Journal: Nucleic Acids Research

Article Title: A supernumerary designer chromosome for modular in vivo pathway assembly in Saccharomyces cerevisiae

doi: 10.1093/nar/gkaa1167

Figure Lengend Snippet: Transformation efficiency and fidelity. ( A ) Transformation efficiencies for the in vivo assembly of 100 and 50 kb synthetic chromosomes. Both neochromosomes have been assembled with 2.5 kb (dark blue), 5 kb (lighter blue) and 10 kb (lightest blue) fragments. The number above each bar indicates the number of assembled fragments. Data were normalized to 10 8 cells using controls on YPD plates, performed in biological duplicates (replicate 1 and 2) in technical triplicates for replicate 1 and technical duplicates for replicate 2. The asterisks indicates that transformation with 5 kb fragments resulted in a significantly different transformation efficiency compared to both neighbouring efficiencies (two-tailed paired homoscedastic t -test P < 0.05). ( B ) Assembly fidelity. In vivo assembly of the 100 kb NeoChr12 from 43 fragments. Eleven colonies that grew on selective medium were tested for mRuby2 and mTurquoise2 fluorescence. Neochromosomes were subsequently screened based on their size on CHEF gel and lastly by Illumina sequencing. The percentage of correct colonies for each screening round is indicated.

Article Snippet: Deletion of mRuby2 (IMF37) or Venus (IMF38), or both mRuby2 and Venus (IMF39) from the 100 kb neochromosome in IMF6 did not increase the growth rate ( ).

Techniques: Transformation Assay, In Vivo, Two Tailed Test, Fluorescence, Illumina Sequencing

Journal: Nucleic Acids Research

Article Title: A supernumerary designer chromosome for modular in vivo pathway assembly in Saccharomyces cerevisiae

doi: 10.1093/nar/gkaa1167

Figure Lengend Snippet: Quantification of neochromosome copy number. ( A ) neochromosome copy number as determined by whole genome sequencing. ( B ) neochromosome copy number estimation based on mRuby2 fluorescence. CEN.PK113-7D with no copies of mRuby2 was used as negative control and IMX2224 with a single copy of mRuby2 integrated in the genome as positive control. ( C ) Schematic overview of the potential segregation of circular neochromosome upon cell division.

Article Snippet: Deletion of mRuby2 (IMF37) or Venus (IMF38), or both mRuby2 and Venus (IMF39) from the 100 kb neochromosome in IMF6 did not increase the growth rate ( ).

Techniques: Sequencing, Fluorescence, Negative Control, Positive Control

Journal: The Plant Journal

Article Title: Live‐cell CRISPR imaging in plants reveals dynamic telomere movements

doi: 10.1111/tpj.13601

Figure Lengend Snippet: Simultaneous visualization of telomeric DNA by CRISPR – dC as9 and the GFP ‐tagged telomeric repeat binding protein 1 ( TRB 1). (a) Immunofluorescence staining against Sp‐ dC as9‐mRuby2. (b) Immunofluorescence staining against TRB 1‐GFP. (c) Overlay showing almost complete co‐localization, except for putative blunt‐ended telomeres (indicated by arrows, nucleus is counterstained with DAPI (in blue). Scale bars: 2 μm.

Article Snippet: Plasmids encoding for eGFP (pSiM24‐eGFP) and mRuby2 (pcDNA3‐mRuby2) were obtained from Addgene ( http://www.addgene.com ). dCas9 and fluorescence protein (FP) sequences were generated with primers containing homologous flanks for subsequent Gibson Assembly cloning into the pCAS9‐TP backbone (for a full list of primers, see Appendix ).

Techniques: CRISPR, Binding Assay, Immunofluorescence, Staining

Journal: The Plant Journal

Article Title: Live‐cell CRISPR imaging in plants reveals dynamic telomere movements

doi: 10.1111/tpj.13601

Figure Lengend Snippet: Comparison of Sa‐ dC as9 and Sp‐ dC as9. Telomeres were visualized by the simultaneous application of two dC as9 orthologues (Sa‐ dC as9 and Sp‐ dC as9). (a) Immunofluorescence staining against Sa‐ dC as9‐eGFP. (b) Immunofluorescence staining against Sp‐ dC as9‐mRuby2. (c) Overlay showing complete co‐localization. Nucleus is counterstained with DAPI (in blue). (d) Quantification of the number of telomere signals observed by two different dC as9 orthologues ( n = 18). Scale bars: 10 μm.

Article Snippet: Plasmids encoding for eGFP (pSiM24‐eGFP) and mRuby2 (pcDNA3‐mRuby2) were obtained from Addgene ( http://www.addgene.com ). dCas9 and fluorescence protein (FP) sequences were generated with primers containing homologous flanks for subsequent Gibson Assembly cloning into the pCAS9‐TP backbone (for a full list of primers, see Appendix ).

Techniques: Comparison, Immunofluorescence, Staining

Journal: PLoS Computational Biology

Article Title: A model of the onset of the senescence associated secretory phenotype after DNA damage induced senescence

doi: 10.1371/journal.pcbi.1005741

Figure Lengend Snippet: Murine dermal fibroblasts (MDFs) are isolated from NEMO-floxed mice. After short expansion in cell culture these MDFs are transfected with pCAG-Cre-T2A-mRuby2 or pCAG-mRuby2, respectively. Because of mRuby2 expression, successfully transfected cells can be sorted by FACS. Cells transfected with pCAG-Cre-T2A-mRuby2 are knocked out for NEMO while pCAG-mRuby2 transfected cells are used as wildtype controls. After transfection cells are treated with 25 μM etoposide for 3 h to induce DNA damage. 24 h after treatment cell culture media is taken for ELISA measurement of secretion and cells are harvested for RNA isolation and subsequent RT-qPCR analysis.

Article Snippet: Recombineering technology [ ] was used to constract plasmids containing CDS of both Cre recombinase and fluorescence reporter, mRuby2 or only mRuby2. pCAG-Cre vector (a gift from Connie Cepko, Addgene plasmid # 13775) was used for the recombineering.

Techniques: Isolation, Cell Culture, Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Imaging plant germline differentiation within Arabidopsis flower by light sheet microscopy

doi: 10.1101/774299

Figure Lengend Snippet: (A) MIPs of a 0.5 mm floral bud expressing ASY1:eYFP (green) and H2B:mRuby2 (magenta) viewed from eight different angles. Scale bar 200 µm. (B,C) Imaris MIP of 3D reconstructed flower. Longitudinal (D) and transversal (E) sections of the 3D reconstructed flower. (F) Surface rendered 3D model of the flower with indicated PMCs. (G) MIP of PMCs from the 3D model. Automated detection of PMCs using Imaris spot detection in one anther lobe is shown (41 PMCs were counted).

Article Snippet: To generate ASY1:eYFP and H2B:mRuby2, the ASY1 (AT1G67370) and H2B (AT3G45980) genomic loci from A. thaliana accession Columbia as well as eYFP from pBlunt-EYFP-TAG and mRuby2 CDS from pcDNA3-mRuby2 (plasmid #40260; Addgene, www.addgene.com ) were PCR amplified.

Techniques: Expressing

Journal: bioRxiv

Article Title: Imaging plant germline differentiation within Arabidopsis flower by light sheet microscopy

doi: 10.1101/774299

Figure Lengend Snippet: (A) MIPs of a flower (upper panel) expressing ASY1:eYFP (green) and H2B:mRuby2 (magenta) and one of its anther lobes (bottom panel) at indicated time points (scale bar 100 µm). (B) Detailed view of the distribution of ASY1 signal in developing PMCs. Scale bar 20 µm.

Article Snippet: To generate ASY1:eYFP and H2B:mRuby2, the ASY1 (AT1G67370) and H2B (AT3G45980) genomic loci from A. thaliana accession Columbia as well as eYFP from pBlunt-EYFP-TAG and mRuby2 CDS from pcDNA3-mRuby2 (plasmid #40260; Addgene, www.addgene.com ) were PCR amplified.

Techniques: Expressing

Journal: bioRxiv

Article Title: Imaging plant germline differentiation within Arabidopsis flower by light sheet microscopy

doi: 10.1101/774299

Figure Lengend Snippet: (A) A 0.3 mm floral bud from the HTA10:RFP reporter line was embedded in media with low melting point agarose within the closed FEP/capillary system and imaged at time point 0 (MIP, upper panel; lower panel: detail of one anther). The bud was imaged again after 96 h of cultivation in the dark at 21 °C. Scale bars 100 µm. (B) A 0.3 mm bud from the HTA10:RFP reporter line was placed into the closed FEP/capillary system and imaged continuously every 1 hour. Time points 0 h, 60 h (onset of first meiotic division) and 96 h were selected to compare developmental progression under regular laser illumination (MIP upper panel; lower panel: detail of one anther). Scale bars 100 µm. (C) Gantt chart depicting the developmental progression of anthers cultivated in closed capillaries. C 01-04 are four independent flowers grown outside of the microscope and imaged only at the beginning and the end of the experiment. Colors of rectangles indicate the most prominent stage of pollen development in individual anther lobes. The experiment was started with 8 individual lobes (indicated by numbers 1-8), but not all of them could be scored due to technical reasons at the end of the experiment. Anther lobes that could not be scored at 96 h are in white. S01, 03, and 04 depict the development of continuously imaged floral buds. S01 is a bud from the HTA10:RFP line, S03 and S04 from the H2B:mRuby2 ASY1:YFP line.

Article Snippet: To generate ASY1:eYFP and H2B:mRuby2, the ASY1 (AT1G67370) and H2B (AT3G45980) genomic loci from A. thaliana accession Columbia as well as eYFP from pBlunt-EYFP-TAG and mRuby2 CDS from pcDNA3-mRuby2 (plasmid #40260; Addgene, www.addgene.com ) were PCR amplified.

Techniques: Microscopy

Journal: bioRxiv

Article Title: Imaging plant germline differentiation within Arabidopsis flower by light sheet microscopy

doi: 10.1101/774299

Figure Lengend Snippet: (A) Chromosome segregation in meiosis I from diakinesis (0 m) to telophase I (64 m) visualized with the HTA10:RFP marker. Images were taken every 30 s, scale bar 10 µm. (B) Restitution mitosis in tapetum cells. Images were taken every 60 s, scale bar 5 µm. (C) Rapid chromosome movements in zygotene. Chromatin axes are visualized with ASY1:eYFP (green), somatic nuclei with H2B:mRuby2 (magenta). Arrowhead points to a chromatin axis that moves within the indicated interval. Images were taken every 5 s, scale bar 5 µm. (D) Asymmetric pollen mitosis I. Chromatin is visualized with H2A:RFP (magenta), 488 nm autofluorescence highlights the pollen wall (green). Images were taken every 5 min, scale bar 10 µm. (E) Female meiosis. MMC is marked with ASY1:eYFP (green), chromatin with HTA10:RFP (magenta). Images were taken every 10 min, scale bar 10 µm.

Article Snippet: To generate ASY1:eYFP and H2B:mRuby2, the ASY1 (AT1G67370) and H2B (AT3G45980) genomic loci from A. thaliana accession Columbia as well as eYFP from pBlunt-EYFP-TAG and mRuby2 CDS from pcDNA3-mRuby2 (plasmid #40260; Addgene, www.addgene.com ) were PCR amplified.

Techniques: Marker