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HORIZON DISCOVERY
New Cellular Reporter Technologies Using X-MAN® Cell Models
Paul Morrill1, Chris Torrance1, Christine Schofield1, Holly Astley1, Sue Rigby1, Tom Henley1, Claire Mahoney1, Paul Gonzales2, Bernardo Chavira2, Mei Cong3, Danette Daniels3, Hélène Benink3, Nancy Murphy3 and Jeff Kelly3
1Horizon
Discovery Ltd, Cambridge, UK
2TGen
Drug Development, Scottsdale, Arizona, USA
Introduction
X-MAN® (gene-X Mutant And Normal) 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 (Figure 1). These models combine X-MAN® cell lines with the
endogenous gene reporting capabilities in the form of NanoLuc® luciferase and HaloTag®
reporter technologies.
3Promega
Corporation, Madison, Wisconsin, USA
X-MAN® HaloTag® Reporter Technology
Mutated gene-X
X-MAN® Nano-Glo®: 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 (Figure 2). Further applications
include investigating changes in protein dynamics in response to the cellular environment or
targeted therapies.
(A)
(C)
(D)
K-Ras
HaloTag® WT
Silver stain
Figure 2. 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.
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e [email protected]
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Horizon Discovery, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom
X-MAN-Glo®
X-MAN® tumorigenic cell lines can be used for in vivo profiling studies. To further extend this
capability, we have introduced a luciferase marker into X-MAN® 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 6. DLD1 PI3Kα (E545K) knock-out xenografts. Firefly luciferase (Luc2) has been introduced into XMAN® 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).
K-Ras
HaloTag® WT
cRaf
65-75
kDa
Hif1α transcription
Cell biomass
Figure 5. 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.
(B)
(B)
Hif1α transcription (Luciferase RLU)
Hif1α transcription (Luciferase RLU)
(A)
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.
SW48 K-Ras HaloTag®
Mutant genotype: Isogenic background
K-Ras G13D
K-Ras G12V
SW48 Parental
Single Missense
Mutation
Figure 3. An overview of the
HaloTag® reporter
technology. Many
functionalities are possible
from a single fusion
construct.
SW48 HaloTag®
Figure 1. An overview of the gene
engineering technology. A virallymediated homologous
recombination based technology
that is highly efficient in performing
gene-targeting in somatic human
cell lines.
Normal genotype: Isogenic background
K-Ras G12D
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 (Figure 3). 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.
• X-MAN® Nano-Glo®: NanoLuc® luciferase reporters, with the key application in primary highthroughput screening and also further applications in protein degradation and stability
studies
• X-MAN® 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
Wild type gene-X
K-Ras G12C
WB against
cRaf
Figure 4. 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) KRas 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.
X-MAN® HaloTag® and
Nano-Glo® lines in
development
A range of reporter lines are currently
being generated and will be available by
Q2/Q3 of 2012.
Genotype
Cell Line
NanoLuc® promoter reporter
Hif1α
NanoLuc® protein reporter
HaloTag® promoter reporter
HaloTag® protein reporter
NanoLuc® promoter reporter
NRF2
NanoLuc® protein reporter
HaloTag® promoter reporter
HaloTag® protein reporter
β-catenin
NanoLuc® promoter reporter
NanoLuc® protein reporter
K-Ras
HaloTag® promoter reporter
p21
NanoLuc®promoter reporter
cMyc
NanoLuc® promoter reporter
Gli1
NanoLuc® promoter reporter