Cellular Reporter Technologies Using X MANâ„¢ Cell Models
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Email: [email protected] Web: www.horizondiscovery.com New Cellular Reporter Technologies Using X‐MAN™ Cell Models Paul Morrill 1 , Chris Torrance 1 , Christine Schofield 1 , Holly Astley 1 , Sue Rigby 1 , Tom Henley 1 , Claire Mahoney 1 , Paul Gonzales 2 , Bernardo Chavira 2 , Mei Cong 3 , Danette Daniels 3 , Hélène Benink 3 , Nancy Murphy 3 and Jeff Kelly 3 1 Horizon Discovery Ltd, Cambridge, UK 2 TGen Drug Development, Scottsdale, Arizona, USA 3 Promega Corporation, Madison, Wisconsin, USA Introduction X‐MAN™ (gene‐XMutant 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 GENESIS™ 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. Figure 1. An overview of the GENESIS™ gene engineering technology. GENESIS™ is a virally‐mediated homologous recombination based technology that is highly efficient in performing gene‐ targeting in somatic human cell lines. 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. 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. X‐MAN™ 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 (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. Figure 3. An overview of the HaloTag® reporter technology. Many functionalities are possible from a single fusion construct. 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. (D) 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) 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. Other in vivo models containing luciferase include the SW48 K‐Ras suite of mutations (including G12D, G12R and G12A). Normal genotype: Isogenic background Mutant genotype: Isogenic background Single Missense Mutation Wild type gene‐X Mutated gene‐X K‐Ras HaloTag® cRaf 65‐75 kDa Silver stain Figure 6. DLD1 PI3Kα (E545K) knock‐out xenografts. Firefly luciferase (Luc2) has been introduced into X‐MAN™ cell lines to allow non‐invasive radiance measurements. Images shown (left) are 11 days post infection. WT Genotype Cell Line Hif1α NanoLuc™ promoter reporter NanoLuc™ protein reporter HaloTag® promoter reporter HaloTag® protein reporter NRF2 NanoLuc™ promoter reporter 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 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. X‐MAN™ Nano‐Glo™: NanoLuc™ luciferase reporters, with the key application in primary high‐ throughput 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 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. K‐Ras HaloTag ® WT (C) (A) (B) 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. WB against cRaf K‐Ras G13D K‐Ras G12V K‐Ras G12D K‐Ras G12C SW48 K‐Ras HaloTag® SW48 Parental SW48 HaloTag® (A) (B) Hif1α transcription (Luciferase RLU) Hif1α transcription (Luciferase RLU) Hif1α transcription Cell biomass
Transcript of Cellular Reporter Technologies Using X MANâ„¢ Cell Models
Email: [email protected]: www.horizondiscovery.com
New Cellular Reporter Technologies Using X‐MAN™ Cell ModelsPaul 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 3Promega Corporation, Madison, Wisconsin, USA
IntroductionX‐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 withintargeted drug discovery with the development of new reporter disease models using our GENESIS™gene engineering technology (Figure 1). These models combine X‐MAN™ cell lines with theendogenous gene reporting capabilities in the form of NanoLuc™ luciferase and HaloTag® reportertechnologies.
Figure 1. An overview of the GENESIS™ gene engineering technology. GENESIS™ is a virally‐mediated homologous recombination based technology that is highly efficient in performing gene‐targeting in somatic human cell lines.
X‐MAN‐Glo™X‐MAN™ tumorigenic cell lines can be used for in vivo profiling studies. To further extend thiscapability, we have introduced a luciferase marker into X‐MAN™ cell lines, to allow non‐invasiveassessment of cancer progression in laboratory animals. Figure 6 shows images of the DLD‐1colon carcinoma line, which contains the heterozygous PI3Kα hinge domain mutation E545K,where the E545K mutation has been reverted to the wild type allele.
X‐MAN™ Nano‐Glo™: NanoLuc™ Luciferase ReportersNanoLuc™ luciferase is a novel, small, bright luciferase reporter. It is one third the size and 100 timesbrighter than the most commonly used luciferase, firefly. These features enable the development ofhomogenous assays for primary and secondary screening, reporting on pathways via theendogenous promoter only, exploited using X‐MAN™ cell lines.
NanoLuc™ luciferase reporter lines allow transcriptional reporter output of endogenouspromoter/response elements that drive NanoLuc™ expression (Figure 2). Further applicationsinclude investigating changes in protein dynamics in response to the cellular environment ortargeted therapies.
X‐MAN™ HaloTag® Reporter TechnologyHaloTag® reporter technology encompasses a recombinant protein tag that allows flexibilitybetween protein purification, expression and localization, protein interaction discovery, screeningand other functional analysis (Figure 3). The technology is based on the formation of a covalentbond between the protein tag and synthetic ligands, and is designed to enable understanding ofprotein function in a cellular and biochemical environment.
Figure 3. An overview of the HaloTag® reporter technology.Many functionalities are possible from a single fusion construct.
We have generated a suite of mutated K‐Ras SW48 cell lines each with a HaloTag® reporter toinvestigate localisation and protein interactions between wild type and K‐Ras G12C, G12V, G12Dand 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 andmutant cell lines by confocal microscopy and protein pull‐down.
(D)
Figure 4. HaloTag® reporter images and pull‐down. (A)‐(C) Confocal experiments using SW48 wild type K‐RasHaloTag® 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 theplasma membrane and cytoplasm whereas cytoplasmic and nuclear staining is seen in SW48 parental cellstransiently transfected with HaloTag® alone. (B) K‐Ras HaloTag® cells labelled with HaloTag® ligand (red) and aDNA stain (green). (C) K‐Ras HaloTag® cells labelled with HaloTag® ligand (red) and CellMask™ plasmamembrane stain (green). (D) Pull‐down experiments show enrichment of proteins for the K‐Ras HaloTag® ascompared to SW48 control cells. Enrichment of the known K‐Ras interactor Raf is detected using an antibodyagainst Raf. Scale bars = 20 µm.
Other in vivo models containing luciferase include the SW48 K‐Ras suite of mutations (includingG12D, G12R and G12A).
Normal genotype: Isogenic background
Mutant genotype: Isogenic background
Single Missense Mutation
Wild type gene‐X
Mutated gene‐X
K‐RasHaloTag®
cRaf65‐75kDa
Silver stain
Figure 6. DLD1 PI3Kα (E545K) knock‐out xenografts. Firefly luciferase (Luc2) has been introduced into X‐MAN™ cell lines to allow non‐invasive radiance measurements. Images shown (left) are 11 days post infection.
WT
Genotype Cell Line
Hif1α
NanoLuc™ promoter reporterNanoLuc™ protein reporterHaloTag® promoter reporterHaloTag® protein reporter
NRF2
NanoLuc™ promoter reporterNanoLuc™ protein reporterHaloTag® promoter reporterHaloTag® protein reporter
β‐cateninNanoLuc™ promoter reporterNanoLuc™ protein reporter
K‐Ras HaloTag® promoter reporter
p21 NanoLuc™ promoter reporter
cMyc NanoLuc™ promoter reporter
Gli1 NanoLuc™ promoter reporter
X‐MAN™ HaloTag® and Nano‐Glo™ lines in developmentA range of reporter lines are currently being generated and will be available by Q2/Q3 of 2012.
X‐MAN™ Nano‐Glo™: NanoLuc™ luciferase reporters, with the key application inprimary high‐throughput screening and also further applications in proteindegradation and stability studies
X‐MAN™ HaloTag®: Reporter disease models for secondary screening, with particularapplications including protein purification, pull‐downs and imaging studies
X‐MAN‐Glo™: For tagging existing X‐MAN™ cell models with luciferase for in vivoimaging applications
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 alinear relationship is observed. (B) Correlation of concentration of Actinomycin D, a non‐specific transcriptionalinhibitor, with luciferase signal. A classical dose response is observed.
K‐RasHaloTag® WT
(C)
(A) (B)
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.
WB against cRaf
K‐Ras G13D K‐Ras G12V K‐Ras G12D K‐Ras G12C
SW48
K‐Ras HaloTag®
SW48
Paren
tal
SW48
HaloTag®
(A) (B)
Hif1
αtran
scrip
tion (Lucife
rase RLU
)
Hif1
αtran
scrip
tion (Lucife
rase RLU
)
Hif1α transcriptionCell biomass
