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Cells and Cell Based Assays

CompoZr® Cellular Reporters



 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).

Bioascend. Achieve results with cell lines shaped for your research.

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).
 

Bioascend. Achieve results with cell lines shaped for your research.

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.
 

Bioascend. Achieve results with cell lines shaped for your research.

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.)
 

Product No. Cell Line Gene Target(s) Human Gene ID Protein Function
CLL1031-1VL U2OS GFP-TUBA1B TUBA1B 10376 Globular protein - microtubule dynamics
CLL1032-1VL U2OS GFP-ACTB ACTB 60 Cytoskeletal protein - cell motility
CLL1033-1VL U2OS GFP-LMNB1 LMNB1 3912 Nuclear protein - nuclear stability & gene expression
CLL1034-1VL U2OS RFP-TUBA1B TUBA1B 10376 Globular protein - microtubule dynamics
CLL1035-1VL U2OS RFP-ACTB ACTB 60 Cytoskeletal protein - cell motility
CLL1036-1VL U2OS GFP-HMGA1 HMGA1 3159 Non-histone protein - regulation of gene expression
CLL1037-1VL U2OS GFP-ACTB RFP-TUBA1B ACTB / TUBA1B 60 / 10376 Cytoskeletal protein - cell motility / Globular protein - microtubule dynamics
CLL1038-1VL U2OS BFP-LMNB1 RFP-ACTB LMNB1 / ACTB 3912 / 60 Nuclear protein - nuclear stability & gene expression / Cytoskeletal protein - cell motility
CLL1039-1VL MCF10A RFP-TUBA1B TUBA1B 10376 Globular protein - microtubule dynamics
CLL1135-1VL SKOV3 GFP-HER2 HER2 2064 EGF receptor 2 – forms heterodimers with other EGF receptors to mediate downstream signaling pathways
CLL1136-1VL U2OS GFP-NUP98 NUP98 4928 Nucleoporin 98kD protein – protein in nuclear pore complex
CLL1139-1VL SKOV3 GFP-STAT3 STAT3 6774 Transcription factor – regulates nuclear gene expression
CLL1140-1VL A549 RFP-STAT3 STAT3 6774 Transcription factor – regulates nuclear gene expression
CLL1141-1VL A549 GFP-EGFR EGFR 1956 EGF receptor 1 – cell surface protein, binds epidermal growth factor, dimerizes and stimulates downstream signaling to promote proliferation
CLL1143-1VL SKOV3 GFP-HER2 / RFP-EGFR HER2 / EGFR 2064 / 1956 EGF receptor 2 – forms heterodimers with other EGF receptors to mediate downstream signaling pathways / EGF receptor 1 – cell surface protein, binds epidermal growth factor, dimerizes and stimulates downstream signaling to promote proliferation
CLL1149-1VL A549 GFP-CTNNB1 / RFP-LMNB1 CTNNB1 / LMNB1 1499 / 3912 Beta-catenin – protein in adherens junction, involved in regulation of cell adhesion and growth / Nuclear protein – nuclear stability & gene expression
CLL1158-1VL A549 GFP-STAT1 STAT1 6772 Transcription factor – regulates nuclear gene expression
CLL1167-1VL A549 GFP-SMAD4 SMAD4 4089 Transcription factor – regulates nuclear gene expression
CLL1218-1VL U2OS LMNB1-TUBA1B-ACTB LMNB1 / TUBA1B / ACTB 3912 / 10376 /60 Nuclear protein – nuclear stability & gene expression / Globular protein – microtubule dynamics / Cytoskeletal protein – cell motility

 

Resources on CompoZr Cellular Reporter Cell Lines

 Human EGFR Live Cell Fluorescent Biosensor Assay

Sigma® Life Science offers 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).
 

Product No. Product Name Gene Symbol Gene/Protein Function
CLL1097-1VL A549 CELLS EGFR SH2 BIOSENSOR EGFR cell surface receptor for EGF, receptor tyrosine kinase regulates signal transduction for proliferation

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.

EGFR Biosensor Utilized in A549 Live Cell Assay
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).

Specificity of EGFR Biosensor Assay
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.
 

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