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CRITERIA FOR SYSTEMS

CRITERIA FOR SYSTEMS. Displays G Protein-Regulated Phenomena Interactions With Other Signaling Systems Mammalian Cell Clonal or Inbred Nonmalignant/Euploid Complete Genome Now or Soon Requisite Mass and Homogeneity Ability to Modify Gene Expression

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CRITERIA FOR SYSTEMS

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  1. CRITERIA FOR SYSTEMS • Displays G Protein-Regulated Phenomena • Interactions With Other Signaling Systems • Mammalian Cell • Clonal or Inbred • Nonmalignant/Euploid • Complete Genome Now or Soon • Requisite Mass and Homogeneity • Ability to Modify Gene Expression • Known and Preferably Bidirectional Inputs • Quantifiable and Interesting Outputs • Developmental and/or Pathological Interest • Access to Normal Counterpart

  2. The Cardiac Myocyte • Regulated Contraction (Gs/Gi-coupled GPCRs) and Secretion in Culture • Adaptive (Hypertrophic) Responses (Gq-coupled GPCRs) • Many Cell-Surface Receptors and Signaling Pathways • Mouse Cardiomyocytes are Problematic • Quantity and Homogeneity of Cells

  3. The Splenic B Lymphocyte • Homogeneous Populations Isolated Easily • (3 Billion/Week) • Many Cell Surface Receptors and Signaling Pathways • Gi-Coupled GPCRs Mediate Directional Motility in Response to Chemokines • Mice with Deletions of Genes Encoding B Cell Signaling Molecules are Often Viable • 1-2 Day Survival in Culture • Availability of Immortal mouse B cell lines

  4. Example – cAMP Double Ligand Screen

  5. LPA Ter Gsa Gsa Gsa Adenylyl Cyclase Adenylyl Cyclase Adenylyl Cyclase Adenylyl Cyclase –Centric Model

  6. Expression of Membrane-Bound Adenylyl Cyclase Isoforms inB Cells IsoformPCR product AC 1 557 bp AC 2 351 bp *AC 3 691 bp *AC 4 293 bp AC 5 1113 bp *AC 6 649 bp *AC 7 393 bp AC 8 524 bp *AC 9 535 bp AC isoform 1 2 3 4 5 6 7 8 9 Gsa

  7. Approaches for Addressing Hypothesis • Pharmacological Inhibitors • Knock Out Mice • AC1, AC3, AC5, and AC8 have been made • Antisense or RNAi • No isoform selective agents are available • AC3, AC4, AC6, AC7, AC9 expressed in B cells • Life of B cells in Culture too Short • BCL-2 Mice • WEHI B Cell Line • Unsuccessful

  8. Experience with AfCS Cell Preparations

  9. Is it Time to Reconsider our Choice of cells? • Are there Better Cells that are More “Experimentally Friendly”? • What are the Criteria for the Choice of a New Cell Type?

  10. Major Criteria for Cell Selection • Possess Interesting (Physiological, Biochemical, or Behavioral) outputs • Possess Richness of Inputs (Ligand Responses) • Ability to Express (Ectopically) Sufficient Amounts of Proteins for Biochemical Analysis • Ability to Alter Gene Expression (RNAi Approaches) • Amenable to High Throughput Assays (Intracellular Second Messengers, Phosphoprotein, Microscopy, Transcript Profiling, etc.) Developed by AfCS Laboratories

  11. Testing New Cell Lines • Exploration of 7 Cell Lines • Assembled from Suggestions and Input by Steering and Systems Committees, as well as the General Membership. • “A” List: J774A.1, Raw 264.7, AtT-20 • “B” List: 3T3-L1, IC-21, Neuro 2a, N1E-115

  12. RAW 264.7 • Mouse Alveolar Macrophage Transformed by Abelson Murine Leukemia Virus • Cells are Capable of Chemotaxis, Pinocytosis, Phagocytosis, and Secretion • Capable of Antibody-Dependent Lysis of Sheep Erythrocytes • Differentiate into Osteoclast-Like Cells • Most Widely Used Macrophage Cell Line (Septic Shock, Lipidomics) • Diversity of Ligand Responses • Cytokines • Chemokines • Toll-Like Receptors • GPCRs • Transfection Studies Published • Transcript Profiles Published (LPS) • RNAi reported to work

  13. J774A.1 • Macrophage Line Derived from Derived Female BALB/c Mice • Cells are capable of Chemotaxis, Phagocytosis, Secretion (Including Autocrine/Paracrine Factors: NO, TNF- Alpha, IL-6, GM-CSF) • Foam Cell Formation • Respiratory Burst • Growth Inhibition • Diversity of Ligand Responses • Cytokines • Chemokines • Toll-Like Receptors • GPCRs • Transfection Studies Published • Infection with Lentivirus • RNAi Reported to Work

  14. AtT-20/ D16v-F2 • Mouse, Pituitary Tumor • ACTH Secretion • Epithelial Morphology • Subclone of AtT-20 – Grows as Monolayer • Reported to be Readily Transfectable • Many Ligand Responses (CRH, INE, IL1, IL2, TNF, IL6, INF and , PACAP, VIP, SST, ACh, Activin, ANP/CNP, LIF, Substance P, Glucocorticoids • Antisense Knock down works

  15. IC-21 • Murine Line Derived SV40 Transformed Peritoneal macrophage • Cells are Capable of Chemotaxis,Phagocytosis • Less Well Studied than the Other 2 Macrophage Cell Lines • Fewer Reports on Ligand Responses

  16. 3T3-L1 • Mouse Fibroblast • Well Studied • Transfection by Electroporation and Lipid-Mediated Delivery • Will Differentiate into Adipocyte (IBMX, Insulin, Glucocorticoids), but Preparation is Heterogenous

  17. Neuroblastoma Lines • N1E-115 • Mouse Neuroblastoma • So-called “Adrenergic Clone” Derived from C1300 Neuroblastoma (Nirenberg). • Will Differentiate into Neuronal Morphology and “Function” • Ligand Responses (GPCRs Plentiful) • Aneuploid (Modal Chromosome # = 192!!) • Neuro 2A • Mouse Neuroblastoma (Ruddle clone) • Diploid • Less Well Studied Neuroblastoma Line • Functional outputs: Ionic conductances

  18. Testing New Cell LinesPlan of Attack • 7 Cell Lines Obtained by Dallas Cell Lab (J774A.1, Raw 264.7, AtT-20, 3T3-L1, IC21, Neuro 2a, N1E-115) • Leading Candidate (J774A.1, Raw 264.7, AtT-20) Cells are Currently Growing at all Locations (SF, Stanford, Cal Tech, Dallas) • Rapid Assessment of “Major Criteria”

  19. Expression of Proteins • Transfection vs. Infection • Promoters, Delivery Vehicle, Approaches • Viral Vectors (Retrovirus, Lentivirus) • Assess Efficiency (% of cells) • Assess Expression Levels (amount/cell) • Is Sorting/Selection Required

  20. Manipulating Gene Expression Will RNAi Work in these Cells? Compare siRNA and Pol III Promoter Constructs Plasmid- vs. Viral-Based Delivery

  21. Richness of Signaling Inputs • Expression of Receptors • Literature Search • Test Ligands for “Commonly Expressed” GPCRs • Affymetrix Chip Analysis, Most Complete Mouse • Chip Available.

  22. Amenable to AfCS Assays • Microscopy • Morphology • Adherence to Desired Substrates • Performance of Subcellular Markers • Ligand-Induced Domain Translocations (PH) • Adequate Transfection Efficiencies and Levels

  23. Amenable to AfCS Assays • Phosphoproteins • Suitability of Existing Antibody Cocktails • Ligand-Induced Changes in Protein Phophorylation • Adequate Transfection Efficiencies and Levels for 32P Labeling/Pulldowns • Sufficient Material for Pervanadate or Ligand Stimulation/Affinity Purification/Protein ID

  24. Amenable to AfCS Assays • Second Messengers • Are Ligand-Induced Responses of Sufficient Magnitude? • Will Current Methodologies Work? • Are Responses Predicted by MA Analysis Present?

  25. Amenable to AfCS Assays • Additional Assays • Lipid Analysis – Are Cells Appropriate? Do Differentiated 3T3-L1 Present Problems? • Protein Traps – is Expression Level of Tagged-Proteins Sufficient? • Yeast 2 Hybrid – Additional Prey Libraries? • Chemotaxis • Phagocytosis, Secretion, Electrophysiology, Others(?)

  26. Is a New Cell Line Better Suited for this Project? How Quickly Can this be Assessed? How Much Infrastructure Need be Redeveloped, and How Long Will this Take? If a Change is Made, When Can We Start the Ligand Screens?

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