nfκb reporter 660 promoter  (Addgene inc)

Journal: Science Advances

Article Title: Scalable and cost-efficient custom gene library assembly from oligopools

doi: 10.1126/sciadv.ady2279

Figure Lengend Snippet: ( A ) Library sequences were expressed as a fusion protein with mKate2, which was kept constant between library variants and indicates level of expression. Cells were sorted into seven bins of GFP fluorescence approximately uniformly distributed across a log-scale within a narrow mKate2 window to control for expression. ( B ) We calculated a LogF for each sequence averaged from two replicates. We calculated an activity threshold by fitting observed library sequences with a mixture model to determine the intersection of functional and nonfunctional populations. We also calculate the LogF for an additional 76 incorrect assemblies that serve as nonfluorescent controls. ( C ) GFP fluorescence plotted by model and sequence identity. Double mutants are two mutations from avGFP. MCMC sampling sequences are plotted by pairwise identity to the closest natural sequence in the UniProt GFP family. Two known FPs are included as references and indicated by green stars.

Article Snippet: We replaced KillerOrange with an mKate2 construct ordered from Twist Bioscience using the Bam HI and Not I restriction sites.

Techniques: Expressing, Fluorescence, Control, Sequencing, Activity Assay, Functional Assay, Sampling

Journal: Nature Communications

Article Title: TREM2 expression level is critical for microglial state, metabolic capacity and efficacy of TREM2 agonism

doi: 10.1038/s41467-026-68706-8

Figure Lengend Snippet: A Schematic of the reporter construct and the proteins expressed in vitro. Created in BioRender. Mühlhofer, M. ( https://BioRender.com/mls61se . B Representative immunofluorescent images of HeLa cells transfected with the reporter construct shown in ( A ). mKate2 and TREM2 stainings are displayed in magenta and green. Scale bars 20 µm and 5 µm. C Immunoblot analysis of time-dependent degradation of TREM2 and mKate2 upon transient overexpression in HeLa cells. Various TREM2 species including the C-terminal fragment (CTF) are indicated. Shown are three technical replicates from a single experiment. Actin was used as a loading control. The asterisk indicates an unspecific signal. Non trans., non-transfected HeLa cells. Uncropped images in Source Data. D Quantification of the immunoblot data shown in ( C ). For TREM2, the immature and mature TREM2 signals were quantified while for mKate2 the two signals below and above the 36 kDa marker band were quantified. E CRISPR strategy to create the Trem2-mKate2 reporter mice. Sites of silent mutations serving genotyping purposes are indicated. F Representative immunofluorescent images of cortical sections of Trem2-mKate2 mice (wt and homozygous knock-in) showing positive labelling of mKate2 in knock-in animals only (magenta). Dashed squares represent the area of the zoom in in the bottom row. Iba1 and DAPI stainings are displayed in green and blue. Scale bars 20 μm and 5 μm. G Breeding scheme used to create the experimental mice for the Trem2-mKate2 KI/wt .APP/PS1 tg/wt strain. Only heterozygous reporter mice were used throughout the study. Non-transgenic littermates are referred to as’healthy’ and APP/PS1 transgenic mice are referred to as ‘disease’. H Representative immunofluorescent images of hippocampal sections of disease mice (4 months). mKate2 is displayed in magenta, Iba1-positive microglia or TREM2 in green and amyloid beta (Aβ) plaques in red. Scale bars 50 μm and 20 μm. I Percentage of mKate2+ signal with decreasing distance to a plaque. Shown is the mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SD. Male and female mice are indicated by triangles and circles. One data point represents averages of multiple images per mouse. Statistical analysis was carried out by repeated measures one-way ANOVA with Tukey’s multiple comparisons test. N = 7 mice, age = 4 months. Created in BioRender. Mühlhofer, M. ( https://BioRender.com/mls61se ). Source data are provided as a Source Data file.

Article Snippet: We obtained the mKate2 plasmid from Addgene and amplified the sequence by standard polymerase chain reaction (PCR).

Techniques: Construct, In Vitro, Transfection, Western Blot, Over Expression, Control, Marker, CRISPR, Knock-In, Transgenic Assay

Journal: Nature Communications

Article Title: TREM2 expression level is critical for microglial state, metabolic capacity and efficacy of TREM2 agonism

doi: 10.1038/s41467-026-68706-8

Figure Lengend Snippet: A Example contour plots showing the rationale for the FACS gating strategy in healthy and disease mice based on the amount of mKate2 positivity as a proxy for TREM2 expression. Subpopulations were sorted and named based on the amount of mKate2 present. B Boxplot of normalized Trem2 expression coloured by condition. Boxplots show the 25%, 50% (median) and 75% percentile; whiskers denote 1.5 times the interquartile range (applies to all following boxplots). For the RNA-seq data of Trem2-mKate2 KI/wt .APP/PS1 tg/wt mice: N = 5 mice for low healthy, mid healthy, and low disease conditions; N = 4 mice for mid disease and high disease conditions; age = 9 months for all conditions. C PCA of the top 5000 most variable genes coloured by condition. D Boxplot of gene set variation analysis (GSVA) enriching for the plaque induced gene signature from Chen et al. coloured by condition. Male and female mice are indicated by triangles and circles. Statistics were calculated with an unpaired, two-sided Wilcoxon test followed by a Benjamini-Hochberg adjustment ( B , D ). Source data are provided as a Source Data file.

Article Snippet: We obtained the mKate2 plasmid from Addgene and amplified the sequence by standard polymerase chain reaction (PCR).

Techniques: Expressing, RNA Sequencing

Journal: Nature Communications

Article Title: TREM2 expression level is critical for microglial state, metabolic capacity and efficacy of TREM2 agonism

doi: 10.1038/s41467-026-68706-8

Figure Lengend Snippet: A Schematic showing the workflow of scRadiotracing following radiolabelled [ 18 F]-FDG injection in 14-months-old Trem2-mKate2 KI/wt .APP/PS1 tg/wt mice. Created in BioRender. Mühlhofer, M. ( https://BioRender.com/mls61se . B Quantification of radiotracer uptake on a per cell basis in sorted microglial subpopulations after radiolabelled FDG injection. Left: proportion of subpopulations; right: quantification of FDG uptake on a per cell basis. N = 6 for healthy mice and N = 5 for disease mice. Shown is the mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SD. Statistical analysis was carried out by one-way ANOVA with Tukey’s multiple comparisons test. Male and female mice are indicated by triangles and circles. Source data are provided as a Source Data file.

Article Snippet: We obtained the mKate2 plasmid from Addgene and amplified the sequence by standard polymerase chain reaction (PCR).

Techniques: Injection

Journal: Nature Communications

Article Title: TREM2 expression level is critical for microglial state, metabolic capacity and efficacy of TREM2 agonism

doi: 10.1038/s41467-026-68706-8

Figure Lengend Snippet: A PCA analysis of 141 analytes measured in microglia sorted by mKate2 levels in healthy and disease mice from the Trem2-mKate2 KI/wt . APP/PS1 tg/wt line. N = 7 and N = 4 for healthy and disease mice, respectively; age = 9 months. B Top 20 highest contributors to PC1 and PC2 (lrl: correlation coefficient). C Heatmap displaying most significantly changed lipids/metabolites within the comparison of high vs. low mKate2 (i.e., TREM2) in disease mice. D Volcano plots showing significantly changed ( p < 0.05 and log2(fold change) > 0.5) lipids/metabolites in sorted microglia by mKate2 levels in healthy and disease mice. For each metabolite, an unpaired, two-sided moderated t-test was performed to assess differential abundance between experimental groups. P values were adjusted for multiple comparisons using the Benjamini-Hochberg FDR procedure. E Concordance analysis of metabolites associated with TREM2 level differences in healthy vs. disease states. Significantly ( p < 0.05 and log2(fold change) > 0.5) changed analytes are highlighted according to disease status, and those modified in both healthy and disease mice are labelled in red. F Abundances of metabolites associated with PPP and positive energetics vs. mKate2 (i.e., TREM2) levels plotted on a per mouse basis. N = 7 and N = 4 for healthy and disease mice, respectively. Male and female mice are indicated by triangles and circles. Source data are provided as a Source Data file.

Article Snippet: We obtained the mKate2 plasmid from Addgene and amplified the sequence by standard polymerase chain reaction (PCR).

Techniques: Comparison, Modification

Journal: Nature Communications

Article Title: TREM2 expression level is critical for microglial state, metabolic capacity and efficacy of TREM2 agonism

doi: 10.1038/s41467-026-68706-8

Figure Lengend Snippet: A Gating on CD11b-positive cells in single cell suspensions of whole brain. B Defining mKate2 low, mKate2 mid and mKate2 high gates in the CD11b-positive cell population. C Representative FACS data from a Trem2-mKate2 KI/wt .APP/PS1 tg/wt mouse showing the pHrodo gates in the mKate2 low, mid, and high subpopulations from which percentages of pHrodo-positive cells were quantified. D , E Quantification of percentage of pHrodo-positive cells ( D ) and median fluorescence intensity ( E ) in the mKate2 low, mid, and high subpopulations in 9-10-months-old Trem2-mKate2 KI/wt .APP/PS1 tg/wt mice (N = 5). F , G Quantification of percentage of pHrodo-positive cells (F) and median fluorescence intensity (G) in the mKate2 low, mid, and high subpopulations in 13-months-old Trem2-mKate2 KI/wt .APP SAA/SAA .hTfR KI/KI mice (N = 4). Male and female mice are indicated by triangles and circles. Shown is the mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. Statistical analyses in ( D – G ) were carried out by one-way ANOVA with Tukey’s multiple comparisons test. Source data are provided as a Source Data file.

Article Snippet: We obtained the mKate2 plasmid from Addgene and amplified the sequence by standard polymerase chain reaction (PCR).

Techniques: Single Cell, Fluorescence

Journal: Nature Communications

Article Title: TREM2 expression level is critical for microglial state, metabolic capacity and efficacy of TREM2 agonism

doi: 10.1038/s41467-026-68706-8

Figure Lengend Snippet: A Schematic of experimental design. Created in BioRender. Mühlhofer, M. ( https://BioRender.com/mls61se ). 8-month-old Trem2-mKate2 KI/wt .APP SAA/SAA .hTfR KI/KI mice were injected with 1 mg/kg ATV antibody monthly until 11 months. At 12 months, mice were injected with 30 mg/kg FDG, perfused 1 h later and then the single cell fractions of isolated brains were sorted into low, mid and high mKate2 expressing subpopulations. Cells were pelleted and frozen for further analysis. N = 6 mice per treatment group. B PCA analysis of 149 analytes measured by LC-MS in sorted microglia. C Top 20 highest contributors to PC1 and PC2 (lrl: correlation coefficient). D Heatmap of top 40 differentially regulated lipids and metabolites. E Volcano plots of treatment comparisons within the same mKate2/TREM2 level. For each metabolite, an unpaired, two-sided moderated t-test was performed to assess differential abundance between experimental groups. P-values were adjusted for multiple comparisons using the Benjamini-Hochberg FDR procedure. F Feature plots of individual lipids/metabolites altered by ATV:4D9 (orange symbols) vs ATV:Iso (grey symbols) within low, mid and high mKate2 subpopulations (N = 6 mice per treatment group). Shown is the mean \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SEM. p values were calculated within each group by unpaired Student’s t-test (two-tailed). Male and female mice are indicated by triangles and circles. Source data are provided as a Source Data file.

Article Snippet: We obtained the mKate2 plasmid from Addgene and amplified the sequence by standard polymerase chain reaction (PCR).

Techniques: Injection, Single Cell, Isolation, Expressing, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test