic DNA PCR analysis identifies monoallelically and biallelically targeted colonies. (B) Southern blot analysis of no. 4 biallelic HR colony shows precise engineering without off-target recombination. (C) Immunostaining of GFP, PAX6, Cre, and NKX2.1 in PAX6-P2A-Cre/AAVS1-LSL-GFP H9 hESC line at day 17 dorsal or ventral NPs reveals that both regional NPs are developed from PAX6 expression NE. Scale bar, 100 μm. (D) H&E and immunolabeling results of adjacent sections from PAX6-P2A-Cre/AAVS1-LSL-GFP H9 hESC line-derived teratomas. GFP labels all existing neural tubes (NT), but it is not present in cartilages (CT) or intestine tissues (IN). Scale bar, 200 μm. (E and F) H&E and immunolabeling results of adjacent sections from PAX6-P2A-Cre/AAVS1-LSL-GFP H9 hESC line-derived teratomas. Both PAX6 + dorsal NPs and NKX2.1 + ventral NPs are labeled with GFP. Scale bar, 100 μm. See also Figure S2 .

Article Snippet: PAX6-P2A-Cre, FOXA2-P2A-Cre, PAX6-Cre ERT2 , and FOXA2-Cre ERT2 donor plasmids were constructed by ligating the left and right homologous arms with Cre or Cre ERT2 cassettes PCR amplified from pCAG-Cre-ERT2 (Addgene, no. 14797) ( ) into Oct4-2a-eGFP-PGK-puro vector (Addgene, no. 31938) ( ) with BamHI and EcoRI.

Techniques: Expressing, In Vitro, In Vivo, TALENs, Southern Blot, Immunostaining, Immunolabeling, Derivative Assay, Labeling

Journal: Stem Cell Reports

Article Title: Genetic Engineering of Human Embryonic Stem Cells for Precise Cell Fate Tracing during Human Lineage Development

doi: 10.1016/j.stemcr.2018.09.014

Figure Lengend Snippet: Improvement of the Tracing Fidelity by Introducing Controllable Recombinases (A) Schematic diagram for generating FOXA2-Cre ERT2 lines. Primer sets for genomic DNA PCR are labeled with arrows. (B and C) Genomic DNA PCR results showing recombination of Cre ERT2 at the ATG region of FOXA2 (B) and CAG-LSL-GFP (C) cassettes within the AAVS1 intron. (D) Southern blot analysis of no. 4 FOXA2-Cre ERT2 /AAVS1-LSL-GFP colony with GFP probe. (E and F) Immunolabeling of GFP, FOXA2, Cre, and PAX6 in dorsal NPs and FP cells with (E) or without (F) 4-OHT treatment at days 6 or 12 post differentiation. 4-OHT was administered 2 days before staining. Scale bars, 100 μm. See also Figure S5 .

Article Snippet: PAX6-P2A-Cre, FOXA2-P2A-Cre, PAX6-Cre ERT2 , and FOXA2-Cre ERT2 donor plasmids were constructed by ligating the left and right homologous arms with Cre or Cre ERT2 cassettes PCR amplified from pCAG-Cre-ERT2 (Addgene, no. 14797) ( ) into Oct4-2a-eGFP-PGK-puro vector (Addgene, no. 31938) ( ) with BamHI and EcoRI.

Techniques: Labeling, Southern Blot, Immunolabeling, Staining

Journal: Stem Cell Reports

Article Title: Genetic Engineering of Human Embryonic Stem Cells for Precise Cell Fate Tracing during Human Lineage Development

doi: 10.1016/j.stemcr.2018.09.014

Figure Lengend Snippet: Temporal Tracing of PAX6 Expression Cells Identifies their Sequential NE and Dorsal NP Identities (A) Schematic diagram for generating PAX6-Cre ERT2 lines. Primer sets for genomic DNA PCR are labeled with arrows. (B and C) Genomic DNA PCR results showing recombination of Cre ERT2 at the ATG region of PAX6 (B) and CAG-LSL-GFP (C) cassettes within the AAVS1 intron. (D) Immunolabeling of GFP, PAX6, Cre, and NKX2.1 in dorsal and ventral NPs at day 17 in PAX6-Cre ERT2 /AAVS1-LSL-GFP H9 hESC line treated with 4-OHT through days 8–10. Scale bar, 100 μm. (E) Immunolabeling of GFP, PAX6, Cre, and NKX2.1 in dorsal and ventral NPs at day 17 in PAX6-Cre ERT2 /AAVS1-LSL-GFP H9 hESC line treated with or without 4-OHT through days 15–17. Scale bar, 100 μm. See also Figure S6 .

Article Snippet: PAX6-P2A-Cre, FOXA2-P2A-Cre, PAX6-Cre ERT2 , and FOXA2-Cre ERT2 donor plasmids were constructed by ligating the left and right homologous arms with Cre or Cre ERT2 cassettes PCR amplified from pCAG-Cre-ERT2 (Addgene, no. 14797) ( ) into Oct4-2a-eGFP-PGK-puro vector (Addgene, no. 31938) ( ) with BamHI and EcoRI.

Techniques: Expressing, Labeling, Immunolabeling

Journal: Stem Cell Reports

Article Title: Genetic Engineering of Human Embryonic Stem Cells for Precise Cell Fate Tracing during Human Lineage Development

doi: 10.1016/j.stemcr.2018.09.014

Figure Lengend Snippet:

Article Snippet: PAX6-P2A-Cre, FOXA2-P2A-Cre, PAX6-Cre ERT2 , and FOXA2-Cre ERT2 donor plasmids were constructed by ligating the left and right homologous arms with Cre or Cre ERT2 cassettes PCR amplified from pCAG-Cre-ERT2 (Addgene, no. 14797) ( ) into Oct4-2a-eGFP-PGK-puro vector (Addgene, no. 31938) ( ) with BamHI and EcoRI.

Techniques:

Journal: Stem Cell Reports

Article Title: Genetic Engineering of Human Embryonic Stem Cells for Precise Cell Fate Tracing during Human Lineage Development

doi: 10.1016/j.stemcr.2018.09.014

Figure Lengend Snippet:

Article Snippet: PAX6-P2A-Cre, FOXA2-P2A-Cre, PAX6-Cre ERT2 , and FOXA2-Cre ERT2 donor plasmids were constructed by ligating the left and right homologous arms with Cre or Cre ERT2 cassettes PCR amplified from pCAG-Cre-ERT2 (Addgene, no. 14797) ( ) into Oct4-2a-eGFP-PGK-puro vector (Addgene, no. 31938) ( ) with BamHI and EcoRI.

Techniques: