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 Spring offer!
KO cell lines available from $12,500 / €11,130 / £9,500

Offer includes:

  • Sequence verified biallelic frameshift mutations*
  • 10 backgrounds to choose from
  • Offer ends 31,May 2019

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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