Sigma® Life Science has applied the revolutionary CompoZr Zinc Finger Nuclease technology to create an unparalleled range of genetically modified mammalian cell lines for use in areas such as basic research, target validation, drug discovery and drug development.

CompoZr Oncology Disease Model Cell Lines

An individual patient’s response to therapy may vary depending on their unique genotype. To better understand the genetics of cancer, we have generated genetically-defined mutations in human cell lines that model patient-relevant genome alterations. Our oncology offerings focus on colorectal carcinoma using the human DLD1 and SW48 cell lines and on lung cancer using the human A549 cell line. These tools will enable researchers to study disease gene targets in an isogenic setting, under the endogenous promoter and enable better avenues for therapeutic research and drug screening.

Knockout Cell Lines Available

Posters

• An Application of Zinc Finger Nuclease Technology to Create Knockout Cancer Lines (914 Kb PDF)
• An Application of Zinc Finger Nuclease Technology to Create Multiple Complete Gene Knockouts in Polyploid Cancer Lines (1.08 Mb PDF)

Cell Culture Media Components

DLD1 cell lines

• R5886 – RPMI-1640 Medium
• F2442 – Fetal Bovine Serum
• G7513 – L-glutamine
• S8636 – Sodium pyruvate solution
• C6164 – Cell Freezing Media-DMSO

A549 cell lines

• N4888 – F-12 Ham’s Nutrient Mixture
• F4135 – Fetal Bovine Serum
• G7513 – L-glutamine
• C6164 – Cell Freezing Media-DMSO

SW48 cell lines

• D5671 – Dulbecco’s Modified Eagle’s Medium – high glucose
• F4135 – Fetal Bovine Serum
• G7513 – L-glutamine
• S8636 – Sodium pyruvate solution
• C6164 – Cell Freezing Media-DMSO

Advantages and Benefits

• Cell lines provide a clean gene knockout in human cells with complete and permanent loss of the protein
• Provided with the parental cell line, the set enables study of gene function in an isogenic setting
• Effective knockout of all alleles for polyploidy cell lines
• Disease-relevant mutations offer “mini-patients” for testing drug response, drug resistance and drug screening
• Accurately model target patient genotype based on literature reports
• Target ID and validation with complete loss of gene
• Allows researchers to conduct complicated genetic studies in human cell systems

Gene Modification

To generate knockout mutations of an endogenous gene, a pair of ZFNs were designed and transduced into the cells to make a specific double-strand break (DSB) in the coding region of the gene of interest. During the process of non-homologus end joining (NHEJ) to repair the DSB, imperfect repair results in cells containing mutations (insertions or deletions of DNA) that result in nonsense transcripts targeted for degradation, yielding a gene knockout phenotype.

Figure 1. (A) Each Zinc Finger Nuclease (ZFN) consists of two functional domains: A DNA-binding domain comprised of a chain of zinc finger modules, each recognizing a unique triplet (3 bp) sequence of DNA. Four to six zinc finger modules are stitched together to form a Zinc Finger Protein (ZFP), with specificity of ≥12 bp. A DNA-cleaving domain comprised of the nuclease domain of FokI is attached to the ZFPs. When the DNA-binding and DNA-cleaving domains are fused together, a highly specific pair of 'genomic scissors' is created that binds with 24-36 bp specificity of the ZFPs and cleaves the DNA. (B) The addition of zinc finger nucleases to the cell results in creation of a double-strand break at the target site. This double-strand break is repaired by one of two endogenous repair pathways, either the non-homologous end joining (NHEJ) or the homologous recombination (HR) pathway. NHEJ is used to create gene knockouts while HR is utilized for targeted integration.

Example of BAX Gene Knockout in all Four Alleles in A549 Lung Carcinoma Cells

Many cell immortalized cell lines (e.g. A549) exhibit polyploidy of gene alleles and the ZFN technology can be use to knockout a specific gene in all alleles of the cell.

Figure 2. Knockout of tetraploid BAX in A549 cells using ZFNs.

(A) Following treatment with a ZFN specific for BAX, a clone was isolated that contained a unique disruption in all four alleles of BAX. The DNA sequence of the wild type and four disrupted alleles are shown above.

(B) BAX protein concentration was measured in wild-type and knockout cell lines using an enzyme immunometric assay specific for BAX. For three unique clones, each with all 4 alleles disrupted, the measurement was below the lower limit of detection demonstrating that no BAX protein is produced when 4 out of 4 alleles are disrupted. Clones with 1, 2 or 3 of four alleles disrupted produce less BAX protein (The lower linear detection limit for the assay is at 15 pg).

Where to use CompoZr Disease Model Cell Lines

• High throughput screening for drug discovery
  • Screen large compound collections for drug candidates
  • Genotype-specific response on drug candidates
  • Novel mechanisms of drug action
• To accelerate the development of personalized medicine
  • Genetically defined cell lines
  • “Mini-patients” for testing drug response
  • Profiling patient genotypes with drug resistance
  • Identification of drug combinations to combat drug resistance
  • Target identification and validation
  • Profiling of target patient genotypes
• Drug repositioning for specific patient profiles
• Validate putative disease causing mutations and SNPs
• Identify new targets in specific patient genotypes
• Mechanistic studies of drug action
• Mechanistic studies of disease development, progression and remission
• Functional assays upon perturbation of specific pathways

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