斑马鱼实验手册

rηf(;nttA~Lrqa2008712.............................................................11........................................................................12..............................23UH--........31.3.131.3.243.3.........................................................................................4164.1Morpholino....61.3Rescue..71.4.481.5.........................91.5.1..................................................................................................95.2..........................................................95.3...101.5..................10............112.1..................................112.1...................................................................................112.1.2............................................................................122.1.3............132.1.4................................132.2..........142.2.1DNA........................................................142.2.2RNA................142.2.3nker-MediatedpeR................................................................................................142.3........172.3.1.2.5.1................................................222.5.2........................................................222.5.3.................................................................................232.6.................................................................242.6.1..........242.6.2......0............#•••••••••••••••••••••I....242.6.3I••••••••••••••••••••••••••I••~•..............................................252.6262.6.5..............................................................................272.7...........28293.1I•~•••••••I••••••••0I••I........................................................293.2I..................303.2.1ZFINZIRe303.2.23.2.33.3323.3.1GenBank.......................323.3.2GenBankRefSeq323.3.3................333UniPro333.5EnsemblVegaBioI\a3.5.1Ensemllbl..e........343.5.2Ensembl................................353.5.3VegaBioMart..................................................................................................................363.6............................................363.6.1..........................363.6.2373.6.3..101(ωf7υJiaωOMrmεmωrriaω0iωOωZzebebfiSP(Actinopterygii)(Cypriniformes)l(Cyprinidae)00ia)(K:mmet19531-1Core1-1103Tubing~nABL344-1.2O.7mml1-2j3(0\A7...8pf1-2Dahmetal.2005)28.5°CU1lpvageperiod2(hpLhourspostferation)516464X82.75hpf(5124.3hpflastulaperiod)5.5hpf(gastrulaperiod)9hpf10hpf(burl)12hpf(segmentationperiod)116hpf(1418hpf18020hpf24hpfo48hpf5dpfKimmelet01.1995.-id2·J1.3tRT-PCRy(expressionpattern)3situ)1.3.1i(insituhybridization)RNA)mRNARNA(DNARNA)(probe)(RNA)Unsitu)(radioisotopelabeling)(non-radioactivelabeling)digoxigeniDIG)(biotin)(fluorochrome)(colloidgold)u~9(DigitalisIf(UTP)DIG-I1-UTPUTPfrllcRNA(complementaryRNA)RNARNA(riboprobe)0RNARNA-RNADNA-RNADNARNA(DNARNADNARNAIvlll(horseradishperoxidaseHRP)DAB/H2024--1-/H202(alkalinephosphataseAPAKP)BCIP/N(BCIP:5--4--3-n5/nx-NBT:RNAp1-33.DIGIAntip(NET1-31.3.21.3.31985(Zhuet01.1985)StuartDNA(Stuartet01.1988)GFPOsRedmRFP1)0GFP(Aequoreavictoria)EGFPmRFP1OsRed(Ocosoma)1-4)4124hpf1-11-1kdrl(flk)flilαetslb(etsrp)sImo2gotolnppop10tryinS||LPα6iRB|35gcgαsstelov3(hue)icl1IJIlodlIkrox20egr2aegr2bII]51.4(knockout)knockdown)(rescue)(over-expression)(ectopicexpression)20085(Menget01.2008;Doyonet01.2008)(morpholino)RNARNAi)morpholino~1.4.1Morpholino(morpholinooligo)GeneTools((1)mQrpholino(translationinitiationinhibition)morpholinoATGillillpholinomorpholinomRNAmRNAmorpholinomorpholino(2)morpholino(splicinginhibition)morpholinomRNARNA(hnRNA)morpholino(3'1-56I!UIt;;j'ftl~5'-IJU-Y5'3'5'3'l1-5morpholinoiIJmorpholinodmorpholinoC3'L(dominantnegative)rnRNAmRNAωmRNAmRNART-PCRmorpholinomorpholinoSNPmorpholino2-3morpholinorllGeneToolsmorpholinomorpholinop53uetor2003)morpholinomorpholino1.4.3Rescueknock-downknock-downmorpholinorescue7knoclownrescuemRNAjknock-downmRNAmRNAknock-downrescuerescuemRNAmRNA5'I]'I'AmbionT3/T7mMESSAGEmMACHINE3'5'-UTR3'-UTRmorpholinorescuemRNAmorpholinomRNArescuemRNAmorpholinoGeneTools25morpholino4ATG5'-UTRKozak4G-3TAATG5'15bpT;-61-9IG-3I-6-9ICATGGCCGCCACC8GmorpholinomRN.L\morpholinorescueImorpholinorescueImorpholino1.4.4mRNABACmRNABACrescue8'1.5i~)txr1.5.1j(ethylmethanesulphonateEMS)(ethylnitrosouraENU)i;)jlIDNApDNA(genetrapping)J.&EGFP)DNAO(promotertrapping)(enhancertrapping)iIIR(splicing-mediatedtrapping)poly(A)(poly{A)trapping)exontrapping)in3(basalpromoter)jtBJ-iintrontrapping;(spliceacceptorSA)(polyA)SA(splicedonorSD)Poly(A)3'poly(A)poly(A)1.5.2Tol2DNADNAo9To/2Oryziaslatipes)rlIhAT(hobolActivatorITam3)(openreadingframeORF)To/2EGFPmRNA(mosaicfounder)1211.5.3To12RNADNA(MoloneymurineleukemiavirusMoMLV)iG(ωVeω5iCωωujlarstoma1latiti1000-2000IpFlII1.5.4EMSENUdimethvlsulfeDMSO)-.d102012.1.1(1)28.5°C(El14h/10h(2)10%NaCI450-500μs/cmpHNaHC037HoltfreiterBufferHoltuffer)E3Hank's(3)(brineshrimp)f4°e-20oe1L34gNaCI3.5g~28°e27~3010minIH7