Journal: Cell metabolism

Article Title: Fibroblasts mobilize tumor cell glycogen to promote proliferation and metastasis

doi: 10.1016/j.cmet.2018.08.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pLKO.1 TRC control , Addgene , Cat#10879.

Techniques: Virus, Recombinant, Mutagenesis, Colorimetric Assay, Bioassay, Phospho-proteomics, Plasmid Preparation, Control, Software

Journal: Scientific Reports

Article Title: Lineage tracing analysis of cone photoreceptor associated cis-regulatory elements in the developing chicken retina

doi: 10.1038/s41598-019-45750-7

Figure Lengend Snippet: Quantification of lineage trace recombination efficiencies mediated by FlpE, Cre and PhiC31. ( a ) Schematic representation of recombinase-based lineage tracing strategy. Red triangles represent recombinase target sequences. ( b – d ) Quantification of FACS-analyzed dissociated retinal cells electroporated with a UbiqC::TdTomato electroporation control, a FlpE, Cre, or PhiC31 recombinase plasmid, and the corresponding responder vector. Retinas were electroporated at E5 and fixed after two days in culture. ( b ) Quantification of basal recombination, with recombinase plasmids driven by a basal promoter (TATA box). Error bars represent 95% confidence intervals, n = 12, p < 0.001 upon Kruskal Wallis test with post hoc Dunn test. ( c ) Quantification of enhancer-driven recombination, with recombinase plasmids driven by the ThrbCRM1 enhancer element. Error bars represent 95% confidence intervals, n = 6. ( d ) Quantification of ubiquitous recombination, with recombinase plasmids driven by the ubiquitous CAG promoter. Error bars represent 95% confidence intervals, n = 6. CRM, cis-regulatory module; bp, basal promoter; pA, polyadenylation sequence.

Article Snippet: Bp::PhiC31 was made by PCR-amplifying the PhiC31 coding region from pPhiC31o (Addgene plasmid #13794 was a gift from Philippe Soriano) using an Xma1-tagged forward primer and a BsrG1-tagged reverse primer (see Supplementary Table for all primer sequences).

Techniques: Electroporation, Control, Plasmid Preparation, Sequencing

Journal: Scientific Reports

Article Title: Lineage tracing analysis of cone photoreceptor associated cis-regulatory elements in the developing chicken retina

doi: 10.1038/s41598-019-45750-7

Figure Lengend Snippet: In ovo lineage trace of 3 Thrb CRMs yields unique patterns of activity. ( a – e ) Representative images of vertically sectioned retinas that were electroporated in ovo at E3 with CAG::nucβgal as an electroporation control, bp::PhiC31 ( a ), CAG::PhiC31 ( b ), ThrbCRM1::PhiC31 ( c ), ThrbCRM2::PhiC31 ( d ), or ThrbICR::PhiC31 ( e ) and CAaNa::GFP. Embryos were grown until E10, and all images are maximum intensity projections. To the right of each image is a schematic of the chick retina with recombined cell types from all counted images colored in green. ( f ) Quantification of the % recombination in each retinal layer, for each of the conditions assessed, n = 3–8. ( g ) Quantification of the % of representation for each retinal cell type among the recombined cells for that particular condition (GFP+ cells of one cell type/all GFP+ cells *100). The negative control had no recombination (f) so it was excluded here. Error bars represent SEM, n = 3–8. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; bp, basal promoter; PRs, photoreceptors; HCs, horizontal cells; ACs, amacrine cells; BCs, bipolar cells; RGCs, retinal ganglion cells.

Article Snippet: Bp::PhiC31 was made by PCR-amplifying the PhiC31 coding region from pPhiC31o (Addgene plasmid #13794 was a gift from Philippe Soriano) using an Xma1-tagged forward primer and a BsrG1-tagged reverse primer (see Supplementary Table for all primer sequences).

Techniques: In Ovo, Activity Assay, Electroporation, Control, Negative Control

Journal: bioRxiv

Article Title: Pooled genome-wide CRISPR activation screening for rapamycin resistance genes in Drosophila cells

doi: 10.1101/2022.12.09.519790

Figure Lengend Snippet: A. Schematic of pooled library cells generation. SAM complex was inserted into attP sites containing S2R+ cells to generate SAM cells. The pooled attB flanking sgRNA library was integrated into attP sites by phiC31 mediated recombination to generate pooled library cells. B. Cell proliferation of S2R+ cells with rapamycin treatment at different concentrations. Cell proliferation was measured by Celltiter Glo. Six replicates are shown as individual circles. C. Pooled focused library screen with DMSO treatment for 15 days and 30 days. Data were analyzed by MAGeCK. Each dot represents a gene. Genes with significant difference (FDR < 0.05) are highlighted. D. Counts of sgRNAs targeting scyl or Cyp12a4 in the initial population and final population after proliferation for 30 days in DMSO. Each dot represents a sgRNA. sgRNAs targeting scyl or Cyp12a4 are highlighted. E. Pooled focused library screens with 0.1 nM and 1 nM rapamycin treatment for 15 days and 30 days. Data were analyzed by MAGeCK. Each dot represents a gene. Gene with significant difference (FDR < 0.05) is highlighted. F. Counts of sgRNAs targeting CG8468 in the DMSO treated population and rapamycin treated population. Each dot represents a sgRNA. sgRNAs targeting CG8468 are highlighted.

Article Snippet: The PhiC31 integrase expressing pBS130 plasmid was obtained from Addgene (#26290).

Techniques:

Journal: eLife

Article Title: Quantitative spatial and temporal assessment of regulatory element activity in zebrafish

doi: 10.7554/eLife.65601

Figure Lengend Snippet: Diagramatic representation of the Q-STARZ pipeline. ( A ). Top: map of the landing pad vector. Bottom: scheme for generating stable transgenic ‘landing lines’. The landing pad vector is co-injected with Tol2 mRNA into one-cell stage wild-type embryos. Tol2-mediated recombination integrates the landing pad containing phiC31-attB sites flanking the tracking cis-regulatory element (CRE)-reporter cassette ( SHH -SBE2, a CRE driving eGFP in the developing forebrain) at random locations in the zebrafish genome. F0 embryos showing mosaic eGFP expression are raised to adulthood. F1 embryos obtained by outcrossing F0 lines with wild-type zebrafish are screened for tracking CRE-driven reporter (eGFP) expression. Embryos where eGFP expression was only observed in the expected activity domain (forebrain) of the tracking CRE were raised to adulthood to establish stable ‘landing lines’. ( B ) Top: map of the dual-CRE dual-reporter vector. Bottom: scheme for replacing the tracking cassette in the landing line with the dual-CRE dual-reporter cassette containing the enhancers to be assayed for spatiotemporal activity. Assay vector and mRNA coding for phiC31 integrase are injected in one cell stage embryos derived from outcrossing F1 landing line with wild-type fish. Injected embryos were selected for loss of tracking CRE ( SHH -SBE2)-driven eGFP fluorescence in forebrain and mosaic expression of both eGFP and mCherry resulting from the test CREs in the assay cassette. F0 transgenic lines were established from selected embryos and eGFP and mCherry expression imaged in F1 embryos derived from outcrossing these lines with wild-type fish. Signals from both reporters were observed in the activity domains of both CREs in F1 embryos bearing the assay constructs with ‘neutral’ sequence between the two CRE-reporter units (yellow signal seen in expressing tissues in the merge channel). However, eGFP and mCherry expression was restricted to tissues where the associated CREs are active upon inclusion of three copies of the chicken β-globin 5′HS4 (3XcHS4) insulator between the two CRE-reporter units.

Article Snippet: Recombinant DNA reagent , pcDNA3.1 phiC31 (plasmid) , Addgene , Plasmid #68310 , .

Techniques: Plasmid Preparation, Transgenic Assay, Injection, Expressing, Activity Assay, Derivative Assay, Fluorescence, Construct, Sequencing

Journal: eLife

Article Title: Quantitative spatial and temporal assessment of regulatory element activity in zebrafish

doi: 10.7554/eLife.65601

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pcDNA3.1 phiC31 (plasmid) , Addgene , Plasmid #68310 , .

Techniques: Cloning, TA Cloning, Plasmid Preparation, Purification, Recombinant, Software

Journal: ACS synthetic biology

Article Title: A Hot-Swappable Genetic Switch: Building an Inducible and Trackable Functional Assay for the Essential Gene MEDIATOR 21.

doi: 10.1021/acssynbio.5c00085

Figure Lengend Snippet: Figure 1. Engineering and rapid prototyping of the integrase eraser approach. (A) Cloning strategy based on (Guiziou et al.20) for the target integrase target with BsaI adapters highlighted for use in golden gate cloning strategies, see Plasmid Maps. (B) Design of the integrase target. The target is composed of two PhiC31 integrase sites (triangles) surrounding a constitutive promoter (pUBQ10), the rescue coding sequence for MED21:6xHA and the fluorescent reporter (mScarlet). In the absence of integrase, MED21:6xHA is expressed. In the presence of integrase, the integrase mediates inversion of the DNA between the integrase sites, inverting the promoter and leading to mScarlet expression. The expression of the integrase is mediated by the selected promoter selected. (C) Rapid prototyping of the MED21 Eraser target in transient transfections of Nicotiana benthamiana at 2 days after injection. On the left side is a control target that switches from mTurquoise to mScarlet alone (top) and with a p35S:PhiC31 construct (bottom). On the right side shows the MED21 target alone (top) and MED21:HA target with a p35S:PhiC31 construct (bottom). The BFP channel for the MED21:HA is shown to demonstrate the level of background expected for the BFP channel in the negative control. Microscopy images were taken on a 20x objective to allow a wide view of switching efficiency, and the 50-μm scale bar in the RFP channels applies to all paired BFP images.

Article Snippet: The Arabidopsis MED21:HA sequence was amplified by PCR with appropriate Golden Gate restriction sites and the construction of integrase targets was performed by Golden Gate reaction in the modified pGreenIIHygr vector containing compatible Golden Gate sites defined in Guiziou et al.20 Plasmids used for construction of the novel targets are available at Addgene: 5 prime side mScarlet cassette - L0-T4 tRBSC_mScarlet (Addgene #195887), Integrase site flanked pUBQ10 - L0-T1 Target PhiC31−(Addgene https://doi.org/10.1021/acssynbio.5c00085 ACS Synth.

Techniques: Cloning, Plasmid Preparation, Sequencing, Expressing, Transfection, Injection, Control, Construct, Negative Control, Microscopy

Journal: ACS synthetic biology

Article Title: A Hot-Swappable Genetic Switch: Building an Inducible and Trackable Functional Assay for the Essential Gene MEDIATOR 21.

doi: 10.1021/acssynbio.5c00085

Figure Lengend Snippet: Figure 2. Cell-type-specific integrase eraser implemented in lateral root primordium results in MED21 loss of function and increases in lateral root initiation. (A) Schematic of the predicted behavior for the MED21 eraser approach. Lateral root primordium cells (red) will lose expression of the rescue construct, rendering them an effective knockout for MED21. (B−J) Confocal microscopy analysis of the wild-type control switch (from Figure 1C) and MED21 eraser lateral root initiation. All scale bars are 50 μm. (J) Still image from Supplemental Movie 1 of the root growth phenotype of MED21 eraser lines compared to the wild type at day 11. (K) Whole seedling epifluorescent image of MED21 eraser lines at day 14.

Article Snippet: The Arabidopsis MED21:HA sequence was amplified by PCR with appropriate Golden Gate restriction sites and the construction of integrase targets was performed by Golden Gate reaction in the modified pGreenIIHygr vector containing compatible Golden Gate sites defined in Guiziou et al.20 Plasmids used for construction of the novel targets are available at Addgene: 5 prime side mScarlet cassette - L0-T4 tRBSC_mScarlet (Addgene #195887), Integrase site flanked pUBQ10 - L0-T1 Target PhiC31−(Addgene https://doi.org/10.1021/acssynbio.5c00085 ACS Synth.

Techniques: Expressing, Construct, Knock-Out, Confocal Microscopy, Control

Journal: ACS synthetic biology

Article Title: A Hot-Swappable Genetic Switch: Building an Inducible and Trackable Functional Assay for the Essential Gene MEDIATOR 21.

doi: 10.1021/acssynbio.5c00085

Figure Lengend Snippet: Figure 3. A chemically inducible MED21 eraser. (A) Schematic of the predicted behavior for the MED21 iEraser. The estradiol inducible integrase construct is composed of p35S:XVE (transcriptional activator composed of a DNA-binding domain of LexA, the transcription activation domain of VP16, and the regulatory region of the human estrogen receptor) and pLexA-minimal 35S driving expression of PhiC31. (B,C) Characterization of the iEraser by fluorescence microscopy shows induction as early as 48 h after treatment. The scale bar represents 50 μm (C). (D) Growth phenotypes were visible in estradiol-induced switched plants grown on 1 μM β-estradiol induction plates for 6 days. Seedlings were first grown for 6 days on LS media lacking β-estradiol before being transplanted to induction media (12 days total growth). (E) Protein expression analysis by Western blot for 12 independent iEraser lines. (F) Protein expression analysis by Western blot for roots and shoots isolated from plants grown with or without β-estradiol. Seedlings were grown on LS media for 6 days, and then transplanted to either control or induction media for another 6 days of growth. The experiment was performed in triplicate for each genotype. (G,H) Lengths of lateral (G) and primary (H) roots were quantified at 6 days from the indicated Integrase Eraser type. Seedlings were grown as in (F). Letters indicate significant difference (ANOVA and Tukey HSD multiple comparison test; p < 0.001).

Article Snippet: The Arabidopsis MED21:HA sequence was amplified by PCR with appropriate Golden Gate restriction sites and the construction of integrase targets was performed by Golden Gate reaction in the modified pGreenIIHygr vector containing compatible Golden Gate sites defined in Guiziou et al.20 Plasmids used for construction of the novel targets are available at Addgene: 5 prime side mScarlet cassette - L0-T4 tRBSC_mScarlet (Addgene #195887), Integrase site flanked pUBQ10 - L0-T1 Target PhiC31−(Addgene https://doi.org/10.1021/acssynbio.5c00085 ACS Synth.

Techniques: Construct, Binding Assay, Activation Assay, Expressing, Fluorescence, Microscopy, Western Blot, Isolation, Control, Comparison

Journal: ACS synthetic biology

Article Title: A Hot-Swappable Genetic Switch: Building an Inducible and Trackable Functional Assay for the Essential Gene MEDIATOR 21.

doi: 10.1021/acssynbio.5c00085

Figure Lengend Snippet: Figure 4. Engineering and rapid prototyping of the “hot-swap” isoform switch approach. (A) Schematic of the predicted behavior for the MED21 Swap. Lateral root primordia will switch the expression of the rescue construct from wild-type (blue) to mutant isoform (red). (B) Design of the integrase target. The target is composed of two integrase sites (triangles) surrounding a constitutive promoter (pUBQ10), and each MED21 isoform is fused to the P2A self-cleaving peptide sequence and a fluorescent reporter (mTurquoise or mScarlet). In the basal state, MED21 and mTurquoise are produced. Once the PhiC31 integrase is expressed, it mediates inversion of the DNA between the integrase sites, inverting the promoter and leading to production of mMED21 and mScarlet. (C) Alignment of the AtMED21 N-terminal mutants. (D−G) Epifluorescence microscopy analysis of wild-type and mMED21 swap lateral root initiation in Arabidopsis primary transformants. Microscopy images were taken on a 20x objective, and the scale bar represents 50 μm. (H) Lateral root density (LRD per mm) and (I) lengths of lateral roots (Lateral root length in mm) from primary transformant lines were quantified at 14 days post germination from the indicated Integrase Swap type. At least 15 independent primary transformant lines were tested for each swap type. Lines were transplanted to soil and PCR genotyped for the med21 genotype. In (H), the LRD data points from med21 homozygous plants are colored in blue, while heterozygotes are colored dark gray, and ungenotyped samples are colored light gray. Letters indicate significant difference (ANOVA and Tukey HSD multiple comparison test; p < 0.001). In (I), data are presented as violin plots with nested boxplots to demonstrate that medians and interquartile ranges are comparable across conditions.

Article Snippet: The Arabidopsis MED21:HA sequence was amplified by PCR with appropriate Golden Gate restriction sites and the construction of integrase targets was performed by Golden Gate reaction in the modified pGreenIIHygr vector containing compatible Golden Gate sites defined in Guiziou et al.20 Plasmids used for construction of the novel targets are available at Addgene: 5 prime side mScarlet cassette - L0-T4 tRBSC_mScarlet (Addgene #195887), Integrase site flanked pUBQ10 - L0-T1 Target PhiC31−(Addgene https://doi.org/10.1021/acssynbio.5c00085 ACS Synth.

Techniques: Expressing, Construct, Mutagenesis, Sequencing, Produced, Epifluorescence Microscopy, Microscopy, Comparison