{ "generated_at": "2026-02-11T20:03:12.795327", "criteria_version": "feasibility_go_no_go_v2", "go_now_problem_count": 6, "notes": [ "These packets are pre-POC designs for external lab discussion, not final SOPs.", "All experiments require institution-specific biosafety, GMO, and permit review before execution.", "Vendor catalogs and availability should be revalidated at ordering time." ], "experiments": [ { "id": "opc-go-001", "title": "Integrated Transcriptomics to Identify Novel TET2-CHIP Inflammatory Targets", "decision_bucket": "go_now", "best_score": 0.72, "best_confidence": 0.75, "maps_to_problem_statement": "The transcriptomic approaches fail to identify and validate novel therapeutic targets beyond already known markers like CXCL2/3 and IL8", "maps_to_sub_question": "What are the novel, previously unidentified molecular targets revealed by integrated transcriptomic analysis of TET2-CHIP-mediated cardiovascular disease?", "objective": "Perform bulk and single-cell RNA-seq on TET2-deficient mouse and human models to identify and validate novel inflammatory mediators beyond known chemokines, followed by orthogonal validation.", "readouts": [ "Primary: Differentially expressed genes (DEGs) in TET2-deficient vs WT macrophages with FDR<0.05 and |log2FC|>1.5, filtered against known CHIP markers", "Secondary: Cell-type-specific expression patterns from scRNA-seq identifying myeloid subpopulations driving novel gene expression", "Secondary: qPCR and Western blot validation of top 10 novel candidates with >2-fold expression change and correlation R\u00b2>0.7 with RNA-seq" ], "design": { "overview": "Generate TET2-knockout and wild-type bone marrow-derived macrophages (BMDMs), perform bulk RNA-seq and scRNA-seq under basal and inflammatory conditions (LPS stimulation), conduct bioinformatic filtering to exclude known markers, then validate top novel candidates by qPCR, Western blot, and ELISA in mouse and human TET2-CHIP samples.", "work_packages": [ "WP1: Generate Tet2-/- and WT mouse BMDMs, culture under basal and LPS conditions (10 ng/ml, 6h), isolate RNA and prepare samples for bulk RNA-seq (n=4/group) and scRNA-seq (n=3/group)", "WP2: Perform sequencing (150bp paired-end bulk RNA-seq at 40M reads/sample; 10X Chromium scRNA-seq targeting 5000 cells/sample), conduct quality control, alignment, and differential expression analysis with DESeq2 and Seurat", "WP3: Bioinformatic prioritization - exclude genes matching literature search for CHIP/TET2/cardiovascular inflammation, prioritize secreted proteins and surface markers with drug target potential using Gene Ontology and druggability databases", "WP4: Validate top 10 candidates by qPCR in independent BMDM samples (n=6/group), Western blot for protein expression (n=4/group), and ELISA for secreted factors in culture supernatants and plasma from Tet2-/- atherosclerosis mice" ], "controls": [ "Positive control: CXCL2, CXCL3, and IL8 expression must show expected upregulation (>3-fold) in TET2-deficient conditions, validating the model", "Negative control: Wild-type BMDMs under identical culture and stimulation conditions to establish baseline expression", "Technical control: ERCC spike-in controls for bulk RNA-seq; housekeeping genes (ACTB, GAPDH, 18S) for qPCR normalization; isotype controls for flow cytometry if surface markers identified" ], "sample_size_plan": "Bulk RNA-seq: n=4 biological replicates per condition (WT basal, WT+LPS, Tet2-/- basal, Tet2-/-+LPS). scRNA-seq: n=3 pooled samples per condition targeting 5000 cells each. qPCR validation: n=6 independent biological replicates. Western blot: n=4. ELISA: n=8 mouse plasma samples per genotype.", "success_criteria": [ "Identification of \u226550 novel DEGs (FDR<0.05, |log2FC|>1.5) in TET2-deficient conditions that are absent from curated list of 200+ known CHIP-associated inflammatory markers", "\u22655 of top 10 prioritized candidates validated by qPCR with concordant direction and >2-fold change (p<0.05), and \u22653 confirmed at protein level by Western or ELISA" ], "estimated_timeline_weeks": 16 }, "materials": [ { "item": "Tet2 knockout mice (C57BL/6 background)", "supplier": "Jackson Laboratory", "catalog_or_id": "Stock No: 017573", "link": "https://www.jax.org/strain/017573", "purpose": "Source of bone marrow for TET2-deficient macrophage generation" }, { "item": "C57BL/6J wild-type mice", "supplier": "Jackson Laboratory", "catalog_or_id": "Stock No: 000664", "link": "https://www.jax.org/strain/000664", "purpose": "Control mice for wild-type BMDM generation" }, { "item": "Recombinant Mouse M-CSF", "supplier": "BioLegend", "catalog_or_id": "576406", "link": "", "purpose": "Differentiation of bone marrow cells into macrophages" }, { "item": "LPS from E. coli O111:B4", "supplier": "Sigma-Aldrich", "catalog_or_id": "L4391", "link": "https://www.sigmaaldrich.com/US/en/product/sigma/l4391", "purpose": "Inflammatory stimulation of macrophages" }, { "item": "RNeasy Plus Mini Kit", "supplier": "Qiagen", "catalog_or_id": "74134", "link": "https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/rna-purification/total-rna/rneasy-plus-mini-kit", "purpose": "RNA extraction from cultured macrophages for bulk RNA-seq and qPCR" }, { "item": "Chromium Next GEM Single Cell 3' Kit v3.1", "supplier": "10X Genomics", "catalog_or_id": "1000269", "link": "https://www.10xgenomics.com/products/single-cell-gene-expression", "purpose": "Single-cell RNA-seq library preparation" }, { "item": "TruSeq Stranded mRNA Library Prep Kit", "supplier": "Illumina", "catalog_or_id": "20020594", "link": "https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/truseq-stranded-mrna.html", "purpose": "Bulk RNA-seq library preparation" }, { "item": "NovaSeq 6000 SP Reagent Kit v1.5 (300 cycles)", "supplier": "Illumina", "catalog_or_id": "20028401", "purpose": "Sequencing reagents for bulk RNA-seq" }, { "item": "ERCC RNA Spike-In Mix", "supplier": "Thermo Fisher", "catalog_or_id": "4456740", "link": "https://www.thermofisher.com/order/catalog/product/4456740", "purpose": "Technical control for RNA-seq quality and normalization" }, { "item": "High-Capacity cDNA Reverse Transcription Kit", "supplier": "Applied Biosystems", "catalog_or_id": "4368814", "link": "https://www.thermofisher.com/order/catalog/product/4368814", "purpose": "cDNA synthesis for qPCR validation" }, { "item": "PowerUp SYBR Green Master Mix", "supplier": "Applied Biosystems", "catalog_or_id": "A25742", "link": "https://www.thermofisher.com/order/catalog/product/A25742", "purpose": "qPCR reagent for candidate validation" }, { "item": "Custom TaqMan Gene Expression Assays", "supplier": "Thermo Fisher", "catalog_or_id": "Custom design", "link": "https://www.thermofisher.com/order/custom-genomic-products/tools/gene-expression/", "purpose": "Probe-based qPCR validation of top 10 novel candidates" }, { "item": "Pierce BCA Protein Assay Kit", "supplier": "Thermo Fisher", "catalog_or_id": "23225", "link": "https://www.thermofisher.com/order/catalog/product/23225", "purpose": "Protein quantification for Western blot normalization" }, { "item": "4-20% Mini-PROTEAN TGX Precast Gels", "supplier": "Bio-Rad", "catalog_or_id": "4561094", "link": "https://www.bio-rad.com/en-us/sku/4561094-mini-protean-tgx-precast-gels", "purpose": "Protein separation for Western blot validation" }, { "item": "Mouse Cytokine Array / Chemokine Array 44-Plex", "supplier": "Eve Technologies", "catalog_or_id": "MD44", "link": "", "purpose": "Multiplex screening of secreted protein candidates in plasma" } ], "estimated_direct_cost_usd": { "low": 28000, "high": 42000, "scope": "Includes RNA-seq (bulk and single-cell) library prep and sequencing, qPCR reagents and custom assays, Western blot supplies, ELISA/multiplex assays, and molecular biology reagents. Excludes mouse housing costs, personnel, bioinformatics compute infrastructure, and institutional core facility access fees." }, "protocol_references": [ { "title": "Fuster et al. (2017) Clonal hematopoiesis associated with TET2 deficiency accelerates atherosclerosis development in mice. Science 355(6327):842-847", "use": "Established protocols for generating TET2-CHIP mouse models and characterizing inflammatory phenotypes in cardiovascular disease context" }, { "title": "Jaiswal et al. (2017) Clonal Hematopoiesis and Risk of Atherosclerotic Cardiovascular Disease. N Engl J Med 377:111-121", "use": "Reference for known CHIP-associated markers to create exclusion list for novel target identification" }, { "title": "Zheng et al. (2017) Massively parallel digital transcriptional profiling of single cells. Nature Communications 8:14049", "use": "10X Genomics scRNA-seq methodology and quality control parameters for immune cell profiling" }, { "title": "Love et al. (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology 15:550", "use": "Statistical framework for differential expression analysis with appropriate FDR correction" }, { "title": "Bustin et al. (2009) The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments. Clinical Chemistry 55(4):611-622", "use": "Best practices for qPCR validation including primer design, normalization strategies, and statistical analysis" } ], "handoff_package_for_lab": [ "Curated exclusion list of 200+ known CHIP/TET2-associated inflammatory genes compiled from literature search (PubMed, GeneCards) including CXCL2, CXCL3, IL8, IL1B, IL6, TNF, and all genes from Fuster 2017 and Jaiswal 2017 studies", "Detailed BMDM differentiation and stimulation protocol with timing, cell density (10^6 cells/well), M-CSF concentration (50 ng/ml for 7 days), and quality control criteria (>95% CD11b+F4/80+ by flow cytometry)", "Bioinformatics analysis pipeline specification: alignment tools (STAR v2.7+), DE analysis parameters (DESeq2, padj<0.05, log2FC>1.5), scRNA-seq clustering strategy (Seurat, resolution 0.5-1.0), and prioritization criteria (secreted proteins, surface markers, druggability scores from DGIdb and ChEMBL)", "qPCR primer design parameters for top candidates: amplicon 80-150bp, Tm 58-60\u00b0C, spanning exon junctions where possible, validated in silico with Primer-BLAST for specificity", "Data deposition plan for GEO submission with metadata templates and FASTQ file organization structure" ] }, { "id": "opc-go-002", "title": "Cell Density and Viability Assessment in Gap Junction Dye Transfer Assay", "decision_bucket": "go_now", "best_score": 0.531, "best_confidence": 0.5, "maps_to_problem_statement": "Lucifer Yellow dye transfer assay lacks essential controls (cell density, viability) and shows unexplained discrepancy where mutant has less dye but comparable migration distance to control", "maps_to_sub_question": "Are cell density and viability comparable between control and mutant groups in the dye transfer assay?", "objective": "Quantify and compare cell density and viability between control and mutant cell lines at the time of Lucifer Yellow dye transfer to identify confounding variables.", "readouts": [ "Primary: Cell density (cells/mm\u00b2) measured by automated nuclear counting (DAPI) at time of dye transfer", "Secondary: Cell viability percentage measured by live/dead fluorescence staining (Calcein-AM/Ethidium homodimer-1)", "Secondary: Metabolic activity measured by MTT assay (absorbance at 570nm)", "Secondary: Apoptotic/necrotic cell percentage by flow cytometry (Annexin V/PI staining)" ], "design": { "overview": "Replicate the existing dye transfer assay conditions with control and mutant cells, but harvest samples immediately before dye injection for comprehensive density and viability measurements using orthogonal methods (imaging, spectrophotometry, flow cytometry).", "work_packages": [ "WP1: Culture preparation - seed control and mutant cells at identical initial densities in 6-well plates and culture dishes, allow to reach confluence state matching original dye transfer experiments (3-5 days)", "WP2: Viability assessment - at time point matching dye transfer assay, perform live/dead staining with confocal imaging, MTT assay in parallel wells, and harvest cells for Annexin V/PI flow cytometry", "WP3: Density quantification - fix parallel samples, perform DAPI nuclear staining, acquire 10 random fields per well using automated microscopy, analyze with CellProfiler for cell counting", "WP4: Statistical analysis and correlation - compare density and viability metrics between control and mutant using t-tests, calculate coefficients of variation, correlate with historical dye transfer data" ], "controls": [ "Positive control for viability assays: cells treated with 0.1% Triton X-100 for 10 min (100% dead control)", "Negative control for viability assays: freshly thawed early passage cells in optimal media (high viability baseline)", "Technical control: cell-free wells with reagents only to measure background fluorescence and absorbance", "Seeding density control: parallel wells seeded at 50%, 100%, and 150% of standard density to establish sensitivity range" ], "sample_size_plan": "N=6 biological replicates per cell line (control and mutant), with 3 technical replicates per assay type per biological replicate, conducted over 2 independent experimental runs. Total: 12 wells per cell line per assay.", "success_criteria": [ "Coefficient of variation for cell density measurements <15% within biological replicates", "Detection sensitivity to identify \u226510% difference in cell density between groups with power \u22650.80 at \u03b1=0.05", "Viability assay concordance: <20% discrepancy between live/dead staining and MTT-based viability percentages", "Flow cytometry viable cell percentage >85% to confirm healthy cultures (unless significant difference detected between groups)" ], "estimated_timeline_weeks": 4 }, "materials": [ { "item": "LIVE/DEAD Viability/Cytotoxicity Kit for mammalian cells", "supplier": "Thermo Fisher Scientific", "catalog_or_id": "L3224", "link": "https://www.thermofisher.com/order/catalog/product/L3224", "purpose": "Simultaneous fluorescent labeling of live cells (Calcein-AM, green) and dead cells (Ethidium homodimer-1, red)" }, { "item": "MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Cell Proliferation Assay Kit", "supplier": "Abcam", "catalog_or_id": "ab211091", "link": "https://www.abcam.com/products/assay-kits/mtt-cell-proliferation-assay-kit-ab211091.html", "purpose": "Colorimetric quantification of metabolic activity and cell viability" }, { "item": "FITC Annexin V Apoptosis Detection Kit with PI", "supplier": "BioLegend", "catalog_or_id": "640914", "link": "", "purpose": "Flow cytometry-based detection of apoptotic and necrotic cells" }, { "item": "DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)", "supplier": "Thermo Fisher Scientific", "catalog_or_id": "D1306", "link": "https://www.thermofisher.com/order/catalog/product/D1306", "purpose": "Nuclear counterstaining for automated cell counting and density determination" }, { "item": "16% Paraformaldehyde (PFA) Aqueous Solution", "supplier": "Electron Microscopy Sciences", "catalog_or_id": "15710", "link": "", "purpose": "Cell fixation for immunofluorescence and nuclear staining" }, { "item": "6-well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate", "supplier": "Corning", "catalog_or_id": "3516", "link": "", "purpose": "Culture vessel for viability assays and density measurements" }, { "item": "96-well Flat Bottom TC-treated Microplate", "supplier": "Corning", "catalog_or_id": "3599", "link": "", "purpose": "MTT assay plate for spectrophotometric readings" }, { "item": "Countess II FL Automated Cell Counter", "supplier": "Thermo Fisher Scientific", "catalog_or_id": "AMQAF1000", "link": "https://www.thermofisher.com/order/catalog/product/AMQAF1000", "purpose": "Initial cell counting and viability assessment during seeding" }, { "item": "Trypan Blue Solution, 0.4%", "supplier": "Thermo Fisher Scientific", "catalog_or_id": "15250061", "link": "https://www.thermofisher.com/order/catalog/product/15250061", "purpose": "Exclusion dye for cell viability during seeding and harvesting" }, { "item": "CellProfiler Software (open source)", "supplier": "Broad Institute", "catalog_or_id": "v4.2.5", "link": "https://cellprofiler.org/releases", "purpose": "Automated image analysis for nuclear counting and cell density quantification" }, { "item": "PBS (Phosphate Buffered Saline), pH 7.4", "supplier": "Gibco", "catalog_or_id": "10010023", "link": "https://www.thermofisher.com/order/catalog/product/10010023", "purpose": "Washing and dilution buffer for all assays" }, { "item": "DMSO (Dimethyl sulfoxide), Cell Culture Grade", "supplier": "Sigma-Aldrich", "catalog_or_id": "D2650", "link": "https://www.sigmaaldrich.com/US/en/product/sial/d2650", "purpose": "Solubilization of MTT formazan crystals" } ], "estimated_direct_cost_usd": { "low": 2800, "high": 4500, "scope": "Includes all reagent kits, consumables (plates, tips), and flow cytometry service fees for 2 experimental runs with 6 biological replicates each. Excludes existing equipment (microscopes, plate readers, cell counter), cell culture media, and personnel costs. Assumes flow cytometry is available as core facility service at $50/sample." }, "protocol_references": [ { "title": "Brauchle E, et al. Cell death stages in single apoptotic and necrotic cells monitored by Raman microspectroscopy. Sci Rep. 2014;4:4698", "use": "Reference protocol for Annexin V/PI flow cytometry gating strategy to distinguish viable, early apoptotic, late apoptotic, and necrotic populations" }, { "title": "Riss TL, et al. Cell Viability Assays. In: Markossian S, et al., editors. Assay Guidance Manual. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004", "use": "Comprehensive guide for MTT assay optimization including cell number linearity, incubation time, and solvent selection" }, { "title": "Carpentier G, et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 2006;7(10):R100", "use": "Automated cell counting pipeline design using nuclear segmentation and intensity thresholding for accurate density measurements" }, { "title": "Wade MH, et al. A fluorescence photobleaching assay of gap junction-mediated communication between human cells. Science. 1986;232(4749):525-528", "use": "Original gap junction assay methodology to ensure density measurements match conditions of dye transfer experiments" } ], "handoff_package_for_lab": [ "Detailed cell culture protocol for both control and mutant lines including passage numbers, seeding densities, media formulations, and growth conditions used in original dye transfer experiments", "Original dye transfer assay timeline and conditions (culture duration, confluence state at time of injection, imaging timepoints) to replicate timing for density/viability measurements", "Historical data on dye transfer results (quantitative fluorescence intensity, migration distance measurements) for correlation analysis with density/viability findings", "Standard Operating Procedure (SOP) for confocal microscopy settings including objective magnification, laser power, gain settings, and z-stack parameters for live/dead imaging", "Flow cytometry compensation matrix file for FITC/PI channels specific to the cytometer that will be used", "CellProfiler pipeline file (.cppipe) pre-configured for nuclear segmentation and cell counting with quality control metrics" ] }, { "id": "opc-go-003", "title": "CRISPR-Based Enhancement of Transformation Competence in Recalcitrant Elite Maize Lines", "decision_bucket": "go_now", "best_score": 0.527, "best_confidence": 0.5, "maps_to_problem_statement": "Genotype-specific transformation efficiency differences are driven by unknown underlying genetic or epigenetic variation, preventing widespread adoption of plant engineering in non-elite lines.", "maps_to_sub_question": "Can identified genetic or epigenetic determinants of transformation competence be edited to convert recalcitrant elite lines into transformable lines?", "objective": "To determine if CRISPR/Cas9 editing of candidate transformation competence loci (BABY BOOM, WUSCHEL2, GRF-GIF) can increase Agrobacterium-mediated transformation efficiency in recalcitrant elite maize inbreds by at least 5-fold.", "readouts": [ "Primary: Transformation efficiency measured as percentage of regenerated T0 transgenic plants per inoculated immature embryo across three independent experiments", "Secondary: Transgene integration events per line assessed by Southern blot and qPCR to confirm stable transformation", "Secondary: Heritability of transformation competence in T1 and T2 generations measured by repeated transformation assays using edited line embryos" ], "design": { "overview": "Edit BBM, WUS2, and GRF5-GIF1 loci in recalcitrant elite maize line B104 using CRISPR/Cas9 to create gain-of-function or overexpression alleles, then compare Agrobacterium-mediated transformation efficiency between edited and parental lines across multiple generations.", "work_packages": [ "WP1: Design and validate CRISPR constructs targeting BBM, WUS2, and GRF5-GIF1 promoter regions to create constitutive or enhanced expression alleles; transform into Hi-II (transformable control line) for validation (Weeks 1-8)", "WP2: Transform B104 elite recalcitrant line with validated CRISPR constructs; select and sequence T0 edited events; advance to T1 generation and confirm edits are heritable; backcross to remove Cas9 if necessary (Weeks 9-32)", "WP3: Conduct standardized transformation efficiency assays on edited B104 lines and wild-type B104 using Agrobacterium strain EHA105 carrying pPHP71539 (bar + uidA); score transformation frequency, regeneration rate, and stable integration (Weeks 33-48)", "WP4: Validate transformation competence heritability in T2 generation; perform molecular characterization of integration sites; statistical analysis comparing transformation rates across genotypes with ANOVA and post-hoc tests (Weeks 49-52)" ], "controls": [ "Positive control: Hi-II maize line transformed in parallel to validate Agrobacterium competence and media conditions (expected efficiency 15-30%)", "Negative control: Wild-type B104 line without editing transformed in parallel (expected efficiency <1%)", "Mock control: B104 line transformed with empty vector CRISPR construct (no gRNA) to control for transformation procedure effects", "Segregation control: T1 null segregants (non-edited siblings) from edited lines to control for genetic background effects" ], "sample_size_plan": "N=3 independent CRISPR constructs per target gene (9 total); minimum 5 independent T0 edited events advanced per construct; transformation efficiency assays with n=200 immature embryos per genotype per replicate across 3 biological replicates (total 600 embryos/genotype); statistical power calculation targets 80% power to detect 5-fold change at \u03b1=0.05", "success_criteria": [ "At least one edited line achieves \u22655-fold increase in transformation efficiency compared to wild-type B104 (e.g., from 0.5% to \u22652.5%) with p<0.05 by one-way ANOVA", "Enhanced transformation competence is heritable and stable through T2 generation with <20% reduction in efficiency compared to T0 edited line", "Edited lines show no major agronomic penalties: germination rate >85%, plant height within 15% of wild-type, and normal seed set (>80% of wild-type)" ], "estimated_timeline_weeks": 52 }, "materials": [ { "item": "pYPQ146 Gateway-compatible Cas9 vector for maize", "supplier": "Addgene", "catalog_or_id": "164544", "link": "https://www.addgene.org/164544/", "purpose": "CRISPR/Cas9 delivery vector with maize-optimized expression cassettes" }, { "item": "GoldenGate MoClo Plant Parts Kit", "supplier": "Addgene", "catalog_or_id": "1000000044", "purpose": "Modular cloning system for assembling gRNA expression cassettes" }, { "item": "Agrobacterium tumefaciens EHA105 competent cells", "supplier": "Teknova", "catalog_or_id": "A1202", "purpose": "Agrobacterium strain for maize transformation" }, { "item": "pPHP71539 binary vector (bar + uidA)", "supplier": "John Innes Centre", "catalog_or_id": "Request from ABRC stock center", "link": "https://abrc.osu.edu/", "purpose": "Standard transformation reporter construct for efficiency assays" }, { "item": "Bialaphos (phosphinothricin, PPT)", "supplier": "Gold Biotechnology", "catalog_or_id": "B-110", "purpose": "Selection agent for bar-expressing transformants" }, { "item": "N6 Basal Medium with Vitamins", "supplier": "PhytoTechnology Laboratories", "catalog_or_id": "N611", "purpose": "Base medium for maize tissue culture and regeneration" }, { "item": "2,4-Dichlorophenoxyacetic acid (2,4-D)", "supplier": "Sigma-Aldrich", "catalog_or_id": "D7299", "link": "https://www.sigmaaldrich.com/catalog/product/sigma/d7299", "purpose": "Auxin for callus induction from immature embryos" }, { "item": "DIG High Prime DNA Labeling and Detection Starter Kit II", "supplier": "Roche", "catalog_or_id": "11585614910", "link": "https://www.sigmaaldrich.com/catalog/product/roche/11585614910", "purpose": "Southern blot analysis to confirm transgene integration" }, { "item": "GUS Histochemical Staining Kit", "supplier": "Sigma-Aldrich", "catalog_or_id": "GUSB-1KT", "link": "https://www.sigmaaldrich.com/catalog/product/sigma/gusb1kt", "purpose": "Visual detection of uidA expression in putative transformants" }, { "item": "Phire Plant Direct PCR Kit", "supplier": "Thermo Fisher Scientific", "catalog_or_id": "F130WH", "link": "https://www.thermofisher.com/order/catalog/product/F130WH", "purpose": "Rapid genotyping of edited events and transgene presence" }, { "item": "Alt-R CRISPR-Cas9 crRNA synthesis", "supplier": "Integrated DNA Technologies", "catalog_or_id": "Custom order", "link": "https://www.idtdna.com/pages/products/crispr-genome-editing/alt-r-crispr-cas9-system", "purpose": "Design validation: in vitro testing of gRNA efficiency before cloning" }, { "item": "Percival AR-95L growth chamber", "supplier": "Percival Scientific", "catalog_or_id": "AR-95L", "purpose": "Controlled environment for regenerating transformants and growing edited lines" } ], "estimated_direct_cost_usd": { "low": 35000, "high": 55000, "scope": "Includes molecular biology reagents, tissue culture supplies, sequencing costs (~$8K), growth chamber space rental, and seed production. Excludes personnel costs, equipment already available (PCR machines, incubators, microscopes), greenhouse rental for backcrossing, and long-term generation advancement costs beyond T2" }, "protocol_references": [ { "title": "Lowe, K. et al. (2016) Morphogenic Regulators Baby boom and Wuschel Improve Monocot Transformation. Plant Cell 28:1998-2015", "use": "Provides validated BBM and WUS2 expression strategies shown to enhance transformation; will adapt promoter and expression level strategies from this seminal work" }, { "title": "Debernardi, J.M. et al. (2020) A GRF-GIF chimeric protein improves the regeneration efficiency of transgenic plants. Nature Biotechnology 38:1274-1279", "use": "Describes GRF5-GIF1 chimeric construct design that enhances regeneration capacity; will create similar constructs or activation of endogenous loci" }, { "title": "Frame, B.R. et al. (2011) Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system. Methods Mol Biol 710:327-341", "use": "Standard protocol for maize transformation efficiency assays; will use this as basis for standardized comparative testing of edited vs. wild-type lines" }, { "title": "Svitashev, S. et al. (2016) Genome editing in maize directed by CRISPR-Cas9 ribonucleoprotein complexes. Nature Communications 7:13274", "use": "Maize CRISPR methodology including delivery methods, selection strategies, and confirmation of edits; informs construct design parameters" }, { "title": "Char, S.N. et al. (2017) An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize. Plant Biotechnology Journal 15:257-268", "use": "Validates Agrobacterium delivery of CRISPR reagents in maize; provides technical details for combining transformation and editing in single step" } ], "handoff_package_for_lab": [ "Validated sequence information for BBM, WUS2, and GRF5-GIF1 loci in B104 background including 5kb upstream promoter regions and full-length CDS obtained through genome sequencing or PCR amplification and Sanger sequencing", "Fresh immature embryo source: maintained B104 and Hi-II nursery plantings with staggered planting dates to ensure continuous supply of 10-14 DAP embryos throughout transformation experiments (minimum 20 donor ears per experiment)", "Completed CRISPR gRNA design with in silico off-target analysis for B104 genome; minimum 3 gRNA candidates per target locus scoring <3 predicted off-targets and >70% predicted efficiency by CRISPOR or comparable tool", "Institutional biosafety and field release approvals for generating and testing CRISPR-edited maize lines; USDA-APHIS regulatory determination if advancing edited lines to field testing", "Baseline transformation efficiency data: at least one pilot transformation experiment completed on wild-type B104 and Hi-II to establish baseline rates and validate tissue culture competency of laboratory (minimum n=100 embryos per line)" ] }, { "id": "opc-go-004", "title": "Functional validation of Type 2 IRES-ribosome structural contacts via mutagenesis and translation assays", "decision_bucket": "go_now", "best_score": 0.527, "best_confidence": 0.5, "maps_to_problem_statement": "Lack of complementary biochemical validation for the structural findings of viral Type 2 IRES recruitment to ribosomal preinitiation complex", "maps_to_sub_question": "Do the structural observations correlate with functional translation activity in cellular or in vitro systems?", "objective": "To validate the functional importance of specific IRES-ribosome structural contacts identified by cryo-EM/X-ray through site-directed mutagenesis and quantitative translation assays.", "readouts": [ "Primary: Relative translation efficiency (luciferase activity normalized to mRNA levels) for wild-type vs. mutant IRES constructs in rabbit reticulocyte lysate (RRL) in vitro translation system", "Secondary: Dual-luciferase reporter ratios (IRES-driven Renilla/cap-dependent Firefly) in transfected HEK293T cells for cellular validation", "Secondary: RNA binding affinity (KD) of wild-type and mutant IRES to purified 40S ribosomal subunits measured by microscale thermophoresis (MST)" ], "design": { "overview": "Generate 8-12 IRES mutants targeting key structural contact residues identified in cryo-EM/crystal structures. Test translation efficiency in parallel using in vitro RRL system and cellular dual-luciferase assays. Correlate functional defects with direct 40S binding affinity measurements.", "work_packages": [ "WP1: Design and generate IRES mutant library (single, double, and compensatory mutations) targeting ribosome contact sites; clone into dual-luciferase bicistronic reporters and in vitro transcription vectors (Weeks 1-3)", "WP2: In vitro transcription of capped, polyadenylated mRNAs; quality control by denaturing gel electrophoresis and capillary electrophoresis; RRL translation assays with luciferase readout and RT-qPCR normalization (Weeks 4-7)", "WP3: Transfection of dual-luciferase constructs into HEK293T cells; measure Renilla/Firefly ratios at 24h and 48h; Northern blot or RT-qPCR to confirm mRNA stability (Weeks 6-9)", "WP4: Purify 40S ribosomal subunits from HEK293T cells; in vitro transcribe fluorescently-labeled IRES RNAs; perform MST binding assays; statistical analysis and correlation with structural contacts (Weeks 8-12)" ], "controls": [ "Positive control: Wild-type EMCV or CrPV IRES (established Type 2 IRES) with known high translation efficiency", "Negative control: Bicistronic construct with no IRES (empty intercistronic spacer) to measure background cap-independent translation", "Technical control: Monocistronic cap-dependent luciferase construct to normalize transfection efficiency and cell viability; mock-transfected cells for background subtraction" ], "sample_size_plan": "8-12 IRES mutants plus wild-type and controls; each construct tested in n=4 biological replicates for RRL assays, n=6 biological replicates for cell-based assays (3 independent transfections \u00d7 2 time points), and n=3 technical replicates for MST binding measurements", "success_criteria": [ "At least 60% of mutations targeting direct contact residues show >2-fold reduction in translation efficiency (p<0.05 by one-way ANOVA with Dunnett's post-hoc test) compared to wild-type IRES", "Pearson correlation coefficient r > 0.6 (p<0.05) between translation efficiency defects and changes in 40S binding affinity (KD) for mutant IRES constructs", "Cellular and in vitro translation assays show concordant results (same direction of effect) for >80% of mutants tested" ], "estimated_timeline_weeks": 12 }, "materials": [ { "item": "Rabbit Reticulocyte Lysate System, Nuclease-Treated", "supplier": "Promega", "catalog_or_id": "L4960", "purpose": "In vitro translation system for testing IRES-driven translation efficiency" }, { "item": "psiCHECK-2 Vector (dual-luciferase bicistronic reporter)", "supplier": "Promega", "catalog_or_id": "C8021", "purpose": "Backbone for cloning IRES variants between Renilla and Firefly luciferase coding sequences" }, { "item": "Dual-Luciferase Reporter Assay System", "supplier": "Promega", "catalog_or_id": "E1960", "link": "https://www.promega.com/products/reporter-assays-and-transfection/reporter-assays/dual_luciferase-reporter-assay-system", "purpose": "Quantify IRES-driven Renilla and cap-dependent Firefly luciferase activities in cell lysates" }, { "item": "HEK293T cells", "supplier": "ATCC", "catalog_or_id": "CRL-3216", "link": "https://www.atcc.org/products/crl-3216", "purpose": "Mammalian cell line for cellular translation reporter assays and 40S ribosome purification" }, { "item": "mMESSAGE mMACHINE T7 Transcription Kit", "supplier": "Thermo Fisher", "catalog_or_id": "AM1344", "link": "https://www.thermofisher.com/order/catalog/product/AM1344", "purpose": "Generate capped, polyadenylated mRNA transcripts for in vitro translation assays" }, { "item": "Q5 Site-Directed Mutagenesis Kit", "supplier": "New England Biolabs", "catalog_or_id": "E0554S", "link": "https://www.neb.com/products/e0554-q5-site-directed-mutagenesis-kit", "purpose": "Introduce point mutations into IRES sequences at structurally-defined contact sites" }, { "item": "Luciferase Assay Reagent (LAR)", "supplier": "Promega", "catalog_or_id": "E1483", "link": "https://www.promega.com/products/reporter-assays-and-transfection/reporter-assays/luciferase-assay-system", "purpose": "Measure luciferase activity from in vitro translation reactions" }, { "item": "Monolith NT.115 Microscale Thermophoresis instrument", "supplier": "NanoTemper Technologies", "catalog_or_id": "NT.115", "purpose": "Measure binding affinity (KD) between fluorescently-labeled IRES RNA and purified 40S subunits" }, { "item": "Monolith His-Tag Labeling Kit RED-tris-NTA 2nd Generation", "supplier": "NanoTemper Technologies", "catalog_or_id": "MO-L018", "purpose": "Fluorescent labeling of RNA or ribosomal proteins for MST measurements" }, { "item": "Label IT Nucleic Acid Labeling Kit, Cy5", "supplier": "Mirus Bio", "catalog_or_id": "MIR3225", "link": "", "purpose": "Fluorescently label in vitro transcribed IRES RNA for MST binding assays" }, { "item": "Sucrose Cushion Ribosome Purification Buffer Set", "supplier": "Custom preparation", "catalog_or_id": "N/A", "purpose": "Buffers for ultracentrifugation-based purification of 40S ribosomal subunits (20 mM Tris-HCl pH 7.5, 100 mM KCl, 5 mM MgCl2, 1M sucrose cushion)" }, { "item": "SuperScript IV Reverse Transcriptase", "supplier": "Thermo Fisher", "catalog_or_id": "18090010", "link": "https://www.thermofisher.com/order/catalog/product/18090010", "purpose": "RT-qPCR to quantify mRNA levels for normalization of translation efficiency" }, { "item": "PowerUp SYBR Green Master Mix", "supplier": "Applied Biosystems", "catalog_or_id": "A25742", "link": "https://www.thermofisher.com/order/catalog/product/A25742", "purpose": "qPCR quantification of luciferase mRNA levels" }, { "item": "Lipofectamine 3000 Transfection Reagent", "supplier": "Thermo Fisher", "catalog_or_id": "L3000015", "link": "https://www.thermofisher.com/order/catalog/product/L3000015", "purpose": "Transfect dual-luciferase reporter constructs into HEK293T cells" }, { "item": "Beckman Coulter Optima XPN-100 Ultracentrifuge with SW41 rotor", "supplier": "Beckman Coulter", "catalog_or_id": "A94471", "link": "https://www.beckman.com/centrifuges/ultracentrifuges/optima-xpn-series", "purpose": "Purification of ribosomal subunits through sucrose cushion ultracentrifugation" }, { "item": "Agilent 2100 Bioanalyzer with RNA 6000 Nano Kit", "supplier": "Agilent", "catalog_or_id": "5067-1511", "purpose": "Quality control of in vitro transcribed mRNA integrity and purity" } ], "estimated_direct_cost_usd": { "low": 8500, "high": 14500, "scope": "Includes molecular cloning reagents, in vitro translation kits, cell culture reagents, transfection reagents, luciferase assay reagents, RT-qPCR supplies, RNA labeling kits, and MST consumables. Excludes major equipment (ultracentrifuge, MST instrument, plate readers, qPCR machine), labor costs, and ribosome purification if commercial 40S subunits are purchased (~$3000 additional)" }, "protocol_references": [ { "title": "Zuker, M. & Stiegler, P. (1981) 'Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information', Nucleic Acids Research, 9(1), pp. 133-148.", "use": "Computational prediction of secondary structure changes introduced by mutations to ensure disruption of tertiary contacts without destabilizing overall IRES fold" }, { "title": "Pestova, T.V. & Hellen, C.U. (2003) 'Translation elongation after assembly of ribosomes on the Cricket paralysis virus internal ribosomal entry site without initiation factors or initiator tRNA', Genes & Development, 17(2), pp. 181-186.", "use": "Established protocol for in vitro translation assays using Type 2 IRES constructs in rabbit reticulocyte lysate" }, { "title": "Deniz, N., et al. (2009) 'Translation initiation factors and their relevance in health and disease', Biological Chemistry, 390(5-6), pp. 331-344.", "use": "Guidelines for normalization strategies in translation assays and interpretation of IRES-mediated translation efficiency" }, { "title": "Khawaja, A., et al. (2015) 'Measuring thermal stability of proteins in cell lysates by microscale thermophoresis', Methods in Molecular Biology, 1261, pp. 253-261.", "use": "Optimized protocols for MST-based measurement of RNA-protein binding affinity including buffer optimization and concentration ranges" }, { "title": "Easton, L.E., et al. (2009) 'A novel method to engineer bicistronic constructs for the expression of influenza A proteins in cell culture and Xenopus laevis embryos', Nucleic Acids Research, 37(10), e68.", "use": "Design principles for bicistronic dual-luciferase reporters to minimize cryptic promoter activity and ensure accurate IRES-dependent translation measurement" }, { "title": "Anger, A.M., et al. (2013) 'Structures of the human and Drosophila 80S ribosome', Nature, 497(7447), pp. 80-85.", "use": "Protocol for purification of mammalian ribosomal subunits from cultured cells using sucrose gradient ultracentrifugation" } ], "handoff_package_for_lab": [ "3D structural coordinates (PDB or CIF file) of the IRES-ribosome complex with annotated contact residues and distances <5\u00c5 for mutagenesis targeting", "Complete nucleotide sequence of the wild-type IRES element with 5' and 3' flanking sequences (50-100 nt) for cloning into expression vectors", "List of 8-12 prioritized residue positions for mutagenesis ranked by structural contact surface area and phylogenetic conservation, including proposed substitutions (e.g., charge reversals, alanine scanning)", "Primer design file with sequences for site-directed mutagenesis of all target positions including compensatory double mutants", "Reference translation efficiency data (if available) for the wild-type IRES from previous studies for benchmark comparison", "Cell culture protocols and media requirements specific to the lab (preferred HEK293T culture conditions, passage numbers, transfection optimization data)" ] }, { "id": "opc-go-005", "title": "Transcriptomic Profiling of Pachytene Genome Activation in Mouse Spermatocytes", "decision_bucket": "go_now", "best_score": 0.527, "best_confidence": 0.5, "maps_to_problem_statement": "The concept of 'pachytene genome activation' (PGS) is introduced without proper characterization of which genes are activated, what defines this process, or what mechanisms underlie it", "maps_to_sub_question": "What is the complete set of genes that are specifically upregulated during pachytene stage (pachytene genome activation)?", "objective": "Identify the complete set of genes specifically upregulated during pachytene stage of meiosis through stage-specific RNA-seq and validation.", "readouts": [ "Primary: Differential gene expression profiles across leptotene, zygotene, pachytene, and diplotene stages by RNA-seq (TPM values, log2 fold-change)", "Secondary: RT-qPCR validation of top 20 pachytene-upregulated candidate genes across meiotic stages (relative expression normalized to housekeeping genes)", "Secondary: Immunofluorescence or RNA-FISH validation of 5-10 candidate genes showing pachytene-specific expression patterns in testis sections" ], "design": { "overview": "Isolate staged spermatocytes (leptotene, zygotene, pachytene, diplotene) from adult mouse testes by STA-PUT velocity sedimentation, perform RNA-seq with 4 biological replicates per stage, identify pachytene-specific upregulated genes (\u22654-fold vs other stages), and validate top candidates by RT-qPCR and RNA-FISH.", "work_packages": [ "WP1: Optimize STA-PUT sedimentation protocol and isolate staged spermatocytes from 8-10 week old C57BL/6J male mice (n=16 mice for 4 replicates per stage); assess purity by flow cytometry (DNA content) and stage markers by RT-qPCR; isolate total RNA with RIN>8.0", "WP2: Perform poly-A selected RNA-seq on all samples (50M paired-end 150bp reads per sample); conduct quality control, read alignment to mm10, and differential expression analysis using DESeq2 to identify genes with pachytene-specific upregulation (\u22654-fold vs all other stages, FDR<0.01)", "WP3: Validate top 20 pachytene-upregulated genes by RT-qPCR across all four meiotic stages using 3 independent biological replicates; design primers, optimize conditions, and quantify relative expression normalized to Gapdh and Actb", "WP4: Perform RNA-FISH on testis sections for 5-10 validated genes to confirm stage-specific expression patterns; analyze images for stage-specific signal; integrate all data and generate final gene list with functional annotation" ], "controls": [ "Positive control: Known pachytene-specific genes (Dazl, Sycp1, Tex19.1) should show expected upregulation pattern", "Negative control: Housekeeping genes (Gapdh, Actb, Rpl19) should show stable expression across all meiotic stages", "Technical control: Pre-meiotic spermatogonia (isolated separately) as out-group to confirm meiotic-specific expression patterns; spike-in controls (ERCC) for RNA-seq normalization" ], "sample_size_plan": "4 biological replicates per meiotic stage (leptotene, zygotene, pachytene, diplotene) for RNA-seq (16 samples total, each from pooled cells from 4 mice); 3 independent biological replicates for RT-qPCR validation; 3 mice for RNA-FISH validation", "success_criteria": [ "RNA quality: RIN \u22658.0 for all samples; Stage purity: \u226585% target stage by flow cytometry DNA content analysis and marker gene expression", "RNA-seq: \u226545M mapped reads per sample, identification of \u2265100 genes with pachytene-specific upregulation (\u22654-fold vs other stages, FDR<0.01); RT-qPCR validation: \u226580% concordance with RNA-seq fold-change direction for top 20 candidates" ], "estimated_timeline_weeks": 14 }, "materials": [ { "item": "C57BL/6J male mice, 8-10 weeks old", "supplier": "The Jackson Laboratory", "catalog_or_id": "000664", "link": "https://www.jax.org/strain/000664", "purpose": "Source of staged spermatocytes for meiotic profiling" }, { "item": "STA-PUT apparatus and chambers", "supplier": "ProScience Inc or custom fabrication", "catalog_or_id": "Custom", "link": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4374907/", "purpose": "Velocity sedimentation separation of staged spermatocytes" }, { "item": "TRIzol Reagent", "supplier": "Thermo Fisher Scientific", "catalog_or_id": "15596026", "link": "https://www.thermofisher.com/order/catalog/product/15596026", "purpose": "Total RNA extraction from isolated spermatocytes" }, { "item": "RNeasy Mini Kit", "supplier": "Qiagen", "catalog_or_id": "74106", "link": "https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/rna-purification/total-rna/rneasy-mini-kit", "purpose": "RNA cleanup and purification after TRIzol extraction" }, { "item": "Agilent RNA 6000 Nano Kit", "supplier": "Agilent Technologies", "catalog_or_id": "5067-1511", "purpose": "RNA quality assessment and RIN determination" }, { "item": "TruSeq Stranded mRNA Library Prep Kit", "supplier": "Illumina", "catalog_or_id": "20020595", "link": "https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/truseq-stranded-mrna.html", "purpose": "Poly-A selected RNA-seq library preparation" }, { "item": "NovaSeq 6000 SP Reagent Kit v1.5 (100 cycles)", "supplier": "Illumina", "catalog_or_id": "20028401", "link": "https://www.illumina.com/products/by-type/sequencing-kits/cluster-gen-sequencing-reagents/novaseq-reagent-kits.html", "purpose": "Paired-end 150bp sequencing for RNA-seq samples" }, { "item": "ERCC RNA Spike-In Mix", "supplier": "Thermo Fisher Scientific", "catalog_or_id": "4456740", "link": "https://www.thermofisher.com/order/catalog/product/4456740", "purpose": "Technical controls for RNA-seq normalization and quality control" }, { "item": "SuperScript IV First-Strand Synthesis System", "supplier": "Thermo Fisher Scientific", "catalog_or_id": "18091050", "link": "https://www.thermofisher.com/order/catalog/product/18091050", "purpose": "cDNA synthesis for RT-qPCR validation" }, { "item": "PowerUp SYBR Green Master Mix", "supplier": "Thermo Fisher Scientific", "catalog_or_id": "A25742", "link": "https://www.thermofisher.com/order/catalog/product/A25742", "purpose": "RT-qPCR quantification of candidate genes" }, { "item": "RNAscope Multiplex Fluorescent Reagent Kit v2", "supplier": "Advanced Cell Diagnostics", "catalog_or_id": "323100", "link": "", "purpose": "RNA-FISH validation of pachytene-specific expression in tissue sections" }, { "item": "Custom RNAscope probes for candidate genes", "supplier": "Advanced Cell Diagnostics", "catalog_or_id": "Custom", "link": "", "purpose": "Gene-specific probes for RNA-FISH validation (5-10 genes)" }, { "item": "Hoechst 33342", "supplier": "Thermo Fisher Scientific", "catalog_or_id": "H3570", "link": "https://www.thermofisher.com/order/catalog/product/H3570", "purpose": "DNA staining for flow cytometry purity assessment and chromosome visualization" }, { "item": "BD FACSAria Fusion flow cytometer (existing equipment)", "supplier": "BD Biosciences", "catalog_or_id": "Facility", "link": "https://www.bdbiosciences.com/en-us/products/instruments/flow-cytometers/research-cell-sorters/facsaria-fusion", "purpose": "Assessment of spermatocyte stage purity by DNA content" }, { "item": "QuantStudio 6 Real-Time PCR System (existing equipment)", "supplier": "Thermo Fisher Scientific", "catalog_or_id": "Facility", "link": "https://www.thermofisher.com/order/catalog/product/4485691", "purpose": "RT-qPCR validation of candidate genes" } ], "estimated_direct_cost_usd": { "low": 18000, "high": 25000, "scope": "Includes: mice ($800), RNA extraction/QC reagents ($1,200), RNA-seq library prep ($3,200), sequencing costs ($8,000-12,000 depending on core facility rates), RT-qPCR reagents and primers ($1,500), RNA-FISH reagents and custom probes ($3,000-5,000), flow cytometry costs ($500), consumables ($1,800). Excludes: personnel costs, existing equipment access fees, bioinformatics server costs, publication fees" }, "protocol_references": [ { "title": "Isolation of specific stages of spermatogenic cells from mouse testis by STA-PUT velocity sedimentation (Bryant et al., J Vis Exp 2013)", "use": "Primary protocol for staged spermatocyte isolation with expected purity and yield metrics" }, { "title": "Transcriptome Dynamics of the Developing Male Mouse Testis (Soumillon et al., Nature 2013)", "use": "Reference dataset for comparison and validation of meiotic stage-specific expression patterns" }, { "title": "RNA-seq workflow: gene-level exploratory analysis and differential expression (Love et al., F1000Research 2015)", "use": "DESeq2 analysis pipeline for differential expression calling with appropriate statistical thresholds" }, { "title": "Single-cell RNA-seq reveals dynamic, random monoallelic gene expression in mammalian cells (Deng et al., Science 2014)", "use": "Quality control metrics and normalization approaches for RNA-seq data" }, { "title": "RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues (Wang et al., J Mol Diagn 2012)", "use": "RNA-FISH protocol for spatial validation of pachytene-specific gene expression in testis sections" } ], "handoff_package_for_lab": [ "IACUC protocol approval for mouse work including euthanasia method and number of animals (minimum 25 male mice total); facility breeding plan if not purchasing", "Bioinformatics pipeline specification: compute resources allocated (\u2265256GB RAM, 32 cores), software installed (STAR v2.7+, DESeq2, Salmon, FastQC, MultiQC), reference genome mm10/GRCm38, and designated bioinformatician contact", "Primer design table for RT-qPCR validation: list of top 20 candidate genes from preliminary literature (e.g., Dazl, Tex19.1, Hormad1, Spo11, Meiob) plus housekeeping genes (Gapdh, Actb, Rpl19); primers pre-designed using Primer3 with validation criteria", "Standard operating procedures (SOPs) documented: STA-PUT apparatus setup and calibration, testis dissociation protocol, flow cytometry gating strategy for meiotic stages, RNA quality acceptance criteria", "Data management plan: designated storage location for raw FASTQ files (\u2265500GB), processed count matrices, and metadata; GEO accession number reserved for public deposition upon publication" ] }, { "id": "opc-go-006", "title": "Identification and Validation of Safe Harbor Loci in Switchgrass and Miscanthus Genomes", "decision_bucket": "go_now", "best_score": 0.527, "best_confidence": 0.5, "maps_to_problem_statement": "How promoters, enhancers, and cis-regulatory elements (CREs) impact position effects of transgene insertions in bioenergy crops is not understood, and sequences with insulator-like function to dampen ectopic interactions are needed.", "maps_to_sub_question": "Can validated safe harbor loci be identified in bioenergy crop genomes for predictable transgene expression across diverse genetic backgrounds?", "objective": "Identify and experimentally validate conserved intergenic genomic loci that support predictable, stable transgene expression across multiple bioenergy crop genotypes and generations.", "readouts": [ "Primary: Transgene expression level (fluorescence intensity and qRT-PCR) across \u22656 independent insertion events per locus, measured as coefficient of variation (CV) between events", "Secondary: Multi-generational expression stability (T0 through T2) measured by qRT-PCR with <20% variation between generations", "Secondary: Chromatin accessibility (ATAC-seq) and histone modification patterns (H3K9ac, H3K4me3) at candidate loci across tissues and genotypes" ], "design": { "overview": "Perform pan-genome comparative analysis to identify conserved intergenic regions, then use CRISPR-mediated homology-directed repair to insert fluorescent reporter cassettes at 4-6 candidate safe harbor loci in switchgrass (Panicum virgatum) and Miscanthus \u00d7 giganteus, followed by multi-generational phenotyping and expression analysis.", "work_packages": [ "WP1: Pan-genome bioinformatics analysis - identify conserved intergenic regions (>2kb) in \u226520 switchgrass and \u226510 Miscanthus accessions with low syntenic variation, open chromatin signatures, and absence of predicted regulatory elements within 5kb; prioritize 6-8 candidate loci based on conservation, accessibility, and distance from genes", "WP2: CRISPR/Cas9 construct design and delivery - design HDR donor templates containing mEGFP-NOS terminator cassette flanked by 800bp homology arms for each candidate locus; transform embryogenic callus of 3 switchgrass cultivars (Alamo, Kanlow, Cave-in-Rock) and 2 Miscanthus genotypes using Agrobacterium-mediated transformation", "WP3: Plant regeneration and molecular characterization - regenerate T0 plants, confirm precise insertion by junction PCR and Sanger sequencing, select 6-10 independent events per locus per genotype, advance to T1 and T2 generations, collect leaf, stem, and root tissues across vegetative and reproductive stages", "WP4: Expression and epigenetic profiling - quantify mEGFP expression by fluorometry and qRT-PCR across tissues, developmental stages, and generations; perform ATAC-seq and ChIP-qPCR for H3K9ac and H3K4me3 at insertion sites; statistical analysis of expression variance, heritability, and chromatin state correlation" ], "controls": [ "Positive control: Random T-DNA insertion events (n=20) generated in parallel transformations to establish baseline expression variability (expected CV >80%)", "Negative control: Non-transformed wild-type plants of each genotype for background fluorescence measurement and normalization", "Technical control: Internal reference gene control (ubiquitin, actin) for qRT-PCR normalization; spike-in controls for ATAC-seq library preparation" ], "sample_size_plan": "6-8 candidate loci \u00d7 5 genotypes (3 switchgrass + 2 Miscanthus) \u00d7 6-10 independent insertion events per locus = 180-400 independent T0 events; advance 3-5 events per locus to T1 (90-200 families) and 2-3 to T2 (60-120 families); 3 biological replicates per tissue type (leaf, stem, root) \u00d7 3 developmental stages \u00d7 3 technical replicates for expression analysis", "success_criteria": [ "At least 2 loci show CV <30% for transgene expression across independent insertion events within same genotype and tissue (compared to >80% for random insertions)", "Selected safe harbor loci maintain expression levels within 2-fold variation across T0, T1, and T2 generations (\u226580% of families) and show consistent chromatin accessibility (ATAC-seq peak present in \u226590% of samples, fold-change <2\u00d7 between genotypes)" ], "estimated_timeline_weeks": 104 }, "materials": [ { "item": "pKSE401 CRISPR-Cas9 plant expression vector", "supplier": "Addgene", "catalog_or_id": "62202", "link": "https://www.addgene.org/62202/", "purpose": "Base vector for CRISPR/Cas9 expression and HDR donor template construction" }, { "item": "mEGFP coding sequence (codon-optimized for monocots)", "supplier": "GenScript", "catalog_or_id": "Custom synthesis", "link": "", "purpose": "Fluorescent reporter for transgene expression tracking" }, { "item": "Agrobacterium tumefaciens strain EHA105", "supplier": "Thermo Fisher Scientific", "catalog_or_id": "A13435", "link": "https://www.thermofisher.com/order/catalog/product/A13435", "purpose": "Agrobacterium-mediated transformation of grass embryogenic callus" }, { "item": "Murashige and Skoog Basal Medium with Vitamins", "supplier": "PhytoTechnology Laboratories", "catalog_or_id": "M519", "link": "", "purpose": "Base medium for tissue culture and plant regeneration" }, { "item": "2,4-Dichlorophenoxyacetic acid (2,4-D)", "supplier": "Sigma-Aldrich", "catalog_or_id": "D7299", "link": "https://www.sigmaaldrich.com/catalog/product/sigma/d7299", "purpose": "Auxin for callus induction from immature embryos" }, { "item": "PowerPlant Pro RNA Isolation Kit", "supplier": "Qiagen", "catalog_or_id": "13427", "link": "https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/rna-purification/total-rna/rneasy-powerplant-pro-kit/", "purpose": "RNA extraction from grass tissues for qRT-PCR" }, { "item": "Luna Universal One-Step RT-qPCR Kit", "supplier": "New England Biolabs", "catalog_or_id": "E3005", "link": "https://www.neb.com/products/e3005-luna-universal-one-step-rt-qpcr-kit", "purpose": "Quantitative RT-PCR for transgene expression measurement" }, { "item": "SYBR Green I Nucleic Acid Gel Stain", "supplier": "Thermo Fisher Scientific", "catalog_or_id": "S7563", "link": "https://www.thermofisher.com/order/catalog/product/S7563", "purpose": "Fluorescent detection for junction PCR validation" }, { "item": "Nextera DNA Library Prep Kit", "supplier": "Illumina", "catalog_or_id": "20018705", "link": "https://www.illumina.com/products/by-type/sequencing-kits/library-prep-kits/nextera-dna.html", "purpose": "ATAC-seq library preparation for chromatin accessibility profiling" }, { "item": "SimpleChIP Plus Enzymatic Chromatin IP Kit", "supplier": "Cell Signaling Technology", "catalog_or_id": "9005", "link": "https://www.cellsignal.com/products/chromatin-immunoprecipitation-chip/simplechip-plus-enzymatic-chromatin-ip-kit-magnetic-beads/9005", "purpose": "Chromatin immunoprecipitation for histone modification analysis" }, { "item": "Anti-Histone H3K9ac antibody", "supplier": "Abcam", "catalog_or_id": "ab4441", "link": "https://www.abcam.com/histone-h3-acetyl-k9-antibody-chip-grade-ab4441.html", "purpose": "ChIP-qPCR detection of active chromatin marks" }, { "item": "Anti-Histone H3K4me3 antibody", "supplier": "Abcam", "catalog_or_id": "ab8580", "link": "https://www.abcam.com/histone-h3-tri-methyl-k4-antibody-chip-grade-ab8580.html", "purpose": "ChIP-qPCR detection of promoter-associated chromatin marks" }, { "item": "SpectraMax iD3 Multi-Mode Microplate Reader", "supplier": "Molecular Devices", "catalog_or_id": "iD3", "link": "", "purpose": "Fluorescence quantification of mEGFP expression in plant extracts" }, { "item": "QuantStudio 5 Real-Time PCR System", "supplier": "Thermo Fisher Scientific", "catalog_or_id": "A28140", "link": "https://www.thermofisher.com/order/catalog/product/A28140", "purpose": "qRT-PCR and ChIP-qPCR analysis" } ], "estimated_direct_cost_usd": { "low": 45000, "high": 75000, "scope": "Includes molecular biology reagents, tissue culture supplies, antibodies, sequencing costs (ATAC-seq for 48 samples at ~$200/sample), and plant growth materials. Excludes personnel costs, major equipment purchases (assumes access to qPCR machine and plate reader), greenhouse space fees, and whole-genome sequencing for pan-genome analysis (assumes publicly available data)." }, "protocol_references": [ { "title": "Xi, Y. et al. (2009) Agrobacterium-mediated transformation of switchgrass and inheritance of the transgenes. BioEnergy Research 2:275-283", "use": "Establish baseline transformation protocol for switchgrass embryogenic callus culture, selection conditions, and regeneration media" }, { "title": "Buenrostro, J.D. et al. (2015) ATAC-seq: A method for assaying chromatin accessibility genome-wide. Current Protocols in Molecular Biology 109:21.29.1-21.29.9", "use": "ATAC-seq protocol adaptation for plant tissues to assess chromatin accessibility at candidate safe harbor loci" }, { "title": "Lee, K. et al. (2019) CRISPR/Cas9-mediated targeted T-DNA integration in rice. Plant Molecular Biology 99:317-328", "use": "Design of HDR donor templates and CRISPR/Cas9 strategy for precise transgene insertion in monocots" }, { "title": "Dong, N. et al. (2020) Validation of reference genes for gene expression studies in switchgrass (Panicum virgatum) by quantitative real-time RT-PCR. PLoS ONE 15:e0233662", "use": "Selection of validated reference genes for normalization of transgene expression across tissues and developmental stages" }, { "title": "Grewal, D. et al. (2021) Identification of safe harbor loci in rice genome by harnessing the property of zinc-finger nucleases to induce DNA damage and repair. Frontiers in Genetics 12:688597", "use": "Bioinformatic criteria for candidate safe harbor locus identification including synteny conservation, intergenic spacing, and regulatory element prediction" } ], "handoff_package_for_lab": [ "Bioinformatics dataset containing: (1) coordinates of 6-8 prioritized candidate safe harbor loci with flanking sequences (\u00b15kb) in switchgrass AP13 and Miscanthus reference genomes; (2) pan-genome alignment data showing conservation across accessions; (3) predicted regulatory element annotations; (4) existing RNA-seq and chromatin accessibility data if available", "Cloned and sequence-verified CRISPR/Cas9 constructs for each candidate locus containing sgRNA expression cassettes, Cas9 driven by maize ubiquitin promoter, and HDR donor templates with mEGFP-NOS cassette and locus-specific homology arms (800bp each side); provided as purified plasmid DNA (\u226510 \u03bcg each) and glycerol stocks of E. coli clones", "Standard operating procedures (SOPs) for: (1) switchgrass and Miscanthus embryogenic callus induction from immature seeds; (2) Agrobacterium-mediated transformation conditions optimized for each genotype; (3) junction PCR primer sequences and validation PCR protocols; (4) tissue sampling schedule across developmental stages with photographic guides for standardization", "Pre-negotiated sequencing service agreement for ATAC-seq (estimated 48-96 samples) including library QC requirements, sequencing depth (50M paired-end reads/sample), and data delivery timeline", "Material transfer agreements (MTAs) or seed acquisition documentation for switchgrass cultivars (Alamo, Kanlow, Cave-in-Rock) and Miscanthus genotypes to be used, with greenhouse space allocation confirmed for multi-generational plant maintenance" ] } ] }