Journal: bioRxiv

Article Title: Cell-cycle dynamics of nascent transcription and mature RNA accumulation are concordant in normal fibroblasts

doi: 10.1101/2025.09.12.675830

Figure Lengend Snippet: (A) BJ-Fucci fibroblasts. BJ-5ta fibroblast cells are transduced with the Fucci4 cell-cycle reporters mKO2-Cdt1 aa30-120 , mTurquoise2-SLBP aa18-126 , Clover-Geminin aa1-110 , and mMaroon1-H1. (B) Nuclear fluorescence signals in a single BJ-Fucci cell (0–19 hr), daughter cell (19–43 hr), and grand-daughter cell (43hr–) measured by time-lapse imaging (duration 48 hr, interval 15 min). Circle, min-max normalized log 10 intensity. Line, Loess regression. Top, empirical cell cycle phase annotations. See for additional cells. (C) FACS of BJ-Fucci cells into early G1 (EG1), G1, early S (ES), and late S (LS)/G2/M phases. Populations indicated in the Clover/mKO2 plot (left) are shown in mTurquoise2 histogram (right). EG1 and ES are subsets of Clover/mKO2 populations defined by mTurquoise2 intensity. (D) Principal component analysis (PCA) of RNA-seq biological replicates by their mature RNA abundance (log 10 RNA-seq TPM) in all genes. (E) Mature RNA abundance (gene-level z-score of log 10 RNA-seq TPM) of select genes known to be dynamic in expression during the cell cycle. (F) K-means clustering of the union of differentially expressed genes by their mature RNA dynamics (gene-level z-score of log 10 RNA-seq TPM). “Icons” are within-cluster mean of log 10 TPM z-score and used in subsequent figures to graphically represent cluster dynamics. (G) Molecular Function and Cellular Component GO terms enriched in dynamic mature RNA clusters. Top 3 most enriched terms from each cluster are selected and their enrichment in each cluster was shown. (H) Mature RNA abundance dynamics (log 10 RNA-seq TPM z-score) of five representative genes in each cluster.

Article Snippet: Independently, we transduced BJ-5ta cells with the two vectors individually (cell ID cc1376-1 for mKO2/Clover and cc1376-2 for mTurquois2/mMaroon1) or with mKO2-SLBP aa18-126 (gift from Michael Lin; Addgene ID 83914; RRID:Addgene_83914; cell ID cc1376-3) or with Clover-Geminin aa1-110 (gift from Michael Lin; Addgene ID 83915; RRID:Addgene_83915; cell ID cc1376-4), to generate references for signal compensation in flow cytometry.

Techniques: Transduction, Fluorescence, Imaging, RNA Sequencing, Expressing

Journal: Cells

Article Title: The Impact of ALS-Associated Genes hnRNPA1 , MATR3 , VCP and UBQLN2 on the Severity of TDP-43 Aggregation

doi: 10.3390/cells9081791

Figure Lengend Snippet: C -terminal domain truncations affect TDP-43 aggregate formation in the cytoplasm of SH-SY5Y cells: ( A ) A schematic representation of mKO2-tagged constructs and their abbreviations. Only TDPwt has nuclear localization signal (NLS), whereas others lack NLS and except dNLS that holds intact C-terminus, they carry deletions of LCD: IDR2 is deleted in dNLSd343 (ends at 343 aa residue), IDR2 and CR are deleted in dNLSd299 (ends at 299 aa residue); whole C -terminal domain is deleted in dNLSd267 (ends in 267 aa residues). A control construct KO2only holds sequence for mKO2 protein alone. ( B ) Western blot analysis of mKO2-tagged constructs. ( C ) Quantification of transfected cells harboring aggregates. ( D ) Average number of aggregates in an individual cell. ( E ) Average aggregate size within individual cells. ( F ) SH-SY5Y cells, expressing mKO2-tagged constructs. Nuclei are counterstained with DAPI. Scale bars: 10 µm.

Article Snippet: TDP-43 constructs: TDP-43 was subcloned from previously published plasmid [ ] into mKO2-C1 plasmid, containing sequence for fluorescent protein mKusabira Orange2 (mKO2) (Addgene #54494, deposited by Michael Davidson and Atsushi Miyawaki [ ]).

Techniques: Construct, Residue, Control, Sequencing, Western Blot, Transfection, Expressing

Journal: Cells

Article Title: The Impact of ALS-Associated Genes hnRNPA1 , MATR3 , VCP and UBQLN2 on the Severity of TDP-43 Aggregation

doi: 10.3390/cells9081791

Figure Lengend Snippet: SH-SY5Y cells co-transfected with mKO2 constructs and wt or mut hnRNPA1. ( A ) Quantification of co-transfected cells harboring aggregates. ( B ) Average number of aggregates in an individual cell. ( C ) Average aggregate size. ( D ) SH-SY5Y cells co-transfected with mKO2 constructs and wt or mut hnRNPA1. Probed for HA-tag and counterstained with DAPI. Scale bars: 10 µm.

Article Snippet: TDP-43 constructs: TDP-43 was subcloned from previously published plasmid [ ] into mKO2-C1 plasmid, containing sequence for fluorescent protein mKusabira Orange2 (mKO2) (Addgene #54494, deposited by Michael Davidson and Atsushi Miyawaki [ ]).

Techniques: Transfection, Construct

Journal: Cells

Article Title: The Impact of ALS-Associated Genes hnRNPA1 , MATR3 , VCP and UBQLN2 on the Severity of TDP-43 Aggregation

doi: 10.3390/cells9081791

Figure Lengend Snippet: SH-SY5Y cells co-transfected with mKO2 constructs and wt or mut MATR3. ( A ) Quantification of co-transfected cells harboring aggregates. ( B ) Average number of aggregates in an individual cell. ( C ) Average aggregate size. ( D ) SH-SY5Y cells co-transfected with mKO2 constructs and wt or mut MATR3. Probed for HA-tag and counterstained with DAPI. Scale bars: 10 µm.

Article Snippet: TDP-43 constructs: TDP-43 was subcloned from previously published plasmid [ ] into mKO2-C1 plasmid, containing sequence for fluorescent protein mKusabira Orange2 (mKO2) (Addgene #54494, deposited by Michael Davidson and Atsushi Miyawaki [ ]).

Techniques: Transfection, Construct

Journal: Cells

Article Title: The Impact of ALS-Associated Genes hnRNPA1 , MATR3 , VCP and UBQLN2 on the Severity of TDP-43 Aggregation

doi: 10.3390/cells9081791

Figure Lengend Snippet: SH-SY5Y cells co-transfected with mKO2 constructs and wt or mut VCP. ( A ) Quantification of co-transfected cells harboring aggregates. ( B ) Average number of aggregates in an individual cell. ( C ) Average aggregate size. ( D ) SH-SY5Y cells co-transfected with mKO2 constructs and wt or mut VCP. Probed for HA-tag and counterstained with DAPI. Scale bars: 10 µm.

Article Snippet: TDP-43 constructs: TDP-43 was subcloned from previously published plasmid [ ] into mKO2-C1 plasmid, containing sequence for fluorescent protein mKusabira Orange2 (mKO2) (Addgene #54494, deposited by Michael Davidson and Atsushi Miyawaki [ ]).

Techniques: Transfection, Construct

Journal: Cells

Article Title: The Impact of ALS-Associated Genes hnRNPA1 , MATR3 , VCP and UBQLN2 on the Severity of TDP-43 Aggregation

doi: 10.3390/cells9081791

Figure Lengend Snippet: SH-SY5Y cells co-transfected with mKO2 constructs and wt or mut UBQLN2. ( A ) Quantification of co-transfected cells harboring aggregates. ( B ) Average number of aggregates in an individual cell. ( C ) Average aggregate size. ( D ) SH-SY5Y cells co-transfected with mKO2 constructs and wt or mut UBQLN2. Probed for HA-tag and counterstained with DAPI. Scale bars: 10 µm.

Article Snippet: TDP-43 constructs: TDP-43 was subcloned from previously published plasmid [ ] into mKO2-C1 plasmid, containing sequence for fluorescent protein mKusabira Orange2 (mKO2) (Addgene #54494, deposited by Michael Davidson and Atsushi Miyawaki [ ]).

Techniques: Transfection, Construct

Journal: Methods in enzymology

Article Title: Chemoenzymatic Synthesis and Applications of Prokaryote-Specific UDP-Sugars

doi: 10.1016/bs.mie.2017.06.003

Figure Lengend Snippet: Plasmids used in this manuscript.

Article Snippet: Transform plasmid into freshly-competent BL21 (DE3) RIL cells, growing transformants on LB-agar selection media containing corresponding vector selection antibiotic (see ) and 30 μg/mL CAM. table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Construct Vector Resistance Reference Addgene ID GST-PglFΔ1–130 ( Cj ) pGEX4T-2 Carb, 50 μg/mL Olivier et al., 2006 89708 His 8 -TEV-PglC ( Ng ) modified pET30b(+) Kan, 30 μμg/mL Hartley et al., 2011 89709 T7-PglD-His 6 ( Cj ) pET24a(+) Kan, 30 μg/mL Morrison et al., 2010 89710 PseB-His 6 ( Cj ) pET-24a(+) Kan, 30 μg/mL this report 89723 His 8 -TEV-PseC ( Cj ) modified pET30b(+) Kan, 30 μg/mL Hartley et al., 2011 89724 His 8 -TEV-PseH ( Cj ) modified pET30b(+) Kan, 30 μg/mL Hartley et al., 2011 89725 T7-WbpB-His 6 ( Pa ) pET-24a(+) Kan, 30 μg/mL Larkin et al., 2009 89711 T7-WbpE-His 6 ( Pa ) pET-24a(+) Kan, 30 μg/mL Larkin et al., 2009 89712 T7-WbpD-His 6 ( Pa ) pET-24a(+) Kan, 30 μg/mL Larkin et al., 2009 89713 T7-AglK-His 6 ( Mv ) pET-24a(+) Kan, 30 μg/mL Larkin et al., 2013 89714 T7-AglC-His 6 ( Mv ) pET-24a(+) Kan, 30 μg/mL Larkin et al., 2013 89726 Open in a separate window Plasmids used in this manuscript.

Techniques: Plasmid Preparation, Modification

Journal: Scientific Reports

Article Title: A Modular Assembly Platform for Rapid Generation of DNA Constructs

doi: 10.1038/srep16836

Figure Lengend Snippet: ( A ) Promoters, genes, and backbones from established GMAP-compatible collections are used in a one step isothermal assembly reaction to produce DNA constructs on demand. ( B ) Schematic of possible orderings of genes and promoters. Promoter A (pA) is flanked by sites 1 and 2, promoter B (pB) is flanked by sites 3 and 4, promoter C (pC) is flanked by sites 1 and 3, gene A (gA) is flanked by sites 2 and 3, gene B (gB) is flanked by sites 4 and 5, and gene C (gC) is flanked by sites 2 and 5. ( C ) Schematic diagram of experiment using GMAP retroviral backbone. Retroviruses expressing GFP driven by different promoters were assembled using GMAP and used to transduce murine 3T6 fibroblasts or murine lung cancer (KP) cells. PuroR, puromycin resistance; pGK, human phosphoglycerate kinase 1 promoter; CMV, cytomegalovirus immediate-early promoter; EFS, elongation factor 1α promoter; SV40, simian virus early 40 promoter; CCSP, clara cell secretory protein promoter; UBC, human Ubiquitin C promoter. ( D ) Bar graph shows flow cytometry measurements of median fluorescence intensity (MFI) of GFP from 3T6 and KP cells transduced with GMAP-generated retrovirus. Data are representative of at least three independent experiments. ( E ) GMAP-generated lentiviruses expressing mTagBFP2-A UTR (i), mKate2-B UTR (ii), or mKO2-C UTR (iii) sensor cassettes were assembled and used to transduce a Cre reporter cell line (3TB). 3TB cells were selected with hygromycin and visualized by confocal microscopy. Insets show higher magnification. Scale bar, 100 μm. ( F-H ) Histograms from 3TB cells expressing mTagBFP2-A UTR ( F ), mKate2-B UTR ( G ), or mKO2-C UTR ( H ) transfected with three inducible hairpin constructs targeting the A, B, or C 3’UTRs assembled using GMAP. After transfection 3TB cells were selected with blasticidin, treated with doxycycline, and knockdown was assessed by flow cytometry analysis on GFP + cells. Grey histograms represent cells lines transfected with an inducible shRNA targeting luciferase. Data are representative of at least three independent experiments.

Article Snippet: Accession Codes : The following plasmids were deposited into Addgene: TOPO 2-5, TOPO 1-4, RV 2-5, LV 1-5, R26TV CAG LSL 2-5, TOPO mTagBFP2, TOPO mKate2, TOPO mKO2, TOPO iRFP670, TOPO CloverCP, TOPO HygroR, TOPO ZeoR, TOPO PuroR, TOPO rtTA3-2A-Cre, TOPO tTRKRAB- rtTA3-Luc, TOPO Cre-2A-GFP, TOPO EFS, TOPO pGK, TOPO CMV, TOPO CCSP, TOPO sv40, TOPO UBC, TOPO pTRE, and TOPO HRE.

Techniques: Construct, Retroviral, Expressing, Transduction, Virus, Ubiquitin Proteomics, Flow Cytometry, Fluorescence, Generated, Confocal Microscopy, Transfection, Knockdown, shRNA, Luciferase

Journal: Heliyon

Article Title: Markerless bacterial artificial chromosome manipulation method by red proteins of phage λ mediated homologous recombination utilizing fluorescent proteins for both positive and counter selection

doi: 10.1016/j.heliyon.2023.e18983

Figure Lengend Snippet: Characteristics of fluorescent proteins used in this study.

Article Snippet: The mKO2 gene was obtained from mKO2-pBAD gifted by Michael Davidson and Atsushi Miyawaki (#54555, Addgene) [ ].

Techniques:

Journal: Heliyon

Article Title: Markerless bacterial artificial chromosome manipulation method by red proteins of phage λ mediated homologous recombination utilizing fluorescent proteins for both positive and counter selection

doi: 10.1016/j.heliyon.2023.e18983

Figure Lengend Snippet: Optimization of template plasmids. pAmp-9mScarlet without the terminator rrnB T1 was constructed (A). mScarlet protein expression in the pelleted HTS16CR cells was observed on an LED transilluminator after the cells were transformed with pAmp-9mScarlet (+) or pAmp-9mScarlet without a terminator (−) following overnight cultivation in 2 mL of LB medium at 37 °C (B) . Additionally, the plasmid yields of 2 mL of HST16CR cultures transformed with pAmp-9mScarlet (+) (with terminator) and pAmp-9mScarlet (−) (without terminator) were compared (C). The fluorescent protein gene (FP), EGFP, mClover3, YPet, mOrange, mKO2, or mScarlet were ligated into a plasmid (D), which has the same backbone as pAmp-9mScarlet, except for the fluorescent gene, as shown in . E. coli , HST16CR, or HST08 were transformed with these plasmids or pAmp-S as the negative control (control) and then cultivated on LB agar plates at either 30 °C or 37 °C overnight under ampicillin selective pressure (E) (Supplementary fig. 3E-1, -2 and -3). Note that HST08 cells transformed with mKO2 did not grow on the plates either at 30 °C or 37 °C. An unpaired t -test was used to determine the statistical significance. The calculated p values are shown above the compared groups.

Article Snippet: The mKO2 gene was obtained from mKO2-pBAD gifted by Michael Davidson and Atsushi Miyawaki (#54555, Addgene) [ ].

Techniques: Construct, Expressing, Transformation Assay, Plasmid Preparation, Negative Control, Control