Zinc Finger Nuclease (ZFN)

Cell Lines for Cancer Research

CompoZr ZFN-modified Breast Cancer Cell Lines

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

Table 1. Human Breast Cancer Gene Knockout Cell Lines Created in MCF10A Cells

Knockout cell line sets for key genes associated with breast cancer and their specific product numbers are listed in the table below. Each set is sold as two vials – one vial of the genetically-modified cells and one vial of the control MCF10A isogenic parental cells. Click on the gene symbol for a link to Sigma’s YFG (Your Favorite Gene) website where you can obtain more detailed information for a specific gene.


Product No. Product Name Gene Symbol Gene/Protein Function
CLLS1042-1SET MCF10A CELLS CDH1 (-/-) CDH1 calcium-dependent cell adhesion
CLLS1044-1SET MCF10A CELLS GSK3B (-/-) GSK3B energy metabolism, neuronal cell development
CLLS1045-1SET MCF10A CELLS HER2 (-/-) ERBB2; HER2 receptor tyrosine kinase, cell signalling
CLLS1046-1SET MCF10A CELLS PTEN (-/-) PTEN tumor suppressor, cell cycle regulation, AKT signalling
CLLS1048-1SET MCF10A CELLS SYK (-/-) SYK immunoreceptor signalling, proliferation, differentiation
CLLS1049-1SET MCF10A CELLS TP53 (-/-) TP53 cell cycle, DNA repair, apoptosis
CLLS1051-1SET MCF10A CELLS EEF2K (-/-) EEF2K calmodulin signalling, regulates protein synthesis
CLLS1053-1SET MCF10A CELLS RICTOR (-/-) RICTOR cell signalling, cell growth regulation
CLLS1059-1SET MCF10A CELLS BCR (-/-) BCR kinase activity; translocation site for Philadelphia chrom
CLLS1060-1SET MCF10A CELLS CDC25B (-/-) CDC25B phosphatase that regulates cell cycle and mitosis
CLLS1061-1SET MCF10A CELLS H2AFX (-/-) H2AFX histone nuclear protein, chromatin compaction
CLLS1063-1SET MCF10A CELLS AKT2 (-/-) AKT2 oncogene, signal transduction for insulin receptor
CLLS1066-1SET MCF10A CELLS PARP2 (-/-) PARP2 regulation of differentiation, proliferation, tumor transformation
CLLS1069-1SET MCF10A CELLS APC (-/-) APC tumor suppressor; apopotosis, cell adhesion and migration
CLLS1075-1SET MCF10A CELLS ESR2 (-/-) ESR2 ligand activated transcription factor

Why use CompoZr Breast Cancer Cell Lines

  • Genetically-modified human cell lines with isogenic controls
    • Targeted gene knockouts, knock-ins and point mutations at endogenous loci
    • Heritable modification of the genome
    • Enables complicated functional genetics in a human cell system
  • Patient-relevant disease mutations
    • Introduce disease-relevant genes and mutations
    • Observe endogenous protein expression
    • Preserve upstream and downstream regulatory elements
  • Robust and reproducible results
    • Permanent gene modifications in genetically stable cell line
    • Consistent results from experiment to experiment

Where to use CompoZr Breast Cancer Cell Lines

  • Enable Basic research
    • Study gene function in a clean genetic system
    • Mechanistic studies of disease development, progression and remission
    • Functional assays upon perturbation of specific pathways
  • Accelerate Drug Discovery
    • Patient models for high throughput testing of drug response, drug resistance and drug screening
    • Target identification and validation
    • Optimization of lead molecules
    • Drug repositioning for application to new disease areas
  • Develop Personalized Therapy
    • Identify drug responsive and resistant genotypes in patients
    • Determine effective drug combinations to address drug resistance
    • Design targeted, less expensive, more successful clinical trials

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Gene Modification with Zinc Finger Nucleases

To generate patient-relevant mutations of an endogenous gene, a pair of CompoZr Zinc Finger Nucleases (ZFN) were designed and introduced into the cells to make a specific double-strand break (DSB) in the coding region of the gene of interest (Figure 1A). As the cell repairs the break through non-homologous end joining (NHEJ), imperfect repair results in mutations (insertions or deletions of DNA). This causes a frameshift mutation in the coding sequence, resulting in nonsense-mediated decay of the transcript, loss of protein expression and a gene knockout phenotype. Alternatively, a donor vector can also be introduced to drive homologus recombination (HR) to yield knock-ins or point mutations (Figure 1B).

Gene Modification using Zinc Finger Nucleases

Figure 1. Gene modification using CompoZr Zinc Finger Nucleases
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.

MCF10A-PTEN Knockout Cells: A Case Study for a Breast Cancer Cell Line

  • PTEN (phosphatase and tensin homolog) is a phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase and plays a key role in cell cycle regulation by dephosphorylating phosphoinositide substrates. It works as a tumor suppressor by negatively regulating AKT/mTOR signaling pathway (Figure 2).
  • PTEN gene mutations result in activation of phosphatidylinositol 3-kinase (PI3K) pathway, loss of cell cycle control and are related to development of cancers, e.g., HER2+ and basal-like subtype of breast cancer.1-3
  • To study the role of PTEN loss in breast cancer, we developed an MCF10A PTEN (-/-) cell line for all aspects of basic research and drug discovery programs.
  • PTEN protein expression is completely lost in the knockout clone (A2A9) vs. isogenic parental cells (Figure 3) providing an ideal cell line to study the PI3K/AKT/mTOR pathway in breast cancer.


  1. Marty B, Maire V, Gravier E, Rigaill G, Vincent-Salomon A, Kappler M, Lebigot I, Djelti F, Tourdès A, Gestraud P, Hupé P, Barillot E, Cruzalegui F, Tucker GC, Stern MH, Thiery JP, Hickman JA, Dubois T. Frequent PTEN genomic alterations and activated phosphatidylinositol 3-kinase pathway in basal-like breast cancer cells. Breast Cancer Res. 2008, 10:R101.
  2. Nahta R and O'Regan RM. Evolving strategies for overcoming resistance to HER2-directed therapy: targeting the PI3K/AKT/mTOR pathway. Clin. Breast Cancer 2010, 10:S72.
  3. Zhou J, Wulfkuhle J, Zhang H, Gu P, Yang Y, Deng J, Margolick JB, Liotta LA, Petricoin E 3rd, Zhang Y. Activation of the PTEN/mTOR/STAT3 pathway in breast cancer stem-like cells is required for viability and maintenance. PNAS 2007, 104:16158

PI3K/AKT/mTOR signaling pathway

Figure 2. PI3K/AKT/mTOR signaling pathway3. PTEN negatively regulates this critical pathway for controlling cell growth and division. Mutations in PTEN are associated with many types of cancer, including HER2+ and basal-like breast cancer.
Treatment of MCF10A cells with a ZFN specific for the PTEN gene

Figure 3. . Following treatment of MCF10A cells with a ZFN specific for the PTEN gene, a clone (A2A9) was isolated that contained a unique biallelic disruption of the genomic sequence. PTEN protein concentration was measured in the wild-type (WT) and knockout cell lines using an enzyme immunometric assay specific for PTEN. Whereas PTEN protein levels increased with increasing amounts of WT lysate added to the assay, PTEN protein remained below background levels in the ZFN-modified cell line. The dotted red line is the lower limit of detection for the PTEN protein.

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We recommend the following culture media components for use with our cell lines:


MCF10A cell lines
  • 51448C – Dulbecco’s Modified Eagle's Medium/Ham’s Nutrient Mixture F-12
  • H1270 – Horse Serum
  • C8052 – Cholera Toxin (Vibrio cholerae)
  • I9278 – Insulin solution human (recombinant)
  • E9644 – Epidermal Growth Factor human
  • H6909 – Hydrocortisone solution
  • G3126 – L-glutamine
  • C6164 – Cell Freezing Media-DMSO
For more information on these or any of our
ZFN-modified cell lines please contact us.

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