2008/3/131楊文欽 (中央研究院生物農業科學研究所籌備處)電話 : 02-278888340 (Office)E-mail : wcyanggate.sinica.edu.twWebsite:http:/abrc.sinica.edu.tw/abrc_C/people/look_1.php?id=wcy實驗藥學實驗藥學The use of mouse models for pharmacological studies(模式鼠在藥學的應用模式鼠在藥學的應用)時間: 96/3/21 (星期三) 早上3-4節(10:10-12:00)及下午5-8節(13:30-17:20) 地點:研究大4458室及各實驗室2008/3/132Mouse models1. Natural occurring mouse models NOD mice; db/db mice (leptin receptor mutation)2. Genetics modified mouse models 2-1. Gene targeting (Knockout, conditional KO, Knockin) : 2-2. Transgenesis (Transgenic mice expressing WT gene, DN gene and siRNA)2-3. ENU mice 2-4. Nuclear Transfer (Stem cell nuclear transfer (SCNT), Fertilized egg nuclear transfer)Reference:1.Animal models: Jackson lab (www.jax.org ) and Charles River Lab, Inc. (http:/www.criver.com/)2.Manipulating the mouse embryo Brigid Hogan et al.3.Mouse genetics and trangenicsI.J. Jackson et al4.Journal of biochemistry and molecular biology (2004) 37:1215.Journal of Immunology (2007) 178:6984-69936.Toxicology and Applied Pharmacology (2007). 219(1). 54-61Outlines2008/3/133Genetics approaches1. Forward genetics (順向遺傳學)1. Forward genetics (順向遺傳學):為傳統的遺傳學，由(突變株的)性去找出控制此性之基因的方法。如ENU mutation, gene trapping, insertional inactivation.2. Reverse genetics2. Reverse genetics (逆向遺傳學逆向遺傳學):為現代分子遺傳學，由已知基因序，再去研究這基因功能的方法。KO, Tg2008/3/134Advantage : 1. easy breeding2. short lifespan3. multiple pulps4. easy husbandry5. ideal mammalian model.Use : 1. Basic studies on gene regulation and function2. Studies on human disease and its progression3. Pharmacological/ therapeutic studies4. Drug discovery and target identificationProperties and applications of mice in medical research2008/3/1351. Gene targetingKnockout, conditional KO, Knockin2. Transgenesis-Transgenic mice expressing WT gene, DN gene and siRNA3. ENU mice 4. Nuclear TransferStem cell nuclear transfer (SCNT)Fertilized egg nuclear transferHow to engineer mice2008/3/136+Day 1 : PMSG injection Day 3 : HCG injectionXXDay 4 : mating foster motherWith vasectomized males+Day 4 :isolation of oocytes andmicroinjection of DNA DNA+Day 5 :oviduct transferto pseudopregnant fostermotherDay 25 :birth ofoffspringDay 46 :tail tippingand Southern blotJackson et al. Mouse genetics and Transgenics p232Generation of transgenic mice (1982)2008/3/137Transgenic vectorpCAS-mCD3-delta-N3flag-LZ6647 bpBGH pAN3flagLZmCD 3-dalta-enhancermCD 3-dalta-promoterNeomycinAmpicillinSV40 pASV40Sp6ColE1Kpn I (40)Not I (2158)Sca I (6205)Xho I (1071)Dra III (312)BamH I (432)BamH I (2004)EcoR I (426)EcoR I (655)Hind III (926)Hind III (1299)Xma I (1056)Xma I (3292)Bgl II (1065)Bgl II (1650)Nde I (25)Nde I (2734)Sac I (1827)Sac I (2206)Sal I (4458)Sal I (6646)Spe I (18)Spe I (2727)Xba I (30)Xba I (2739)Pst I (1609)Pst I (1660)Pst I (3534)2008/3/138Transgenic procedure2008/3/139Transgenic mice expressing RNAi (2003)promoterRNAi2008/3/1310ES cellsElectroporation G418 selectionX+/+Chimeric+/-+/-+/+/+-/-Generation of knockout mice (1985-1986)KO construct2008/3/1311Knockout construct2008/3/1312Homologous recombination2008/3/1313Conditional knockout (1992)2008/3/1314Cre LoxP systemFigure 1. A model of Cre Function. The loxP sites recognized by Cre are represented with thick arrows and their DNA sequence is shown at the bottom.2008/3/1315Cre LoxP systemThe Cre-LoxP system. The loxP site can be inserted on either side of a piece of DNA. (A) If Cre recombinase is then expressed in the cell, the loxP site will be cut and joined together, removing the piece of DNA between the two sites. (B) Since the loxP site is directional, it can also be used to invert pieces of DNA which are between it. Here two loxP sites which are inserted on either side of a piece of DNA in opposite directions. Once Cre is expressed, the loxP sites are both cut and the piece of DNA inverted and reattached.2008/3/1316Cre/lox regulation of gene expressionFigure 2. Cre/lox Regulation of a Protein Expression. The gene lacZ has LoxPsequences containing a stop signal that prevent the gene from being expressed. When exposed to the Cre protein the LoxP and stop signal are excised and the gene is expressed. Conditions in which the cre is present thus regulated the expression of the lacZ gene.2008/3/1317Tissue-specific gene KOFigure 3. Cre/lox Mouse Breeding. Mice with the Cre protein expressing in a specific cell type are bred with mice that contain a target gene surrounded by loxP sites. When the mice are bred, the cells carrying Cre will cause those cells to lose the target gene.2008/3/1318Knockin constructCell, Vol. 97, 767777, June 11, 19992008/3/1319ENU mice 2008/3/1320Stem cell nuclear transfer (SCNT)Fertilized egg nuclear transferNuclear Transfer2008/3/1321Nuclear Transfer2008/3/1322Nuclear transfer2008/3/1323Usefulness of Tg animalProducing pharmaceuticals, useful enzymes and proteinsGene farm to produce meatsBreeding of high-quality animalsEngineering grafted organs for humans : hDAF tgScientific researchesUsefulness of ko animalProducing pharmaceuticalsProducing useful enzymes and proteinsGene farm to produce meatsBreeding of high-quality animalsEngineering grafted organs for humansScientific researchesUsefulness of nuclear transferProducing exact clones (for medical research,)Producing tissues or organs with identical genesStudies on diseasesApplications of genetically modified animals2008/3/1324Example 1Evaluation of Bu-Zhong-Yi-Qi-Tang on the protection against ListeriaImmunopharmacology (1998) 215-1132008/3/1325Listeria invasionMolecular Medical Microbiology (2002) chapter 69 by Max Sussman2008/3/1326Immunity to ListeriaMAct. MEpitheliaCD8 TKillingCD4 TMNKIFN EpitheliaBloodIntestinesListeria2008/3/1327HOT enhances Listeria clearanceHOT Oral Adm.7 daysLMPlating assay for bacteria countBALB/c2008/3/1328+HOT Oral Adm.7 daysBALB/cKilling activity(phagocytosis)2008/3/1329Up-regulation of IFN- in IELs2008/3/1330Decrease in bacteria number of PP and MLN by HOT is abolished in IFN-g KO mice2008/3/1331Example 2Evaluation of Celebrix using TRAMP miceA prostate mouse model which can spontaneously develops prostate cancer. Better than tumor-bearing mouse model (Current Opinion in Immunology 2004, 16:143150).TRAMP miceTransgenic adenocarcinoma of mouse prostate (TRAMP) model is transgenic for the SV40 large T Ag (Tag) under the control of the rat probasin regulatory elements. Test-tube for anti-prostate cancer drugsCancer Research (2004) 64: 3334-33432008/3/1332Safety/toxicity testCancer Research (2004) 64: 3334-33432008/3/1333Cancer Research (2004) 64: 3334-33432008/3/1334Cancer Research (2004) 64: 3334-33432008/3/1335Cancer Research (2004) 64: 3334-33432008/3/1336Cancer Research (2004) 64: 3334-33432008/3/1337Example 3 XenoMouseJ. Immunol. Methods 231:11-23Mice can generate human antibody in mice. These antibody can be used as prophylactic or therapeutic antibody2008/3/1338Figure 7-4 Germline organization of human Ig loci.Downloaded from: StudentConsult (on 16 December 2005 11:16 AM) 2005 Elsevier Germline organization of human Ig loci2008/3/1339Wen-Chin YangBCR repertoire2008/3/1340J. Immunol. Methods 231:11-23Generation of XenoMouseDouble KODouble Tg2008/3/1341J. Immunol. Methods 231:11-232008/3/1342Generation of mAbs against human protein 2008/3/1343Biologicals are 12% of prescription drugsB: biological drugs (protein or peptides); N: natural products; ND: derivatived from natural products; S: synthetic; S/NM :synthetic using natural product mimic; S* : totally synthetic but pharmacophore is from a natural productNewman et al. J. natural product. 66: 1022 (2003)2008/3/1344Example 4 : Discovery for best-selling drugs using KO mouse modelsNature Review : drug discovery (2003) 2:382008/3/1345Innovator targets/proteins2008/3/13462008/3/1347Vioxx (Merck) withdrawn from market in 20042008/3/13482008/3/1349Bidens pilosa2008/3/1350Natural products and its derivatives comprise 28% of prescription drugsB: biological drugs (protein or peptides); N: natural products; ND: derivatived from natural products; S: synthetic; S/NM :synthetic using natural product mimic; S* : totally synthetic but pharmacophore is from a natural productNewman et al. J. natural product. 66: 1022 (2003)2008/3/13512008/3/1352The role of a novel polyacetylenic glucoside, cytopiloyne, in preventing the onset of non-obese diabetesWen-Chin Yang (楊文欽楊文欽)Institute of BioAgricultural ScienecsAcademia Sinica2008/3/1353Lab directionsI. Characterization of immunomodulatory phytocompounds from herbal medicines.1 flavonoid : 2 patents pending3 polyacetylenes : 1 patent pending, Planta Medica, JBSII. Role of signaling molecules in T cells.Wed like to identify novel genes involved in T cell function and immunity using a combination of methods, including microarray, 2D/MS, immunology tools etc. On the flip side, these significant genes may be used to screen immunomodulatory compounds .2008/3/1354Why develop autoimmune diseases drugs ?1. Autoimmune diseases are the third largest category of illness in the industrialized worldbehind heart disease and cancer.Autoimmune diseases include rheumatoid arthritis, asthma, psoriasis, Type I diabetes, multiple sclerosis, asthma, lupus, Graves and inflammatory bowel diseases, Sjogrens syndrome and scleroderma.2. Over 20 million people are afflicted with autoimmune diseases. The medical expense in USA per year is estimated to $20 billion per year and grow at a rate of over 15%. 3. Few drugs (immunosuppressants and immunomodulatros) are specific for autoimmune diseases.1. http:/www.the-infoshop.com/study/tv10893_autoimmune_disease.html2. http:/www.aegis.com/news/bw/1996/bw961114.html 3. New Eng. J.Med.340, 253 (Jan. 28, 1999).Autoimmunity is a specific cellular or humoral immunity to constituents of the bodys own tissues.2008/3/1355Example 1 : characterization of phytocompounds against intracellular bacteria.MAct. MEpitheliaCD8 TKillingCD4 TMNKIFN EpitheliaBloodIntestinesListeriaImmunity to listeria2008/3/1356Strategy1. To screen phytocompounds which can boost IFN expression.2. to test if this compound can prevent or treat listeria infection.pIFNJurkat cellsPHALuc 024681012141618NSPHACHM1CHM2DLRphytocompoundA flavonoid , Centaurein2008/3/1357Flavonoid from Bidens pilosa can increase IFN transcription and its upstream regulator, T-bet, in cord blood CD4+ T cellsGAPDHIFN-T-betGAPDHIL-4GATA-3NPHAcentaureinNPHAcentaureinA.B.1 3.26 3.971 2.61 4.811 3.92 1.191 0.86 1.632008/3/1358020406080100012345678910Days after infection% Cumulative survival rate01020Centaurein(g/i.p.)Centaurein can protect C57BL mice from Listeria infection 2008/3/1359Conclusions We identified an flavonoid compound, centaurein, from Bidens pilosa using T cell-based transcription. This flavonoid can upregulate transcription of IFN and T-bet. This flavonoid can protect mouse from Listeria infection.2008/3/1360Example 2 : characterization of phytocompounds for autoimmune diseasesTNF/IL-2IFN Cellular immunityImmunopathologye.g. Autoimmunity(NOD, RA, EAE, colitis)Humoral immunityImmunopathologye.g. asthma, allergy, atropyTh0Th1Th2IL-4IL-5IL-10IL-13IL-4T, NK, MastEosinophilIL-12M, DCTh1-mediated NOD Th2-mediated asthm2008/3/1361Type I diabetes/IDDM/NOD1. 194 million of DM patients. 2 to 5 % (4 -10 million) of DM patients = type I diabetes or IDDM.2. an autoimmune disease whose autoreactive T cells can destroy insulin-producing islet cells is characteristic of hypoinsulinoma and hyperglycemia. 3. IDDM in patients share many immunopathological and genetic features with NOD mice, a useful mouse model for IDDM pathogenesis.4. CD4+Th1 cells are the key players for NOD though CD8+T, B, M, and DC cells may be involved. Depletion (CD3 mAb) or inactivation (Immunosuppressants) of T cells can prevent NOD.5. Current treatments for IDDM: insulin injection, gene therapy, cell therapy and a combinatorial treatment ( cells & FK506).1. Habeck, M. Nat Med 9, 1228 (2003).2. Boyle, J.P. et al. Am J Epidemiol 149, 55-63 (1999).3. Kikutani, H. et al. Adv Immunol 51, 285-322 (1992). 4. Atkinson, M.A. et al. & Maclaren, N.K. N Engl J Med 331, 1428-1436 (1994); Nat Med 5, 601-4 (1999); Lancet 358, 221-9 (2001). 2008/3/1362Treatments for Type I diabetesImmunotherapy - immunosuppressant (CsA, FK506) - T cell depletion (-CD3)- NFB-deficient DC1. Atkinson, M.A. et al., Lancet 358: 221 (2001). 2. Obrien, B. et al., J. path. 191:86 (2000).3. Miller, B.J. et al. JI 140:52 (1988) 4. Kawamoto, S. et al. Intl. Immunol. 13:685 (2001)5. Ma, L.L, Diabetes 52:1976 (2003) 6. Rabinovitch, A. et al. Diabetes 51:638 (2002)7. Peck, A.B. et al., Ann. Mes. 33:186 (2001) 8. Bach, J.F. Arthritis Res. 4:S3-S15 (2002) cellsImmune system (T, NK,.)Insulin therapyCell therapy (stem cell, cell, islet, pancreas) Gene therapy (IL-4, IL-10, Insulin)Combinatorial therapy -pancreas transplantation + immunosuppressantsChemical therapy :- Nicotinamide ( cell maturation)- Rapamycin/sirolimus (an inhibitor for IL-2 signaling) +IL-2- Tenideap (anti-inflammation)- Ciamexon (B cell inhibitor)- Linomide (immunomodulator), - Vanadate (PTK inhibitor)- Vitamin D32008/3/1363Cytopiloyne was identified from Bidens pilosa L.1. Asteraceae herbs2. Distributed in tropical or subtropical areas.3. Folk medicine for diabetes, gastrointestinal diseases, inflammation and bacterial infection or malaria.Bidens pilosa L.Marles, R.J. & Farnsworth, N.R.,. Phytomedicine 2, 137-189 (1995).Chih HW, Lin CC, Tang KS. Am J Chin Med 1995; 23: 273-278.Brandao MG, Krettli AU, Soares LS, Nery CG, Marinuzzi HC. J Ethnopharmacol 1997; 57: 131-138.Geissberger P, Sequin U. Acta Trop 1991; 48: 251-261.Pereira RL, Ibrahim T, Lucchetti L, da Silva AJ, Goncalves de Moraes VL. Immunopharmacology 1999; 43: 31-37.2008/3/1364T cell differentiationPHA +IL-12+-IL-4PHA + IL-4+-IL-12IFN-FITCIL-4-PE010210410210401021041021040102104102104Th0Th2Th1PHA + IL-12 +-IL4PHA + IL-4 +-IL12IFN-producing cellsIL-4-producing cells10-12%45-60%phytocompound2008/3/1365Q1 : Can cytopiloyne prevent the onset of non-obese diabetes in NOD mice?2008/3/1366048121630InsulitisDiabetesBirthDeathWkNOD mouse modelInsulitis (4 wk)Diabetes (12 wk)-Urine (polyuria)-Hyperglycemia & glucosuria-Water drinking (polydypsia)NODTh1-mediated autoimmune disease-Hunger(polyphasia)Castano L, Eisenbarth GS. Annu Rev Immunol 1990; 8: 647-679.2008/3/13670102030405060701 2 3 4 5 6 7 8 9 101112131415161718192021222324252627282930Control (10/15)CP(ip) (0/12)CP(im) (0/6)FK 506(ip)(1/6)Diabetes incidence(%)Age of mice (weeks)048121630InsulitisDiabetesBirthDeathCytopiloyne, IP, dieb. alt.Wk202428Cytopiloyne can prevent the onset of non-obese diabetes (NOD)2008/3/1368050010001500Serum glucose(mg/dl)PBS(DM) CP(ip) CP(im) *0100020003000Serum insulin (pg/ml)*PBS(DM) CP(ip) CP(im) Cytopiloyne maintains normal levels of glucose and insulin in the serum2008/3/1369Cytopiloyne decreases cell death and the infiltration of CD4 cells into cell islets8 wk 12 wk 30 wkPBS (DM-)PBS (DM+)CP 2008/3/1370How can cytopiloyne prevent NOD ?ThImpairment-kiss of death(up-regulation of death ligands)Killing -Th1 cells-Th1 cytokines cells2008/3/1371Q2 : Can cytopiloyne skew T cell differentiation ?2008/3/1372TNF/IL-2 IFN NOD/IDDMRA, Crohns colitisTh0Th1Th2IL-4IL-5IL-10IL-13IL-4T, NK, MastEosinophilIL-12M, DCHelper T cell subsets and autoimmune diseasesAsthmaAllergyKatz JD, Benoist C, Mathis D. Science 1995; 268: 1185-1188.Toyoda H, Formby B. Bioessays 1998; 20: 750-757 Abbas AK Murphy KM Sher A Nature 1996; 383: 787-793T-bet, Egr GATA-3, c-Maf, NFAT, Stat6, JunB, NIP45 and Itk2008/3/1373Cytopiloyne suppresses Th1 cell differentiation but promotes Th2 cell differentiationTh1 differentiationTh2 differentiation0 1011030 1011030 1011030 101103IL-4-PEIFN- -FITC5 g5 g7.5 g7.5 g10 g10 g10110110110110110110110110310310310310310310310360%45%26%15%38%31%13%49%0.3%0.3%0.1%0%0.3%0.4%0.1%0.2%0 g0 g0 1011030 1011030 1011030 1011032008/3/1374IFN NOD/IDDMTh0Th1Th2IL-4 ?The possible mechanism by which cytopiloyne drives Th0 cell differentiation into Th2 cells IgEIgG1T-bet GATA-3 ?IgG2c/IgG2aCytopiloyne2008/3/1375Q3 : Can cytopiloyne induce T cell apoptosis ?2008/3/1376CD4CD80 101 1030 101 1030 101 1030 101 10339.2%6.9%21.8%18.9%Cell numberCell numberPBSCPCPPBSCytopiloyne depletes the proportion of CD4+T cells but not CD8 +T cells of the pancreatic lymph nodes0 101 1030 101 1033.3%0.8%Cell numberPBSCP01020304050PBSCPPercentage(%)*CD4CD8CD4 (PI)2008/3/1377Cytopiloyne depletes the proportion of CD4+T cells but not CD8 +T cells in the spleensPercentage(%)051015202530PBSCPCD4 CD8*2008/3/1378The mechanism by which cytopiloyne induces T cell apoptosisCD4+ T cells cells/other ?CytopiloyneFasLTNF 2008/3/1379Q4 : Can cytopiloyne down-regulate theexpression level of CD3 and CD4 but not CD8 molecules?2008/3/13800 100 101 102 103CD3P+I0 100 101 102 1030 100 101 102 1031 g0 100 101 102 1032.5 g5 gCell numberMocktreatment2008/3/13810 100 101 102 1030 100 101 102 103CD4P+I0 100 101 102 1035 gCell number2.5 g0 100 101 102 1031 gMockTreatment2008/3/1382P+I0 100 101 102 1032.5 g0 100 101 102 1030 100 101 102 105 gCD8Cell numberMockTreatment2008/3/1383Q5 : Can cytopiloyne enhance insulin production?2008/3/1384Cytopiloyne can enhance insulin expression in the cell line.01234LG2.5 g10 g15 gPromoter activity (AU)Insulin promoterControl5 gCell numberInsulin23.4%55.7%0 100 101 102 1030 100 101 102 103CP2008/3/13850 101103 0 101103 0 1011030 101103 0 101103 0 101103Cell numberInsulin2.33.55.192.511.6DMSOLGHG2.5 g5 g10 gLG: 3.6 mM glucose; HG: 16.7 mM lCytopiloyne can enhance insulin expression in mice primary -islet cells.2008/3/1386Cytopiloyne can lower blood sugar in the fasting mice.02040608010001246hrBlood glucose (mg/dl)Control25 g/ml CP10 mg Glibenclamide2008/3/1387The possible mechanism by which cytopiloyne can prevent NODCytopiloyneTh1 cellsTh2 cellsDepletion of CD4+T cellsNOD development cells