mcherry coding sequence  (New England Biolabs)

Journal: PLOS Biology

Article Title: Canonical and noncanonical roles of Hop1 are crucial for meiotic prophase in the fungus Sordaria macrospora

doi: 10.1371/journal.pbio.3002705

Figure Lengend Snippet: ( A ) Top: domain diagram of WT Hop1. Bottom: diagram of the deleted or mutated Hop1 sites in the 6 analyzed mutants (). ( B ) Protein localization and phenotypes of the hop1-HORMAΔ mutant. Top: In the hop1Δ background, the mutant protein tagged with mCherry (mC, left) is not visible along the axes, marked by Spo76-GFP (middle) and merge (right). Bottom: colocalization with Ecm11-GFP + Hei10-GFP (middle) indicates that only few SC segments (containing Hei10 foci, arrows) are formed in this mutant; right corresponding DAPI. ( C, D ) hop1-HORMAΔ SCs exhibit the same length ( C ) and Hei10 foci number ( D ) as hop1Δ . Mean and error bar (SD) are indicated for each set. Significance between WT, hop1Δ and hop1-HORMAΔ nuclei was established by Brown–Forsythe ANOVA test: ns = not significant, P -value > 0.05; n = 38 and 42 nuclei. ( E ) hop1-only-HORMA . Top: the HORMA domain alone is sufficient for axis localization of the protein but only as discontinuous segments (arrows) that colocalize with Spo76-GFP (arrows middle and merge right) in contrast to the continuous lines seen in WT with Hop1-mCherry (right). Bottom: colocalization of Hop1-only-HORMA-mC (left) with Ecm11-GFP and Hei10-GFP (middle) and merge (right) indicates that the visible mutant segments correspond to SCs. ( C ) The mutant SCs have the same length as hop1Δ SCs ( C ) but exhibit a slightly lower number of Hei10 foci ( D ): Brown–Forsythe ANOVA test, ns = not significant; n = 31 and 42 nuclei. Scale bars: 2 μm. The raw data underlying panels 6C and 6D are available in . SC, synaptonemal complex; WT, wild-type.

Article Snippet: GFP and mCherry coding sequences (p-EGFP-1, Clontech; pRsetB-mCherry) were fused to the C-terminus of HOP1 under the control of the HOP1 promoter.

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Oligodendrocyte dependent synchronized activity orchestrates circuit maturation and brain functionalization

doi: 10.1101/2024.05.06.590880

Figure Lengend Snippet: a, Diagram illustrating the injection of AAV-hMAG-mCherry and AAV-hMAG-DTA into the mouse cerebellum at early postnatal days (P6-7). b, Fluorescence microscopy image showing mCherry expression (red) in the section of cerebellum at P14 following AAV-hMAG-mCherry injection at P7. Scale bar, 300 µm. c, Specificity of mCherry expression (red) in ASPA-positive oligodendrocytes (green) at P14 by AAV-hMAG-mCherry injection at P7. Scale bar, 100 µm. d, Scatter plot graph depicting the percentage of RFP and ASPA double-positive cells among RFP-positive populations (from 2 mice). e, Sequential visualization of oligodendrocyte deletion over time, indicated by ASPA staining in cerebellar sections from control (AAV-hMAG-mCherry) and DTA-treated (AAV-hMAG-DTA) mice at P10, P14, P21, and P78. Scale bar, 100 µm. f, Quantification of ASPA-positive cell density at each stage (from 3 mice per group per each stage). Bars and dots indicate mean and data from individual fields of view, respectively. g-i, Decrease in correlation coefficients (CCs) indicating reduced synchrony of spontaneous activities among PC population at P13-15 following DTA-mediated oligodendrocyte ablation. g, Representative time-course of spontaneous calcium transients by extracting relative fluorescence changes by in vivo calcium imaging captured from 25 regions of interest (ROIs) in the cerebellum of control and DTA-expressing mice at P14. Scale bar = 20 s. Y-axis is ΔF/F0 = (F - F0)/(F0). h, Correlation coefficient matrices for spontaneous calcium transient activity across multiple ROIs in CTL (upper panel) and DTA (lower panel) mice, with the color scale indicating the strength of the correlation between pairs of ROIs. i, Scatter plot graph of CCs for ROI pairs, categorized by separation distances of 0–80 mm and 120–200 mm (analyzed from four mice per group). Lines and plots indicate mean and data from individual separation distances, respectively. j-l, Quantification of the frequency, amplitude, and integrated area of calcium transients in control (AAV-hMAG-mCherry) and DTA-treated (AAV-hMAG-DTA) mice at P13-15 (data from four mice per group). Bars and dots indicate mean and data from individual ROIs, respectively. **** p < 0.0001 (Mann-Whitney U test).

Article Snippet: For AAV-hMAG-DTA, we amplified the DTA coding sequence from the pAAV-mCherry-flex-dtA (a gift from Naoshige Uchida, Addgene plasmid # 58536) using primer pairs (Table.

Techniques: Injection, Fluorescence, Microscopy, Expressing, Staining, Control, In Vivo, Imaging, Activity Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Oligodendrocyte dependent synchronized activity orchestrates circuit maturation and brain functionalization

doi: 10.1101/2024.05.06.590880

Figure Lengend Snippet: a , Representative traces of CF-EPSCs in PCs from control (CTL) and AAV-hMAG-DTA-injected (DTA) mice at P23. Scale bar, 0.5 nA, 10 ms. b , Frequency distributions of the number of CFs innervating each PC during P23 to P47 for control (orange columns, n = 58 cells, 7 mice) and AAV-hMAG-DTA-injected (cyan, n = 66 cells, 9 mice) mice. c , Average total amplitude of CF-EPSCs (summation of all CF-EPSC in each PC) for control (orange columns) and AAV-hMAG-DTA-injected mice (cyan), with individual data points (from each cell) overlaid on the bars. d , Fluorescence microscopy images showing PCs labeled with Car8 (magenta) and climbing fiber terminals with vesicular glutamate transporter type 2 (VGluT2, green) in control and AAV-hMAG-DTA-injected mice at P21 and P72. Scale bars: 20 µm. e, Frequency distributions of the number of perisomatic CF terminals on PCs for control (orange, n = 253 cells, 3 mice at P12; n = 159 cells, 3 mice at P72-P76) and AAV-hMAG-DTA-injected mice (cyan, n = 157 cells, 3 mice at P12; n = 160 cells, 3 mice at P72-P76). f , Immunofluorescence images showing VGluT2 (green) in control and AAV-hMAG-DTA-injected mice at P21 and P72, highlighting the top dots of VGluT2 in the molecular layer (arrows) and the demarcation between dendrites and soma of each PC (dotted line). Scale bars: 20 µm. g , Bar graph summarizing the relative positioning of CF terminals to the molecular layer thickness in control versus AAV-hMAG-DTA-injected mice at P21 and P72-P82 (n = 13-18 measurements per group, from 3 mice each). Statistical significance denoted as **p < 0.01 and ****p < 0.0001, according to Mann-Whitney U tests.

Article Snippet: For AAV-hMAG-DTA, we amplified the DTA coding sequence from the pAAV-mCherry-flex-dtA (a gift from Naoshige Uchida, Addgene plasmid # 58536) using primer pairs (Table.

Techniques: Control, Injection, Fluorescence, Microscopy, Labeling, Immunofluorescence, MANN-WHITNEY