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

Start your project now with our new Knock Out cell line offer
KO cell lines available from 10 weeks, starting at $9,950 / £7,500 / €8,750

Offer includes:

  • Sequence verified biallelic frameshift mutations*
  • 10 backgrounds to choose from

Get your quote by sending your enquiry to sales@horizondiscovery.com

Please include:

  • Your name, company name and shipping address
  • Gene ID (Ensembl)
  • Cell line chosen from: A549, CT26.WT, HCT116, HEK 293, Jurkat, K562, MCF7, RKO, SW48, THP-1

*The option to KO a gene with 3n is available for a supplemental fee (+10%). Higher copy numbers are outside of this offer.

T&Cs apply.

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

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