Journal: Stem Cells Translational Medicine

Article Title: A Rapid Pipeline to Model Rare Neurodevelopmental Disorders with Simultaneous CRISPR/Cas9 Gene Editing

doi: 10.1002/sctm.16-0158

Figure Lengend Snippet: Simultaneous CRIPSR/CAS9 genome editing and iPSC induction. (A) : Schematic illustrating the transfection, selection, and induction process. (B) : Bright field and fluorescent images of an iPSC colony 7 days and 25 following transfection. Successfully induced colonies are initially RFP+, but become RFP− due the episomal nature of the vector. Scale bars represent 30 μm. (C) : Details of the CRIPSR/CAS9 and iPSC induction episomal vectors used. (D) : Gel showing untransfected, homozygous KO, and heterozygous generated iPSC lines for the gene GRIN2B . (E) : Expression of the GRIN2B gene as assessed via quantitative polymerase chain reaction in forebrain neurons derived from the iPSC lines shown in (D) 28 days after the initiation of differentiation. Expression levels normalized to GAPDH expression. (F) : Sanger sequencing results from the GRIN2B locus of the polymerase chain reaction products shown in (D). (G) : Representative chromatogram plots illustrating the deletion found in one allele of the GRIN2B knockout. All chromatogram plots can be found in the supplement. Abbreviations: iPSC, induced pluripotent stem cell; KO, knockout; RFP, red fluorescent protein.

Article Snippet: A double nickase CRIPSR/Cas9 gene editing system with gRNA (DNA2.0) targeting a 51 base pair (bp) exonic sequence of GRIN2B was generated with a Paprika RFP reporter (DNA 2.0).

Techniques: Transfection, Selection, Plasmid Preparation, Generated, Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, Sequencing, Polymerase Chain Reaction, Knock-Out

Journal: eLife

Article Title: Co-regulation and function of FOXM1 / RHNO1 bidirectional genes in cancer

doi: 10.7554/eLife.55070

Figure Lengend Snippet:

Article Snippet: Genetic reagent ( Homo sapiens ) , pCW57-RFP-P2A-MCS , Addgene , 78933 , Plasmid.

Techniques: Magnetic Beads, Purification, Single Cell Gel Electrophoresis, Bicinchoninic Acid Protein Assay, DNA Methylation Assay, Gel Extraction, TA Cloning, Reporter Assay, Reporter Gene Assay, Plasmid Preparation, Software

Journal: Scientific Reports

Article Title: CRISPR/Cas9 nickase-mediated disruption of hepatitis B virus open reading frame S and X

doi: 10.1038/srep13734

Figure Lengend Snippet: ( A ) Schematic representation of the hepatitis B virus genome. Relaxed circular DNA (rcDNA) of the HB virion (thin continuous line), which is converted to cccDNA (thin continuous and dotted line) following hepatocyte infection, is indicated in the centre of the map. The four viral transcripts of the core (C), polymerase (P), and surface (S) and X proteins are indicated around the outside. Regions targeted by Cas9n via guide RNA (gRNA) specific to S and X sequences are indicated by arrows and scissors (scissors were drawn by Niklas Beschorner). ( B ) DNA sequence and sequence conservation of the regions targeted by Cas9n within the S and X gene of HBV. The sequence shown is based on genotype A consensus. Target sequences in ORF S and X are depicted (S1 and S2, or X1 and X2), each encompassing proto-spacer adjacent motifs (PAM, bold and boxed), 2 × 20 nucleotides complementary to gRNA (boxed) and offset distance between the two sequences complementary to gRNA (underlined).

Article Snippet: Cultures were grown to ~ 70–80% confluence and subsequently transfected with a total of 1.5 μg plasmid DNA (i.e. RFP-GFP reporter constructs together with the expression plasmids Cas9n/sgRNA-S1 and Cas9n/sgRNA-S2, or Cas9n/sgRNA-X1 and Cas9n/sgRNA-X2, respectively) using Lipofectamin 2000 (Invitrogen) according to the manufacturer’s protocol.

Techniques: Virus, Infection, Sequencing

Journal: Scientific Reports

Article Title: CRISPR/Cas9 nickase-mediated disruption of hepatitis B virus open reading frame S and X

doi: 10.1038/srep13734

Figure Lengend Snippet: ( A ) T7EI assays were performed using PCR primers (indicated by arrows) flanking the HBV S or X sequence in the respective reporter plasmid. ( B ) Detection of Cas9n-specific activity was visualized by gel electrophoresis. HEK293 cells were transfected as before and total genomic DNA was isolated at 72 h post transfection for subsequent T7EI cleavage. Arrows depict the sizes of wild-type and Cas9n-mutagenized DNA fragments. ( C ) T7EI assay using genomic DNA from GFP + HEK293 cell cultures at 24 h post transfection. ( D ) Sequence analysis of corresponding DNA samples. Alignment to the wild-type ORF S reporter sequence is shown. gRNA sequences (boxed), PAM (boxed and bold) and Cas9n-mediated deletions are indicated.

Article Snippet: Cultures were grown to ~ 70–80% confluence and subsequently transfected with a total of 1.5 μg plasmid DNA (i.e. RFP-GFP reporter constructs together with the expression plasmids Cas9n/sgRNA-S1 and Cas9n/sgRNA-S2, or Cas9n/sgRNA-X1 and Cas9n/sgRNA-X2, respectively) using Lipofectamin 2000 (Invitrogen) according to the manufacturer’s protocol.

Techniques: Sequencing, Plasmid Preparation, Activity Assay, Nucleic Acid Electrophoresis, Transfection, Isolation, T7EI Assay

Journal: Autophagy

Article Title: Follicular lymphoma-associated mutations in the V-ATPase chaperone VMA21 activate autophagy creating a targetable dependency.

doi: 10.1080/15548627.2022.2050663

Figure Lengend Snippet: Figure 2. Follicular lymphoma-associated VMA21 mutations activate autophagic flux. (A) HEK293T cells transfected with empty vector, WT, or mutant (93X) VMA21 treated -/+ bafilomycin A1. Immunoblots of the indicated antigens. (B) Densitometry quantification (LC3-II:ACTB) of n = 4 independent experiments from representative panel A. Statistical comparisons: i) VMA21 93X versus WT (A; lanes 5–6). ii) VMA21 93X versus WT plus -/+ bafilomycin A1. (A; lanes 8–9), using unpaired 2-tailed t-testing and Bonferroni corrections (*: p < 0.05; **: p < 0.01). Bars: standard deviations. (C) Electron microscopy and enumeration of autophagosomes (AP), autolysosomes (AL) and late endosomes/lysosomes (LE/LY) in HEK293T cells expressing VMA21 WT or MUT 93X. Representative images of N = 70–90 each. (D) Statistical comparisons of data generated in panel C using unpaired 2-tailed Mann-Whitney U-test and Bonferroni corrections. ***: p < 0.001). Bars: Standard eviations. (E-K) S. cerevisiae cells expressing either Vma21 (WT) or Vma21[∆66-77] were analyzed for autophagy. The cells were grown in YPD (-N, 0 min) to mid-log phase, and shifted to nitrogen starvation (SD-N) (E) The GFP-Atg8 processing assay. Pgk1 was used as a loading control. (F) The ratio of free GFP to Pgk1 after 45 min SD-N treatment was quantified. Mean ± SD of n = 4 independent experiments. Unpaired, 2-tailed t-test; *: p < 0.05. (G) Pho8∆60 enzymatic activity was measured under growing and nitrogen-starvation conditions. Mean ± SD of n = 5 independent experiments. Two-way ANOVA; ***: p < 0.001. (H) The prApe1 processing assay. Dpm1 was used as a loading control. (I) The ratio of Ape1 to prApe1 after 15 min SD-N treatment was quantified. Mean ± SD of n = 3 independent experiments. Unpaired, 2-tailed t-test; *: p < 0.05. (J) The Atg8 lipidation assay. Pgk1 was used as a loading control. (K) The ratio of Atg8–PE to Pgk1 after 45 min of nitrogen starvation was quantified. Mean ± SD of n = 4 independent experiments. Two-way ANOVA; *: p < 0.05, **: p < 0.01.

Article Snippet: Autophagy assays using the GFP-LC3-RFP reporter in VMA21-inducible cell lines Generation of GFP-LC3-RFP reporter cell lines HEK293T cells were transfected with pMRX-IP-GFP-LC3RFP (Addgene, 84573; deposited by Dr. Noboru Mizushima) and packaging plasmids and virus-containing supernatant media collected 48 h after transfection.

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Electron Microscopy, Expressing, Generated, MANN-WHITNEY, Control, Activity Assay

Journal: Autophagy

Article Title: Follicular lymphoma-associated mutations in the V-ATPase chaperone VMA21 activate autophagy creating a targetable dependency.

doi: 10.1080/15548627.2022.2050663

Figure Lengend Snippet: Figure 3. Adaptation of an autophagic reporter assays to measure VMA21 MUT activated autophagy. (A) Schema of the autophagy reporter fusion protein GFP-LC3 -(ATG4 cleavage site)-RFP used to make stable HEK293T cells also carrying inducible VMA21 WT and MUT. The less acidic lysosomes in VMA21 MUT cells (see Results) allow for persistence of green fluorescence autolysosomes as a quantitative readout for autophagy. (B-C) Detection of free GFP generated in lysosomes in stable HEK293T or stable OCI-LY1 lymphoma cells also carrying inducible VMA21 WT and MUT. Immunoblots for the indicated antigens. (D) Confocal microscopy images of stable HEK293T cells also carrying inducible empty vector, or VMA21 WT and MUT. (-) empty vector, SAR405: a PIK3C3/VPS34 inhibitor, MRT68921: a ULK1 inhibitor. (E) Densitometry quantification (free GFP:ACTB) of n = 3 independent experiments from representative panel B. Statistical comparisons: i) ATP6V1B2 MUT 371C or 400Q versus WT (B: lanes 8–10) or VMA21 93X versus WT (B: lanes 11–12), using unpaired 2-tailed t-testing and Bonferroni corrections (*: p < 0.05; **: p < 0.01, ***: p < 0.001). (F) Densitometry quantification (free GFP:ACTB) of three independent experiments from representative panel C. Statistical comparisons: i) ATP6V1B2 MUT 400Q versus WT (B: lanes 7–8) or VMA21 93X versus WT (C lanes 9–10). Statistical comparisons as in panel E. (G) Mean GFP-positive vesicles per cells from images as in panel D. (-) vector, VMA21 WT or MUT 93X, or VMA21 p.93X pretreated with the autophagy inhibitors SAR405 or MRT68921. Statistical comparisons using unpaired 2-tailed t-testing and Bonferroni corrections (*: p < 0.05; **: p < 0.01, ***: p < 0.001). Bars: tandard deviations in panels E-G.

Article Snippet: Autophagy assays using the GFP-LC3-RFP reporter in VMA21-inducible cell lines Generation of GFP-LC3-RFP reporter cell lines HEK293T cells were transfected with pMRX-IP-GFP-LC3RFP (Addgene, 84573; deposited by Dr. Noboru Mizushima) and packaging plasmids and virus-containing supernatant media collected 48 h after transfection.

Techniques: Fluorescence, Generated, Western Blot, Confocal Microscopy, Plasmid Preparation

Journal: Autophagy

Article Title: Follicular lymphoma-associated mutations in the V-ATPase chaperone VMA21 activate autophagy creating a targetable dependency.

doi: 10.1080/15548627.2022.2050663

Figure Lengend Snippet: Figure 4. HA-VMA21 MUT 93X proteins aberrantly localized to lysosomes. (A) Confocal microscopy and merged images of cells stained for CANX (ER), GOLGA2 (Golgi) and LAMP1 (lysosomes) and HA-VMA21 WT or MUT. (B) Pearson correlation coefficients for colocalization of VMA21 WT or MUT proteins from (A). (C) Representative results for lysosomal-IPs (lyso-IPs; see Materials and Methods) out of stable HEK293T cells expressing the fusion protein bait 3xHA-TMEM192 or 2xFLAG-TMEM (negative control) and inducible VMA21 WT and MUT. Detection of protein in direct cell lysates and in the retained lysosomal IP preparations for the indicated antigens. (D) Densitometry quantification (VMA21 MUT:WT protein) of n = 3 independent experiments from representative panel C and quantification of V-ATPase components from either lysates or enriched lysosomes. Statistical comparisons using unpaired 2-tailed t-testing (*: p < 0.05). Bars: Standard deviations. (E-H) S. cerevisiae cells expressing either full-length Vma21 (WT) or the truncation mutant Vma21[∆66-77] were analyzed for assembly of V-ATPase subunits. (E) The cellular and vacuolar Vma2 protein levels were monitored: The cells were grown in YPD to mid-log phase, and vacuoles were isolated. The levels of Vma2 were analyzed by western blot. Ponceau S staining was used for total protein normalization. (F) The cellular and vacuolar Vma4 protein levels were monitored as in (E). (G) Vma2 and (H) Vma4 protein levels. Either cellular or vacuolar Vma2 and Vma4 protein levels in WT cells were set to 100%, and the corresponding levels in Vma21[∆66-77] cells were normalized. Mean ± SD of n = 3 independent experiments are shown. Unpaired, 2-tailed t-test; *: p < 0.05, ns: not significant.

Article Snippet: Autophagy assays using the GFP-LC3-RFP reporter in VMA21-inducible cell lines Generation of GFP-LC3-RFP reporter cell lines HEK293T cells were transfected with pMRX-IP-GFP-LC3RFP (Addgene, 84573; deposited by Dr. Noboru Mizushima) and packaging plasmids and virus-containing supernatant media collected 48 h after transfection.

Techniques: Confocal Microscopy, Staining, Expressing, Negative Control, Mutagenesis, Isolation, Western Blot

Journal: Autophagy

Article Title: Follicular lymphoma-associated mutations in the V-ATPase chaperone VMA21 activate autophagy creating a targetable dependency.

doi: 10.1080/15548627.2022.2050663

Figure Lengend Snippet: Figure 6. Follicular-lymphoma associated VMA21 mutations reduce the ability of the V-ATPase to acidify lysosomes because of reduced lysosomal V-ATPase activity. (A) Stable HEK293T cells carrying inducible WT or MUT VMA21 were induced with doxycycline and loaded with the pH indicator dextran-conjugated LysoSensor Blue/ Yellow. Untransfected cells were treated with EMS buffer calibrated to pH 3.5, pH 4.5 or pH 5.5. The fluorescence intensity of cell suspensions was read using flow cytometry. The mean fluorescence intensity of the yellow dye signal is a measure of lysosomal pH. (B) Estimation of lysosomal pH from data generated as in panel A with n = 3. (C) Vacuolar pH was measured using a pH-sensitive Pho8-SEP protein that exhibits stronger fluorescence with higher pH. Upper images show the fluorescence signal of Pho8-SEP; lower images show the corresponding light microscopy. (D) The quantitative analysis of fluorescence intensity of Pho8-SEP in WT, Vma21[∆66-77], and vma21∆ cells. Mean ± SD of n = 3 independent experiments. Unpaired, 2-tailed t-test with Bonferroni correction; *: p < 0.05, ***: p < 0.005. (E) Lysosomal V-ATPase activity (see Materials and Methods) in lysosomal preparations isolated via lyso-IP from cells carrying no lyso-IP bait (negative control), (-) empty vector, VMA21 WT, VMA21 93X, an shRNA targeting VMA21 or bafilomycin A1. n = 4, unpaired, 2-tailed t-test; ***: p < 0.005. (F) Growth of yeast strains (WT, Vma21 [∆66-77], and vma21∆) on YPD plates buffered to pH 5.0 and on YPD plates buffered to pH 7.5. (G) The quantitative V-ATPase activity of WT, Vma21[∆66-77], and vma21∆ vacuoles were measured. After vacuole isolation, the release of inorganic phosphate arising from hydrolysis of ATP was determined. Mean ± SD of n = 3 independent experiments. Unpaired, 2-tailed t-test; ***: p < 0.005.

Article Snippet: Autophagy assays using the GFP-LC3-RFP reporter in VMA21-inducible cell lines Generation of GFP-LC3-RFP reporter cell lines HEK293T cells were transfected with pMRX-IP-GFP-LC3RFP (Addgene, 84573; deposited by Dr. Noboru Mizushima) and packaging plasmids and virus-containing supernatant media collected 48 h after transfection.

Techniques: Activity Assay, Fluorescence, Flow Cytometry, Generated, Light Microscopy, Isolation, Negative Control, Plasmid Preparation, shRNA

Journal: Autophagy

Article Title: Follicular lymphoma-associated mutations in the V-ATPase chaperone VMA21 activate autophagy creating a targetable dependency.

doi: 10.1080/15548627.2022.2050663

Figure Lengend Snippet: Figure 7. Untargeted metabolomic analyses of lysosomes and the cytosolic fraction from cells expressing VMA21 WT or MUT. Intact lysosomes were isolated from HEK293T cells stably expressing the protein bait 3xHA-TMEM192 and inducible VMA21 WT and MUT using the lyso-IP method [35]. An aliquot of cells was treated with bafilomycin A1 for 1 h and processed in parallel. The lysosomes and the post-IP supernatant were processed for untargeted metabolomics using tandem mass spectrometry. (A) LEFT: Pearson correlation of lysosomal amino acid ratios. X-axis: log2 MUT:WT and Y-axis: log2 BAF:WT. RIGHT: X-axis: log2 BAF:WT from this study and Y-axis: log2 BAF:WT from the study by Abu-Remaileh et al., Science 2017. (B) Volcano plot display for lysosomal di- and tripeptides. Left. X-axis: log2-fold ratios of di- and tripeptides in cells carrying MUT:WT VMA21. Y-axis: log10 FDR MUT:WT VMA21. Right. Data for bafilomycin A1-treated cells compared with VMA21 WT cells. (C) Volcano plot display for lysosomal amino acids. LEFT: X-axis log2-fold ratios of amino acids in cells carrying MUT:WT VMA21. Y-axis: log10 FDR. RIGHT: Data for bafilomycin A1-treated cells compared with VMA21 WT cells. (D) Volcano plot display for amino acids in the post-IP supernatant. LEFT: X-axis log2-fold ratios of amino acids in cells carrying MUT:WT VMA21. Y-axis: log10 FDR. RIGHT: Data for bafilomycin A1-treated cells compared with VMA21 WT cells. (E) Heatmap and numerical display of identified amino acids mass ratios for the indicated comparisons on top. Empty cells indicates the amino acid was not identified unequivocally for that condition. The mean results from three biological replicates are shown.

Article Snippet: Autophagy assays using the GFP-LC3-RFP reporter in VMA21-inducible cell lines Generation of GFP-LC3-RFP reporter cell lines HEK293T cells were transfected with pMRX-IP-GFP-LC3RFP (Addgene, 84573; deposited by Dr. Noboru Mizushima) and packaging plasmids and virus-containing supernatant media collected 48 h after transfection.

Techniques: Expressing, Isolation, Stable Transfection, Mass Spectrometry

Journal: Autophagy

Article Title: Follicular lymphoma-associated mutations in the V-ATPase chaperone VMA21 activate autophagy creating a targetable dependency.

doi: 10.1080/15548627.2022.2050663

Figure Lengend Snippet: Figure 9. Results from a high-throughput chemical screen for compounds inhibiting VMA21-MUT induced autophagy. (A) UPPER: Images of HEK293T cells stably expressing the autophagy reporter fusion protein GFP-LC3-(ATG4 cleavage site)-RFP and inducible VMA21 MUT protein (p.93X). High-power inserts show the accumulation of GFP+ autophagosomes (APs) and autolysosomes (ALs). LOWER: Transformed image showing detection and quantification of APs and ALs. (B) Screening flow schema. (C) Overview of assay results for the entire compound library. Displayed is the percentage of cells with > 5 GFP puncta (APs + ALs) in compound-treated cells divided by untreated cells. Individual assay results for cell expression of VMA21 MUT as follows: Blue dots, treated with DMSO only (assay baseline); black dots, treated with the autophagy inhibitor MRT68921 targeting ULK1 (positive control); red dots, treated with various compounds showing autophagy inhibiting properties (primary screen hits); gray dots, treated with various inactive compounds. (D) Pathway enrichment and target analysis for compounds with autophagy inhibitory properties. (E) Dose-response curve of selected autophagy inhibiting compounds including MRT68921 (targeting ULK1) and SAR405 (targeting PIK3C3/VPS34), CDK inhibitors and homoharringtonin

Article Snippet: Autophagy assays using the GFP-LC3-RFP reporter in VMA21-inducible cell lines Generation of GFP-LC3-RFP reporter cell lines HEK293T cells were transfected with pMRX-IP-GFP-LC3RFP (Addgene, 84573; deposited by Dr. Noboru Mizushima) and packaging plasmids and virus-containing supernatant media collected 48 h after transfection.

Techniques: High Throughput Screening Assay, Stable Transfection, Expressing, Transformation Assay, Drug discovery, Positive Control

Journal: bioRxiv

Article Title: MTOR as a selectable genomic harbor for CRISPR-engineered CAR-T cell therapy

doi: 10.1101/2023.09.14.557485

Figure Lengend Snippet: ( a ) Schematic representation of CD19-CAR-2A-EGFP targeting to the reverse DNA strand of the MTOR locus using an AAV6 donor. The F2108L mutation is introduced via the left homology arm (HA) and transgene expression is driven by a human PGK1 promoter. ( b ) Representative timeline for nucleofection and ex vivo rapamycin selection. ( c ) Out-Out PCR for CD19-CAR-2A-EGFP knock-in detection at MTOR . Whole blood (WB) primary CD3 + T cells from WB donor 1 (WB 1) were transfected with a SpCas9-G4 RNP and transduced with an AAV6 vector with the indicated multiplicity of infection (MOI). T cells were treated (rapamycin) or not (vehicle) with 25 nM rapamycin 3 days post-transfection for 8 days. The percentage of edited alleles was determined by TIDER from Sanger sequencing. n = 1 experiment. ( d ) Same as in ( c ), but FACS-based quantification of targeted CD19-CAR-2A-EGFP integration. ( e ) Same as in ( c,d ) with a MOI of 5x10 and primary CD3 + T cells isolated from WB or a leukocyte reduction system (LRS) from three additional healthy donors. n = 3 independent biological replicates performed in triplicate at different times with CD3 + T cells from the indicated healthy donor. Each data point represents a technical replicate. ( e ) Luciferase-based cytotoxicity assay. Following rapamycin selection, CD19-CAR-T cells were incubated with the indicated effector to target (E:T) ratio with NALM6 cells stably expressing firefly luciferase (FLUC) and 25 nM rapamycin. Rapamycin-resistant NALM6 cells harboring the MTOR -F2108L mutation (NALM6-RapaR) were used as a control to analyze the combinatorial impact of rapamycin. Luminescence was measured after 18 hours of incubation. n = 2 independent biological replicates performed at different times with CD3 + T cells from two different healthy donors (WB donor 1 and LRS donor 2). Each data point represents the average of three technical replicates. hPGK1 , human phosphoglycerate kinase 1 promoter. PA, polyadenylation signal. HA, homology arm.

Article Snippet: K562 cells were obtained from the ATCC (CCL-243) and NALM6 cells stably transduced to express a FLUC-T2A-RFP-IRES-Puro reporter gene cassette (Biosettia) were gently provided by Scott McComb (National Research Council of Canada).

Techniques: Mutagenesis, Expressing, Ex Vivo, Selection, Knock-In, Transfection, Transduction, Plasmid Preparation, Infection, Sequencing, Isolation, Luciferase, Cytotoxicity Assay, Incubation, Stable Transfection

Journal: bioRxiv

Article Title: MTOR as a selectable genomic harbor for CRISPR-engineered CAR-T cell therapy

doi: 10.1101/2023.09.14.557485

Figure Lengend Snippet: ( a ) Timeline and experimental setup for the pre-B acute lymphoblastic leukemia xenograft mouse model. Male and female NSG mice were challenged with 0.5 x 10 NALM6-RFP-FLUC cells and daily rapamycin treatment (4 mg/kg) started three days later. Four days after tumor inoculation, 2 x 10 untransduced (UT) T cells or CD19-CAR-T cells were injected (Donor ID WB2, see ). ( b ) Bioluminescence (BLI) quantification and Kaplan-Meier survival plot. Leukemia engraftment, bio-distribution, and tumor progression were assessed by BLI imaging two times per week. The radiance (photons/s) of the regions of interest (ROIs) corresponds to the area containing the whole back side of the body. n = 8 mice per group. ( c ) BLI and tumor bio-distribution of all mice from ( b ) over seven weeks. The color barcode represents the radiance scale (photons/s/cm /sr).

Article Snippet: K562 cells were obtained from the ATCC (CCL-243) and NALM6 cells stably transduced to express a FLUC-T2A-RFP-IRES-Puro reporter gene cassette (Biosettia) were gently provided by Scott McComb (National Research Council of Canada).

Techniques: Injection, Imaging

Journal: Molecular Metabolism

Article Title: The MicroRNA miR-696 is regulated by SNARK and reduces mitochondrial activity in mouse skeletal muscle through Pgc1α inhibition

doi: 10.1016/j.molmet.2021.101226

Figure Lengend Snippet: Pgc1α was targeted by miR-696 . (A) The schematic interaction between miR-696 and Pgc1α 3′UTR. (B) Primary rat-cultured cells were transfected with full-length Pgc1α plasmid on day 3 of differentiation and then transfected with miR-696 mimic on day 4 of differentiation. After 24 h, protein was collected and Western blotting was performed to detect Pgc1α content (100 μg of protein was loaded). β-actin was used as internal control (n = 2). (C) C2C12 cells were co-transfected either with RFP- miR-696 and GFP- Pgc1α -3′UTR or GFP- Pgc1α -3′UTRmt. GFP fluorescence signals were measured to quantify the interactions between miR-696 and Pgc1α 3′UTR (n = 3 experiments). (D) Quantitative analysis of Figure 4 C. Scramble oligonucleotide plus GFP (NC) was used as a negative control (white bar), RFP- miR-696 plus GFP- Pgc1α -3′UTRmt (black bar), and RFP- miR-696 plus GFP- Pgc1α -3′UTR (gray bar). (E–F) Cells were transfected with an empty vector and a scrambled oligonucleotide (EV-Scr), Pgc1α vector and a scrambled ( Pgc1α -Scr), Pgc1α vector and a miR-696 mimetic ( Pgc1α -MiR696), an empty vector, and a miR-696 mimetic (EV-MiR696) (n = 4). (E) The oxygen consumption rate at a basal state and in the presence of oligomycin, FCCP, and rotenone. (F) Bars indicate the variations in the oxygen consumption rate (ΔOCR, basal = basal − rotenone; uncoupled = oligomycin − rotenone; maximal = FCCP − rotenone) from kinetics depicted in Figure 2 E. ∗∗p < 0.01 and ∗∗∗p < 0.001 vs black bar (one-way ANOVA and Tukey's post-test). Each bar indicates mean ± SEM.

Article Snippet: Full-length Pgc1α plasmid, GFP, and RFP reporter plasmids were purchased from Addgene (cat. #4, #35625, and #35626).

Techniques: Cell Culture, Transfection, Plasmid Preparation, Western Blot, Control, Fluorescence, Negative Control

Journal: Molecular Metabolism

Article Title: The MicroRNA miR-696 is regulated by SNARK and reduces mitochondrial activity in mouse skeletal muscle through Pgc1α inhibition

doi: 10.1016/j.molmet.2021.101226

Figure Lengend Snippet: miR-696 reduced glucose uptake in C2C12 cells. (A–B) C2C12 cells were transfected with RFP (control) or RFP- miR-696 (miR-696) (n = 6). (A) Gene expression of Pgc1α and Glut4, in both the presence and absence of miR-696 (β-actin was used as a reference gene). (B) Glucose uptake was measured with or without insulin (100 nM) in the control and miR-696-transfected groups. (C) C2C12 cells were transfected with scrambled oligonucleotide (control), scrambled oligonucleotide and treated with 700 μM of palmitic acid (palmitic acid), miR-696 antisense oligonucleotide and treated with 700 μM of palmitic acid (palmitic acid + miR-696 ASO), and miR-696 full-length oligonucleotide sequence ( miR-696 mimic). All the groups were treated with vehicle (ethanol in 1% BSA) (n = 5). Western blotting of total Akt (Akt) and phosphorylated Akt on serine 473 (pAkt Ser 473) with or without insulin (100 nM) for 10 min. β-actin (30 μg) was used as an internal control. (D) The bars are a quantification of the Western blotting for the treatment groups represented in panel (C) (n = 3). ∗p < 0.05 vs control (Student's t test). Each bar indicates mean ± SEM.

Article Snippet: Full-length Pgc1α plasmid, GFP, and RFP reporter plasmids were purchased from Addgene (cat. #4, #35625, and #35626).

Techniques: Transfection, Control, Gene Expression, Sequencing, Western Blot

Journal: Access Microbiology

Article Title: Unigems: plasmids and parts to facilitate teaching on assembly, gene expression control and logic in E. coli

doi: 10.1099/acmi.0.000596.v3

Figure Lengend Snippet: Unigems plasmids

Article Snippet: GFP and RFP reporter genes, placed under the control of either a strong or a weak promoter (pFAB4026 and pFAB4282 for GFP and pFAB4005 and pFAB4024 for RFP, respectively) (BIOFAB collection [ ]) were analysed using a flow cytometer on three replicate samples each (separate colonies from the same transformation event).

Techniques:

Journal: Access Microbiology

Article Title: Unigems: plasmids and parts to facilitate teaching on assembly, gene expression control and logic in E. coli

doi: 10.1099/acmi.0.000596.v3

Figure Lengend Snippet: Primers for the unigems plasmids

Article Snippet: GFP and RFP reporter genes, placed under the control of either a strong or a weak promoter (pFAB4026 and pFAB4282 for GFP and pFAB4005 and pFAB4024 for RFP, respectively) (BIOFAB collection [ ]) were analysed using a flow cytometer on three replicate samples each (separate colonies from the same transformation event).

Techniques: Sequencing

Journal: Access Microbiology

Article Title: Unigems: plasmids and parts to facilitate teaching on assembly, gene expression control and logic in E. coli

doi: 10.1099/acmi.0.000596.v3

Figure Lengend Snippet: GFP fluorescence of three replicates of each p006-strongGFP and p006-weakGFP and RFP yellow fluorescence of p005-strongRFP and p005-weakRFP. Note the difference in scale in GFP vs RFP: the lack of differences between strong and weak RFP fluorescence is due to a mismatch between its optimal excitation wavelength (550 nm) and the 488 nm excitation laser in the flow cytometer (see Discussion).

Article Snippet: GFP and RFP reporter genes, placed under the control of either a strong or a weak promoter (pFAB4026 and pFAB4282 for GFP and pFAB4005 and pFAB4024 for RFP, respectively) (BIOFAB collection [ ]) were analysed using a flow cytometer on three replicate samples each (separate colonies from the same transformation event).

Techniques: Fluorescence, Flow Cytometry

Journal: Access Microbiology

Article Title: Unigems: plasmids and parts to facilitate teaching on assembly, gene expression control and logic in E. coli

doi: 10.1099/acmi.0.000596.v3

Figure Lengend Snippet: Images of p005-kan-RFP (left column) and p006-kan-GFP (right column) in daylight ( a ) and on a benchtop UV transilluminator (excitation wavelength 395 nm) ( b ). Transformed DH5α cells were grown with 50 µg ml −1 of kanamycin and induced with 100 mM IPTG. The single RFP-expressing colony on the top right plate is due to a mix of plasmids used by the students in this transformation. Photographs were taken with a mobile phone by JB.

Article Snippet: GFP and RFP reporter genes, placed under the control of either a strong or a weak promoter (pFAB4026 and pFAB4282 for GFP and pFAB4005 and pFAB4024 for RFP, respectively) (BIOFAB collection [ ]) were analysed using a flow cytometer on three replicate samples each (separate colonies from the same transformation event).

Techniques: Transformation Assay, Expressing