线虫

秀丽隐杆线虫的显微注射生殖腺阴孔肛门咽头肠交尾针交接蘘线Posteriorgonad(ventralnervecord,runsalongtherightofthemidline)AnteriorintestineLarvalstagesofdevelopment.A.L1larva.Theanteriorventralpairofcoelomocytes(cc)andtheMcellarelocatedontheright.Rectalepithelialcellsareinthemiddleplane,andVNCmotorneuronsarelocatedattheventralmidline.Theremainingcellsareasseenfromtheleftlateralside.VNCmotorneuronsaremorenumerousthanshown.(HSN)Hermaphrodite-specificneuron.B.DICimagesofeachstagelarva.Bar0.1mm.C-H.EnlargedDICimagesofgonadsofL1-adultstageanimalsrespectively.Sizesarenottoscale.ArrowspointtogonadsinD-F.(v)Vulva:(u)uterus.C.FourprimordialgonadcellsarelabeledinL1.D.GermcellsincreaseinnumberinL2.E.ThegonadissimilartoanearlyL2stagegonadindauer.Arrowheadpointstodauer-specificcuticle.F.ThegonadhasextendedalongtheventralbodyinL3.G.Hermaphroditesomaticstructureshaveformedbymid-L4stage.H.Vulvaisopentotheoutsideandtheuterusisfulloffertilizedeggsinadult.I.DICimageofL4tail,whichretainsship-likemorphologyinthehermaphrodite.AginginC.elegansYoungadultOldadult步骤准备挑虫匙准备NGMOP50平板（0.1~0.2ml/皿，涂布时勿接触皿边）观察卵、各期幼虫，成虫（可处死后观察）小量去污染/同步化法（2NNaOH9ml：NaClO1Ml，1滴）传代热激法产生雄虫挑取10只L4期雌雄同体个体放入培养皿30℃温箱8-12hr20℃3-4d挑取5-6只雄虫和1-2只雌雄同体放入杂交培养皿中20℃3-4d验证dpy-5的遗传规律操作步骤day1挑取4-5条野生雄虫，置于3.5mm培养皿内，与1条dpy雌虫交配day220h后，将雌虫置于另一培养皿中产卵6h，弃雌虫。day4约60h后，F1代发育成熟，挑取单条雌虫，接种于3.5cm的NGM上自交产卵day6产卵24h后，弃雌虫，继续培养3dday9所有F2代均发育至L4或成虫阶段，在解剖镜下，分别统计dpy和野生表型的数量，计算分离比dpy-5(e61)dyp：dumpy染色法鉴别秀丽隐杆线虫生命状态高级红墨水1.5mL离心管吸管或（200uL移液器和配套枪头）由于活细胞具有选择透过性，曙红、番红花和亚甲基蓝等染液以及红墨水等对细胞有害的生物染料不能透过细胞膜对活细胞进行染色，而死细胞的细胞膜遭到了损害，失去了选择透过性因此能被染色。利用200μLM9缓冲液冲洗NGM培养基，洗下秀丽隐杆线虫，分别置于1.5mL的EP管中，于1000r/min离心1min，弃上清。添加200μL红墨水，混匀，染色30min。于1000r/min离心1min，弃上清。M9缓冲液冲洗、离心，重复3次后，置于体式显微镜下观察并拍照。显微注射固定载片的准备4%琼脂糖置于65度水浴中。25uL置于载片中央。盖上另一片盖片或载片，不要产生气泡。轻压使得琼脂糖充满整个载片下方。1-2min后揭开载片。65度烘箱干燥1hr。注射针针尖直径约5um。装好注射针，小心使针尖与盖片边缘撞击，打开合适的开口。吸取注射液。固定线虫载片上滴加1滴矿物油将youngadult线虫放到油中轻压使其沾到固定垫上。动作要快。低倍镜下找到线虫调整线虫位置40×镜下聚焦生殖腺注射平面，将针尖插入生殖腺加压注射，可以看到液体进入生殖腺管内在线虫上滴加1滴M9，3-5min后，在解剖镜下挑取从矿物油中游到M9中的线虫，置于OP50培养皿中RescuingamutantphenotypeDNAthecytoplasmofthegonadsmicroinjection(ThomasC.Evans,ed.)thecytoplasmiccoreofthedistalgermlineinjectionb.movMicroinjection1)IdentificationofgenesbyrescuingamutantphenotypeusingaWTcopyofthemutatedgene2)Expressionpatternusingthegeneofinterestwithreporter(lacZorfluorescent)3)InterferenceofabiologicalprocessbyoverexpressionofWTormutatedgene4)RNAiofselectedgenes