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Developing Cellular Reporter Technologies Using Horizon's Cell Models

Jun 10, 2016 2:20:39 PM No Comments

Isogenic cell lines provide genetically defined, patient-relevant, predictive in vitro models of genetic disease. We are further extending their application within targeted drug discovery with the development of new reporter disease models using our gene engineering technology. These models combine Horizon's cell lines with the endogenous gene reporting capabilities in the form of NanoLuc® luciferase and HaloTag® reporter technologies.

  • NanoLuc luciferase reporters, with the key application in primary high-throughput screening and also further applications in protein degradation and stability studies
  • Halotag Reporter disease models for secondary screening, with particular applications including protein purification, pull-downs and imaging studies
  • X-MAN-Glo: For tagging existing X-MAN cell models with luciferase for in vivo imaging applications

NanoLuc Luciferase Reporters

NanoLuc luciferase is a novel, small, bright luciferase reporter. It is one third the size and 100 times brighter than the most commonly used luciferase, firefly. These features enable the development of homogenous assays for primary and secondary screening, reporting on pathways via the endogenous promoter only, exploited using X-MAN cell lines.

NanoLuc luciferase reporter lines allow transcriptional reporter output of endogenous promoter/response elements that drive NanoLuc expression. Further applications include investigating changes in protein dynamics in response to the cellular environment or targeted therapies.

Figure 1. NanoLuc® luciferase detection at endogenous expression levels using HCT116 Hif1α NanoLuc® promoter fusion cell lines. (A) Correlation of cell number with luciferase signal. Even at low cell numbers a linear relationship is observed. (B) Correlation of concentration of Actinomycin D, a non-specific transcriptional inhibitor, with luciferase signal. A classical dose response is observed.

HaloTag® Reporter Technology

HaloTag® reporter technology encompasses a recombinant protein tag that allows flexibility between protein purification, expression and localization, protein interaction discovery, screening and other functional analysis. The technology is based on the formation of a covalent bond between the protein tag and synthetic ligands, and is designed to enable understanding of protein function in a cellular and biochemical environment.

We have generated a suite of mutated K-Ras SW48 cell lines each with a HaloTag® reporter to investigate localisation and protein interactions between wild type and K-Ras G12C, G12V, G12D and G13D isogenic cell lines. These cell lines are identical except for the mutation of interest, which is expressed at the endogenous level. Figures 4 and 5 show analysis of the wild type and mutant cell lines by confocal microscopy and protein pull-down.

 

Figure 2. HaloTag® reporter images and pull-down. (A)-(C) Confocal experiments using SW48 wild type K-Ras HaloTag® cell lines show that K-Ras HaloTag® is present at the plasma membrane and in the cytoplasm. (A) K-Ras HaloTag® cells labelled with HaloTag® ligand show that the K-Ras HaloTag® fusion protein is localised to the plasma membrane and cytoplasm whereas cytoplasmic and nuclear staining is seen in SW48 parental cells transiently transfected with HaloTag® alone. (B) K-Ras HaloTag® cells labelled with HaloTag® ligand (red) and a DNA stain (green). (C) K-Ras HaloTag® cells labelled with HaloTag® ligand (red) and CellMask™ plasma membrane stain (green). (D) Pull-down experiments show enrichment of proteins for the K-Ras HaloTag® as compared to SW48 control cells. Enrichment of the known K-Ras interactor Raf is detected using an antibody against Raf. Scale bars = 20 µm.

 

Figure 3. SW48 mutant K-Ras HaloTag® variants. Confocal images of live cells labelled with HaloTag® ligand (red) and a DNA stain (green) show a similar pattern of localisation as for the wild type K-Ras HaloTag® fusion protein. Scale bars = 20 µm.

X-MAN-Glo®

Horizon's tumorigenic cell lines can be used for in vivo profiling studies. To further extend this capability, we have introduced a luciferase marker into Horizon's cell lines, to allow non-invasive assessment of cancer progression in laboratory animals. Figure 6 shows images of the DLD-1 colon carcinoma line, which contains the heterozygous PI3Kα hinge domain mutation E545K, where the E545K mutation has been reverted to the wild type allele.

Figure 4. DLD1 PI3Kα (E545K) knock-out xenografts. Firefly luciferase (Luc2) has been introduced into Horizon's cell lines to allow non-invasive radiance measurements. Images shown (left) are 11 days post infection.

Other in vivo models containing luciferase include the SW48 K-Ras suite of mutations (including G12D, G12R and G12A).

Click here to download the original poster version of this data

#Cell lines

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