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MCB 130L Lecture 7

MCB 130L Lecture 7. Signal Transduction. This week’s lab. Objective : To examine the effects of various inhibitors and activators on IL-2 expression. Activators and/or inhibitors. CPRG. Assay for -galactosidase cleavage of CPRG. T-cell line containing NFAT Z. Cell Signaling.

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MCB 130L Lecture 7

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  1. MCB 130L Lecture 7 Signal Transduction

  2. This week’s lab Objective: To examine the effects of various inhibitors and activators on IL-2 expression Activators and/or inhibitors CPRG Assay for -galactosidase cleavage of CPRG T-cell line containing NFAT Z

  3. Cell Signaling • Synthesis of signaling molecule • Release/display of signaling molecule • Transport of signal to target cell • Detection of signal by receptor protein • Change in cell growth, differentiation, or metabolism Credit: Nicolle Rager, National Science Foundation

  4. Intercellular signaling Fig.20-1

  5. The Immune System

  6. Cytotoxic recognize antigens produced by the cell (e.g. viral proteins) interact with antigen-MHCI express glycoprotein CD8 kill infected cells Helper* recognize exogenous or ingested antigens interact with antigen-MHCII express glycoprotein CD4 stimulate T- and B-cell and macrophage proliferation and activity T cells T cell response is mediated by activation of the T cell receptor after binding the antigen-MHC complex with the help of CD4 or CD8

  7. T Cell Receptor (TCR) Signaling SIGMA-ALDRICH Ca2+ CRAC

  8. Reporter Genes Reporter genes can "report" many different properties and events: • strength of promoters • efficiency of gene delivery systems • intracellular fate of a gene product, a result of protein traffic • efficiency of translation initiation signals • success of molecular cloning efforts. Transcriptional reporter Translational reporter http://www.wormbook.org/chapters/www_reportergenefusions/reportergenefusions.html

  9. Commonly used reporter genes • LacZ: b-galactosidase • Indicator plates (X-gal) • Colorimetric assays • GFP • Fluorescence of live cells • Luciferase (firefly) • chemiluminescence • CAT: chloramphenicol acetyl-transferase • ELISA • Enzyme assay • Selection for resistance

  10. IL-2 Gene +1 +47 -286 -257 -256 -242 -208 -188 -158 -145 -93 -66 TATA NF-AT NFIL-2D NFIL-2C NFIL-2B NFIL-2A NFAT Z Construct -70 +1 +47 TATA lac Z TK promoter Hygromycin Resistance gene NF-AT NF-AT NF-AT IL-2

  11. Detection of ß-Galactosidase: ß-galactosidase hydrolyzes chlorophenol red galactoside (CPRG), turning it from yellow to purple at alkaline pH. The color changeprovides a quantitative measure of gene transcription during T cell activation.

  12. T-cell activators and inhibitors Activators Concavalin A (ConA)* - plant lectin that binds mannose residues - crosslinks T-cell receptors to activate PMA* - activates protein kinase C pathway Ionomycin* - Ca2+ ionophore Cyclosporin (CsA)* - interacts with cyclophilin - blocks calcineurin FK506 - interacts with FKBP - blocks calcineurin Rapamycin* - inhibits p70 S6 kinase phosphorylation and activation EGTA* - Ca2+ chelator Inhibitors

  13. T Cell Receptor (TCR) Signaling SIGMA-ALDRICH ConA Ca2+ EGTA CRAC PMA ionomycin CsA FK506 Rapamycin = inhibits IL-2 response

  14. Use of NFAT Inhibitors in HIV Research CsA and FK506 Inhibit Completed HIV-1 Reverse Transcription. HIV-1 challenge of CD4+ T cells was performed at 3 days post treatment and assayed after 6 days. Kinoshita et al. Cell 1998

  15. Counting cells with a hemacytometer yellow box = 1 mm x 1 mm x 0.1 mm = 0.1 mm3 =0.1 ml # cells per box = # cells/0.1 ml # cells/0.1 ml x 104 = # cells/ml *Calculate average of 4 boxes *Multiply by dilution factor 1 mm Trypan blue stains dead cells Depth = 0.1 mm 1 mm

  16. Aseptic Cell Culture Technique • Laminar Flow Hood • 70% Ethanol • Surface of hood • Bottles • Gloves • Autoclaved glass • Flamed metal • Opening of bottles, pipetting, etc. • Placement of equipment Contaminated cells

  17. Cell culture gone bad

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