CompoZr^® Cellular Reporters

• CompoZr Cellular Reporter Cell Lines
• Human EGFR Live Cell Fluorescent Biosensor Assay

 CompoZr Cellular Reporter Cell Lines

The study of gene function has long been limited by the use of over-expression systems and by tedious staining procedures. To enable scientists to study protein function and protein localization in a live cellular system, we have generated engineered human cell lines where important proteins are fluorescently tagged at the endogenous locus through the use of CompoZr ZFN technology.

For example, CompoZr Zinc Finger Nucleases were used to fluorescently tag the α-tubulin isoform 1b (TUBA1b) in MCF10A cells. In the example, we show how we tagged the TUBA1b gene (Figure 1), the result of tagging the TUBA1b gene (Figure 2) and functional data showing the result of treating these live cells with paclitaxel (Figure 3).

Figure 1. Schematic of the tagged locus showing ZFN binding sites / ZFN cut site with respect to the targeted integration site. The Donor (top) has the homology arms of indicated length and the fluorescent protein (FP) sequence (green).
 

Figure 2. (A) MCF10A immortalized breast epithelial knock-in cell line expressing the TUBA1B gene (α-tubulin isoform 1b) endogenously tagged with RFP at the N-terminus. Cells were imaged live in Hanks Balanced Salt solution, (Cat. No. H8264), supplemented with 2% Fetal Bovine Serum, (Cat. No. F2442), using RFP filterset (ex 530-560/em 590-650) and 40x/1.3 oil objective. (B) Differential interference contrast (DIC) microscopy of the same cells seen in the fluorescence microscopy image.
 

Figure 3. Using MCF10A RFP-TUBA1B cell line to study Paclitaxel effect on microtubules. Paclitaxel is a mitotic inhibitor used in cancer chemotherapy. Paclitaxel is thought to stabilize microtubules and as a result, interfere with the normal breakdown of microtubules during cell division. RFP tagged TUBA1B MCF10A cells were imaged by differential interference contrast (DIC) and fluorescence microscopy while applying 20 µM Paclitaxel. As time progressed, typical tubulin bundles are formed.
 

Product Offering for Cellular Reporter Cell Lines

(For a shipping request outside of the USA, please contact us.)
 

Resources on CompoZr Cellular Reporter Cell Lines

• Frequently Asked Questions
• Posters

 Human EGFR Live Cell Fluorescent Biosensor Assay

We offer a novel, robust live cell assay for measuring endogenous human EGFR activity using a fluorescent GFP-tagged EGFR-specific SH2 domain protein. This modified A549 cell line provides an innovative and sensitive research tool for studying the molecular mechanism and kinetics of endogenous receptor activation and internalization for EGFR (ERBB1).
 

• Benefits of EGFR Biosensor Cell Line
  
• Applications to Your Research
  
• Live Cell Imaging of the Human EGFR Biosensor
  
• Poster
• Publication

Benefits of EGFR Biosensor Cell Line

• Live cell assays to study EGFR kinetics
• Specific to EGFR activity
• Robust and rapid assays with high sensitivity
• Economical for rapid cell processing and data analysis
• Adaptable to multiple HCS platforms including 384-well plates
• Stable expression of GFP-tagged biosensor
• Endogenous EGFR expression and activity

Applications to Your Research

• Live high content screening for both wild-type or mutant EGFR activity
• Live cell imaging to follow receptor kinetics of activation and internalization
• Ideal for high throughput screens

Live Cell Imaging of the Human EGFR Biosensor

The fluorescent EGFR Biosensor is stably expressed in A549 human lung carcinoma cells and is ideal for real-time live cell imaging of ligand binding and receptor activation and internalization. Activity is easily quantified by image analysis to count cytoplasmic granules following receptor internalization. These cells also provide a rapid and sensitive method for high throughput screening of drug libraries to identify compounds that modulate EGFR activity – both activators or inhibitors.

Figure 1. EGFR Biosensor Utilized in A549 Live Cell Assay – A. The structure of the EGFR biosensor construct consisting of the TagGFP green fluorescent protein and two tandem SH2 domains from adapter protein Grb2. B. EGFR activation and internalization in A549 cells. The EGFR biosensor plasmid was transfected into the A549 cell line, the transfected cells treated with 100 ng/mL EGF to activate EGFR and then the internalization process was observed in real time by fluorescence microscopy. The increasing internalization of the biosensor-EGFR complex over time can be quantitated using granularity analysis.
 

The EGFR Biosensor assay is highly selective for ligands that bind to ERBB1. EGF stimulation activates the receptor-biosensor interaction as initially observed by clustering of the biosensor at the cell surface within minutes of treatment. This is followed by receptor internalization which appears as dense fluorescent granules in the cytoplasm of the cell. Receptor activation can be blocked by specific inhibitors of ERBB1 (e.g., AG1478) and shows no activity for ligands specific for HER2 and HER 3 (e.g., heregulin-1β).

Figure 2. Specificity of A549 EGFR Biosensor Activity – EGF (100 ng/mL) stimulated EGFR (ERBB1) internalization while Tyrphostin AG1478 (1 µM), a selective inhibitor of EGFR, blocked receptor translocation to the plasma membrane and internalization. Hepatocyte growth factor (HGF, 100 ng/mL), a ligand specific for HGFR has low affinity for EGFR and showed much less activity than EGF. Bar = 10 µm.
 

Poster

• Genetically Encoded, SH2 Domain-Based Fluorescent Reporters of Endogenous Receptor Tyrosine Kinase Activity in Living Cells (PDF)

Publication

• Domain-based biosensor assay to screen for epidermal growth factor receptor modulators in live cells.
• Dmitry Malkov, John Fetter, Nathan Zenser, and Keming Song. A Novel Biosensor Assay for Detecting Activation of Endogenous EGFR in Living Cells. Biowire, Spring 2011, p.24-27.

For more information on these or any of our modified cell lines please contact us.