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Creating Better Gene-Edited Cell Lines with the FAST-HDR System
This document outlines the Fast-HDR system developed by ExpressCells for creating gene-edited cell lines, detailing its phases from design to cell culture. It emphasizes improved efficiency and customization in generating knock-in and knock-out cell lines using CRISPR techniques and proprietary plasmid systems. Case studies demonstrate the application of these advanced cell lines in oncology and drug candidate impact analysis.
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Creating Better Gene-Edited Cell Lines with the FAST-HDR System
- 1. Welcome! Creating Better Gene-Edited CellLines with the FAST-HDR System Jon Weidanz, MPH, PhD North Texas Genome Center University of Texas at Arlington Founding Director Oscar Perez-Leal, MD Assistant Professor Pharmaceutical Sciences Department Temple University School of Pharmacy
- 2. Better knock-ins, bettercell lines, better science.
- 3. Types of Projects •Core technology: knock-in cell lines built through antibiotic selection and proprietary plasmid system • C-terminal tagging • Overexpressing cell lines • Point mutations—knock-out target portion of gene and replace with no sequence • Can also produce knock-outs using standard CRISPR techniques
- 4. Phase 1: DesignWork The process starts by analyzing the customer request to ensure it is feasible. Factors that we analyze are: • Size of the genetic insertion • Location in the gene • Is the gene expressed by the particular cell line? • Is the gene essential? If the project is feasible, a molecular biologist will design both a CRISPR and the insertion plasmid, which will include the targeted insertion, an antibiotic selection gene, and often, a gene coding for a tag. 3
- 5. Phase 1: DesignWork Phase 2: Bacteria Work ExpressCells uses E. coli to produce the CRISPR and insertion cassettes 4
- 6. Phase 1: DesignWork Phase 2: Bacteria Work CRISPR Antibiotic Cell Pool (Polyclonal) Single Cell Dilution Phase 3: Cell Culture Homozygous Heterozygous Heterozygous We then culture the cells. This includes: • Insertion of the plasmids into the cell (via electroporation or transfection) • Introduction of the antibiotic to remove cells without the genetic change to create a cell pool • Single cell dilution to identify clones
- 7. Phase 1: DesignWork Phase 2: Bacteria Work CRISPR Antibiotic Cell Pool (Polyclonal) Single Cell Dilution Homozygous Heterozygous Monoclonal ? ? Phase 3: Cell Culture Homozygous Heterozygous Heterozygous
- 8. C-tagging Knock-in CellLines in < 14 Days Create donor vector: 1 d Isolate pure, modified cells: ≈ 10 d FAST-HDR Create donor vector: 21 d Isolate pure, modified cells: 4 – 8 weeks Conventional methods
- 9. FAST-HDR Plasmid VectorSystem Tag P2A Res. gene Left recombination arm Right recombination arm Left recombination arm Right recombination arm Synthetic or PCR-generated DNA Synthetic or PCR-generated DNA ccdB Tag P2A Res. gene ccdB Directional cloning site Directional cloning site + donor HDR vector Tag marker P2A Resistance gene Puromycin 6- H I S mClover 3 6- H I S mRuby3 6- H I S mTagBFP2 6- H I S Luciferase Zeocin™ Blastocidin C B A D
- 10. ExpressCells Live cells Cellmicroscopy Direct image analysis (0 min) Traditional Screening Live cells Additional staining (20 – 60 min) 1st Ab (1 – 16 h) Cell fixing Cell permeabilization Ag blocking (60 – 90 min) Cell microscopy 2nd Ab (1 – 6 h) Triple-Labeling With FAST-HDR Obviates the Need for Immunofluorescence and Staining
- 11. Endogenous, Single GeneLabeling Tagging With mRuby3, Selected With Zeocin™, Day 14 Following Transfection
- 12. Double Gene Labeling TaggingWith mRuby3 + mClover, Selection With Zeocin™ + Puromycin
- 13. Triple Gene Labeling ATP5BTagging With mTagBFP2, β-tubulin With mClover3, Histone H3.3 With mRuby3
- 14. Flexible Approach Mitochondria Red: H3.3-mRuby3 Green:β-tubulin Spindle poles Red: H3.3-mRuby3 Green: β-tubulin Nucleus Red: H3.3-mRuby3 Green: β-tubulin
- 15. Two Case Studies Problem:Does a New Oncology Agent Inhibit Mitosis? Solution: Create cell line where cell division is readily identified via blue tag for protein expressed only during mitosis Problem: Identify Impact of a Drug Candidate on Organelles 14 Solution: Insert three genes that clearly identify the cytoskeleton, nucleus, and mitochondria
- 16. Better High-Content ImagingWith the FAST-HDR Plasmid Vector System • Longitudinal compound library screening • Better target- or phenotype-based hit identification • Longitudinal cell-based toxicology assays • Longitudinal study of inhibitors of intracellular signaling pathways • Discrimination among protein sequence variants • Defining protein–membrane and protein–protein interactions without fixation and staining • Rapid homozygous tagging of target genes
- 17. Better knock-ins, bettercell lines, better science. xpresscells.com info@xpresscells.com +1 (484) 483-6759
- 18.