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CRITERIA FOR Frameworks. Shows G Protein-Directed Wonders Cooperations With Other Flagging Frameworks Mammalian Cell Clonal or Ingrained Nonmalignant/Euploid Complete Genome Now or Soon Imperative Mass and Homogeneity Capacity to Adjust Quality Expression

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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 as well as Pathological Interest Access to Normal Counterpart

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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

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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

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Example – cAMP Double Ligand Screen

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LPA Ter Gs a Gs a Gs an Adenylyl Cyclase Adenylyl Cyclase Adenylyl Cyclase Adenylyl Cyclase –Centric Model

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Expression of Membrane-Bound Adenylyl Cyclase Isoforms in B Cells Isoform PCR item 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 Gs a

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Approaches for Addressing Hypothesis Pharmacological Inhibitors Knock Out Mice AC1, AC3, AC5, and AC8 have been made Antisense or RNAi No isoform particular operators are accessible AC3, AC4, AC6, AC7, AC9 communicated in B cells Life of B cells in Culture too Short BCL-2 Mice WEHI B Cell Line Unsuccessful

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Experience with AfCS Cell Preparations

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Is it Time to Reconsider our Choice of cells? Are there Better Cells that are More "Tentatively Friendly"? What are the Criteria for the Choice of a New Cell Type?

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Major Criteria for Cell Selection Possess Interesting (Physiological, Biochemical, or Behavioral) yields 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, and so on.) Developed by AfCS Laboratories

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

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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 answered to work

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J774A.1 Macrophage Line Derived from Derived Female BALB/c Mice Cells are equipped for 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

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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

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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

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

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

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Testing New Cell Lines Plan 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 "Significant Criteria"

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Expression of Proteins Transfection versus Contamination Promoters, Delivery Vehicle, Approaches Viral Vectors (Retrovirus, Lentivirus) Assess Efficiency (% of cells) Assess Expression Levels (sum/cell) Is Sorting/Selection Required

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Manipulating Gene Expression Will RNAi Work in these Cells? Think about siRNA and Pol III Promoter Constructs Plasmid-versus Viral-Based Delivery

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Richness of Signaling Inputs Expression of Receptors Literature Search Test Ligands for "Ordinarily Expressed" GPCRs Affymetrix Chip Analysis, Most Complete Mouse Chip Available.

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

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

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

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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(?)

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Is a New Cell Line Better Suited for this Project? How Quickly Can this be Assessed? The amount Infrastructure Need be Redeveloped, and How Long Will this Take? In the event that a Change is Made, When Can We Start the Ligand Screens?