Cell Line Engineering

Cell Line Engineering

Custom gene-edited cell lines with your chosen genotype

Overview

With nearly a decade of experience planning and executing nearly 3,000 gene editing experiments, access to a multiple genome editing technologies, and cell line development service options to suit a variety of experimental needs and budgets, Horizon is the global leader in cell line engineering.

  • Strong IP position to support your freedom to operate
  • Substantial experience with over 100 cell lines and 200 target genes
  • Emphasis on design to test your hypothesis, not just project execution
  • A collaborative approach including a dedicated project manager to ensure the project meets your needs
  • Development of genomically validated isogenic cell line pairs (all cell lines are provided with a non-edited control)
  • Initial characterization of parental cell line prior to gene-editing (if required)
Case Studies

Case study I: CRISPR-Mediated Homozygous Knockout

Purpose

Disruption of the gene X in the human melanoma A375 cell line (copy number = 3).

Workflow
  • Transfection with plasmids expressing Cas9 nuclease and a validated gRNA
  • Derivation of single cell clones
  • Screening of the clones
  • Genotype analysis by Sanger sequencing
Targeting Strategy

Targeting

  • Sequencing of individual alleles of 7 clones (via molecular cloning) demonstrated that 3 clones had out of frame deletions on all 3 alleles
  • Sequencing of transcripts from individual alleles indicated out of frame deletions were also present at the RNA level for two of the three clones

Clone 1

Case Study II: Heterozygous Knockin in Leukemia Cells

Purpose

To create a heterozygous point mutation K700E (AAA -> GAA) in SF3B1 in chronic myelogenous leukemia (CML) K562 cells using rAAV.

Workflow

  • Design of rAAV targeting vectors
  • rAAV infection
  • Plating of infected clones
  • PCR and Sanger sequencing to identify KI pools
  • Derivation of single cell clones
  • Screening of the clones by PCR
  • Genotype analysis by Sanger sequencing

Vector Design

Targeting

Targeted allele-specific sequencing shows that both clones contain the K700E mutation. Non-allele specific sequencing shows a ratio of 50:50 of the mutant to wild-type peaks, as would be expected for a 2N locus.

Project Plan

Phase I:

Cell Line Characterization

  • Revival, quality control and establishment of the cell line in routine culture
  • Single cell dilution test
  • Transfection/infection tests
  • Gene copy number analysis

Phase II:

Gene-editing Reagent Design

  • Designing, manufacturing and testing CRISPR gRNAs
  • Designing and manufacturing a suitable donor (e.g. AAV, oligonucleotide, plasmid) if needed
  • Designing a suitable screening strategy to identify correctly targeted cells

Phase III:

Cell Line Engineering

  • Introduction of the targeting event
  • Enrichment of the targeted cell population
  • Assessment of targeting in the pools of cells
  • Plating the cells to 96-well plates and growing them for screening
  • Screening followed by in depth genetic assessment to select correctly targeted populations
  • If required, single cell dilution of targeted populations to get targeted clones
  • Expansion of the selected clones and banking

Phase IV:

Cell Line Validation

  • Final sequence validation
  • Recovery viability testing
  • Sterility testing (bacteria and mycoplasma)

Phase V:

Project Delivery

  • Data pack summarizing the process and analysis of generated cell lines; up to 2 clones per cell line

Contact Us

iPSC

Create translational in vitro disease relevant models

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Immuno-oncology

See our extensive panel of pre-characterized immune cell lines

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CHO

Customized gene editing of the CHO genome to optimize your bioproduction platform.

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