Cells and Cell Based Assays

CompoZr® Disease Models



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

Product No. Product Name
DLD1 Cell Line
Gene Symbol Gene/Protein Function
CLLS1001-1SET DLD1 CELLS BAX -/- BAX pro-apoptotic protein
CLLS1002-1SET DLD1 CELLS HIF1A -/- HIF1A transcription factor induced by hypoxia
CLLS1003-1SET DLD1 CELLS PIK3CA +/- PIK3CA class 1 PI 3-kinase catalytic subunit
CLLS1004-1SET DLD1 CELLS PTEN -/- PTEN tumor suppressor
CLLS1005-1SET DLD1 CELLS BAX/BAK (-/-,-/-) BAX / BAK pro-apoptotic proteins
CLLS1096-1SET DLD1 CELLS SMAD4 -/- SMAD4 transcription factor for TGF beta signaling
CLLS1127-1SET DLD-1 CELLS TP53 (-/-) TP53 tumor suppressor
CLLS1132-1SET DLD-1 CELLS AKT1 (-/-) AKT1 oncogene, serine-threonine protein kinase
CLLS1133-1SET DLD-1 CELLS AKT2 (-/-) AKT2 oncogene, serine-threonine protein kinase
 
Product No. Product Name
SW48 Cell Line
Gene Symbol Gene/Protein Function
CLLS1006-1SET SW48 CELLS BAX -/- BAX pro-apoptotic protein
CLLS1007-1SET SW48 CELLS TP53 -/- TP53 tumor suppressor
CLLS1009-1SET SW48 CELLS PTEN -/- PTEN tumor suppressor
CLLS1010-1SET SW48 CELLS SMAD4 (-/-) SMAD4 transcription factor for TGF beta signaling
CLLS1011-1SET SW48 CELLS BAX/BAK (-/-,-/-) BAX / BAK pro-apoptotic proteins
CLLS1098-1SET SW48 CELLS HIF1A -/-/- HIF1A transcription factor induced by hypoxia
 
Product No. Product Name
A549 Cell Line
Gene Symbol Gene/Protein Function
CLLS1013-1SET A549 CELLS SMAD4 -/- SMAD4 transcription factor for TGF beta signaling
CLLS1014-1SET A549 CELLS HIF1A -/-/- HIF1A transcription factor induced by hypoxia

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.

Disease Model Cell Lines Figure 1
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.

Disease Model Cell Lines Figure 2Figure 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

 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 Modified Cell Lines Created in MCF10A Cells

Modified 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. These cells are suitable for HTS/HCA and be used to identify lead compounds in the drug discovery process.
 

Product No. Product Name Gene Symbol Gene/Protein Function
CLLS1107-1SET
MCF10A CELLS B-RAF V600E B-RAF proto-oncogene, mutation associated with melanoma and colon cancer
CLLS1110-1SET
MCF10A CELLS SMO W535L SMO Hedgehog pathway, mutations associated with oncogenesis
CLLS1118-1SET
MCF10A CELLS MAP3K8 P461L MAP3K8 oncogene, C-terminal point mutation associated with breast cancer
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
CLLS1214-1SET
MCF10A CELLS HER2 (-/-) EXON 2 HER2 receptor tyrosine kinase, cell signalling
CLLS1046-1SET MCF10A CELLS PTEN (-/-) PTEN tumor suppressor, cell cycle regulation, AKT signalling
CLLS1199-1SET
MCF10A CELLS PTEN (-/-) PIK3CA H1047R (+/-) PTEN; PIK3CA proto-oncogene, mutation associated with breast and other cancers
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
CLLS1211-1SET
MCF10A CELLS PVRL4 (-/-) PVRL4 cell adhesion, tumor-associated antigen

 

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

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.

References

  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.