the cre luc reporter m ouse m odel

The CRE Luc Reporter M ouse M odel A transgenic bioimaging mouse - PowerPoint PPT Presentation

The CRE Luc Reporter M ouse M odel A transgenic bioimaging mouse model to assay ligand activation of GPCRs Greg Polites Immuno-Inflammation TSU, Sanofi Pharmaceuticals Inc. Bridgewater, NJ Keystone Symposia: G Protein-Coupled Receptors

  1. The CRE Luc Reporter M ouse M odel A transgenic bioimaging mouse model to assay ligand activation of GPCRs Greg Polites Immuno-Inflammation TSU, Sanofi Pharmaceuticals Inc. Bridgewater, NJ Keystone Symposia: G Protein-Coupled Receptors February 20, 2012

  2. The CRE Luc mouse model background and objectives Gi Gq CRE-luciferase reporter system Gs ai b CRE promoter is responsive to the activation of CREB via aq b g as b g - g the cAMP or PLC pathway + PLC AC Luciferase reporter expression is modulated to reflect cAM P DAG IP3 GPCR activity through a transcriptional readout Ca 2 + PKC PKA Assay can be used for all 3 GPCR classes: Gs ,Gi and CREB luci CaM K indirectly Gq Caliper IVIS Lumina Bioimaging Real-time in vivo imaging utilizes the light emitted by a bioluminescent reporter gene (luciferase) expressed in vivo Allows for quantification of the signal non-invasively Temporal and spatial data can be collected from the same animal which reduces variation and allows each animal to be CCD its own control Optics Bioluminescent Source 2

  3. The CRE Luc mouse model background and objectives M odel goal: Combine a GPCR reporter system with real-time in vivo bioimaging to assay GPCR ligand receptor interactions in primary cells, tissues or live animals. Same reporter system utilized for both in vitro and in vivo assays Profiling of compounds selected from in vitro assays for rapid PK/PD CRE Luc mouse models support rapid application to ligand:receptor pharmacological assays in vitro GPCR ligand interactions can be assayed in a native system avoiding difficult to transfect primary cells and engineered cell lines M odel application: The CRE Luc model has broad applications to GPCR ligand and receptor interactions. Addresses the transition from cells to animal model profiling of leads in GPCR drug development Activity of selected compound in vivo (PK, PD), Source of cells for tissues or whole body imaging in vitro assays Clinical Development In vitro Assay Dev In vivo Target ID 3

  4. CRE Luc Reporter Mouse Model Application Strategy Starting with a variety of luciferase expression profiles, pilot studies defined the model’s potential impact on drug development projects. Typical pilots started with CRE Luc primary cell responses followed by in vivo experiments CRE-Luc Feasibility Liver Adipose T,B,macs. Kidney Lung CNS Pancreas profiles No-Go Go Specific pilots to reach project application decision No-Go Go No-Go 64 187 229 219 175 11, 16 Go Project Tg lines for model applications application 4

  5. Studying the GPCR cAMP signaling pathway using CRE Luc mouse CRE-Luc In vivo In vitro Ex vivo Baseline imaging Compound dosing Compound dosing Tissue homogenates Re-imaging Luciferase assay Radiance/RLU IVIS bioimaging Microplate reader Fold induction Whole live animal imaging Sensitive, accurate Simple, quick Better organ resolution Limited resolution Time-consuming Next set of slides demonstrates this diversity of data with isoproterenol 5

  6. Isoproterenol in vivo response in CRE Luc Response to Isoproterenol in line 187 with CNS predominate luci expression Treatment: isoproterenol, 10MPK, ip Imaging at T=0 and 5 hours Statistically significant increase in quantitative CNS response over baseline BRAIN brain 15X *** 500000 400000 p/s/cm2 300000 T=0 200000 100000 0 baseline isoproterenol treatment ROI SPINAL CORD spinal cord 15X 200000 *** 150000 T=5hrs p/s/cm2 100000 50000 0 baseline isoproterenol treatment 6

  7. Isoproterenol ex vivo (brain slice) response in CRE Luc line 187 Compound induced changes in luciferase levels in brain slices can be detected and quantified by bioimaging Gs agonist: isoproterenol signal is diminished by Gi agonist AM N087 Strategy to identify the region specific expression of the transgene and drug interaction CH1_DATA(cpm) Forskolin 50uM 16000 15000 14000 13000 photo photo Slice 1 12000 1.93x 11000 10000 9000 8000 7000 6000 0 1440 2880 4320 5760 7200 8640 10080 11520 12960 14400 baseline baseline CH1_DATA(cpm) Vehicle (DMSO) 20000 18000 16000 14000 12000 Slice 2 10000 8000 6000 ISO 1um ISO + AMN 1um 4000 2000 0 0 1440 2880 4320 5760 7200 8640 10080 11520 12960 14400 7

  8. Isoproterenol response of CRE Luc primary neurons (and Gs or Gi agonist profiles) Gs: ADR β 1/ 2 Gs: ADR 1/ 2 , Gs: DRD, Gs: DRD, • E18, d 3 cor t i ca l • E14 , d 4 st r i a t a l n e u r on s n e u r on s • t -t e st v s m e d i a dopamine isoproterenol • t -t e st v s DMSO • 5 h ou r t r e a t m e n t • 4 h ou r t r e a t m e n t 3X 11X 4000 * * * * * 550 500 * * p<0.01 450 3000 400 * * * p<0.0001 350 cps cps 300 2000 250 200 1000 150 100 50 0 0 DMSO 10uM media 3uM 10uM [isoproterenol] [dopamine] •E18, d3 cortical neurons •E18, d3 cortical neurons Gi : Gi : mGluR7 , A , AMN082 N082 Gi : CB1 , Gi : , •10µM forskolin/ 10µM rolipram •10µM forskolin •8 hour treatment •4 hour treatment CP5 5 ,94 0 CP5 5 , 4 0 •t-test vs Forskolin Rolipram •t-test vs Forskolin 10uM F 10uM F/ 10uM R 35000 ns * * p<0.005 30X 20% ↓ 30000 ns 9X 150000 25000 * p<0.05 39% ↓ 38% 40% 45% ↓ * * p<0.005 20000 cps 100000 * * * * * * * p<0.0005 cps 15000 10000 50000 5000 0 0 media DMSO F 1nM 10nM 100nM 1uM DMSO F/R 100nM 1uM 10uM [AMN082] [CP55940] 8

  9. Pancreatic specific induction of luciferase by a GLP1 agonist Pancreatic specific induction of luciferase by the GLP1 agonist is blocked by streptozotocin treatment due to the destruction of β -cells 700 STZ increases blood glucose Glucose (mg/ dL) 600 500 Control 400 Basal Basal 300 STZ 200 100 0 0 2 4 Days Induction by Induction by GLP1 agonist Forskolin/ STZ (day 4) blocks the induction of luci rolipram by GLP1 agonist 1400 Fold induction 1200 Control Induction by GLP1 1000 STZ 800 after STZ treatment 600 400 200 0 Before After STZ treatment Control STZ 9

  10. GLP1 agonist induces luciferase expression mainly in the pancreas GLP1R found in multiple tissues, however Ex vivo assay on tissue homogenates compound activity is only seen in pancreas. Vehicle CRE Luc model defines the site of action for a Luciferase (RLU/ μ g protein) 700 GLP1 agonist (0.1 mpk) compound in vivo (rapid PK/PD). 600 Forskolin (5 mpk), rolipram (10 mpk) 500 Isoproterenol (20 mpk) 400 Glucose (1 g/kg) 300 200 100 0 10 6 *P<0.05, one-way ANOVA ** * 10 5 Fold induction * 10 4 * * 10 3 * * * Compound dependent patterns of luciferase * * * 10 2 * * * expression, suggesting that pancreas- * 10 * specific activity of the GLP1 agonist is 1 unlikely an transgenic artifact. 0 Strong induction in the pancreas by the GLP1 agonist, isoproterenol, and forskolin plus rolipram was observed. 10

  11. Pancreatic luciferase response in CRE Luc-Ins2 Akita mice Ins2 Akita is an autosomal dominant mutation that causes early onset hyperglycemia in the absence of obesity, due to a missense mutation resulting in mis-folding of proinsulin and death of β cells. Crossed CRE Luc with Ins2 Akita (FVB/ N background) to see if CRE-Luc induction is correlated with β cell function in this T1DM model. 8-week old mice were subject to baseline imaging on day 1 and treatment with GLP1 agonist (0.1mpk, sc) followed by re-imaging at 4 hr on day 2. Akita/+ Akita/+ WT Akita/+ Akita/+ 600 500 Fold induction 400 WT 300 Akita/+ 200 * 100 0 Male Female *P < 0.05, Akita/+ vs WT Females Males Two-way ANOVA model. Decreased CRE Luc induction by the GLP1 agonist (0.1 mpk, sc, 4 hrs) in the highly diabetic male mice. This effect was not significant in the less diabetic female littermates. In vivo signals were confirmed by ex vivo luciferase assay in a subset of animals. 11

  12. Summary From initial studies, we have demonstrated the utility of the CRE Luc model to profile compounds in whole animals, tissue extracts, slices, and primary cells in vitro. Profiling responses for various GPCRs have been tested in the following combinations Gs agonist: In vitro with microglia, neurons, cardiomyocytes, M EFs and brain slices In vivo in the pancreas, brain, spinal cord Gs antagonists: In vitro: microglia, neurons, and T cells In vivo: brain, spinal cord Gi agonists: In vitro: neurons, Tcells, brain slices Gi antagonists: In vitro: neruons, Tcells, brain slices 12 12

  13. Characterization of the CRE Luc lines Details of the profiling assays with the CRE Luc transgene have been summarized in a single table (available upon request) Eight CRE Luc lines are available through Taconic Biosciences 13

  14. Acknowledgements and Model Availability Immunology Experimental Pharmacology Holly Dressler (PTL, model generation, development, and applications) Fernando Camacho (psoriasis) Kyriakos Economides (psoriasis) Andy Giovanni (brain slices) Sarah Favara (linage profiling, CNS) Zhen Pang (diabetes, Metabolism) Nancy Wu (dibaetes, Metabolism) CRE-Luc model information Greg Polites: or CRE-Luc model availability Taconic Biosciences email: 14 14


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