斑馬魚是研究神經系統發育、功能和組織修復的理想模型,尤其是特異性表達熒光蛋白的轉基因品系,幫助我們標記目標組織和細胞,可以清晰直觀地觀察到活體神經細胞的動態生物學過程。
國家斑馬魚資源中心現有各類研究用品系1400多種,其中轉基因品系近200種。本文為大家推薦其中常用的幾種神經系統標記品系。

雙色熒光標記的斑馬魚神經系統
斑馬魚中樞神經系統自前段向后依次為:嗅球(olfactory bulb)、端腦(telencephalon)、間腦(diencephalon)、下丘腦(hypothalamus)、視頂蓋(optic tectum)、小腦(cerebellum)、后腦(rhombencephalon)和延髓(medulla)等基本結構,如下圖所示。幼年期的斑馬魚大腦組織透明,且結構簡單,可以作為優良的神經系統動物模型(Shang et al. 2015)。

斑馬魚神經系統 (Grandel et al. 2006)
能全面標記中樞神經系統的品系首推CZ417(ihb304Tg, Tg(gad1b:mCherry)),該品系由gad1b啟動子驅動紅色熒光蛋白mCherry的表達。gad1b是編碼生成谷氨酸脫羧酶(Glutamic Acid Decarboxylase,GAD)亞型GAD67的基因。GAD負責催化谷氨酸(glutamate)的脫羧反應,產生γ-氨基丁酸(Gamma Amino Butyric Acid,GABA)和CO2。GABA是起抑制性作用的神經遞質,因此gad1b啟動子通常是作為抑制性神經元的特異性啟動子。gad1b基因的表達譜很廣,從嗅球(olfactory bulb)、視頂蓋(optic tectum)、眼睛(eye)、延髓(medulla oblong)和脊髓(spinal cord)都可以觀察到紅色熒光蛋白的表達(Song et al., 2017)。

CZ417, ihb304Tg, Tg(gad1b:mCherry) (Song et al., 2017)
CZ413(tsu33Tg, Tg1(elavl3:YFP))和CZ160(knu3Tg, Tg(elavl3:EGFP))分別使用神經發育標記基因elavl3的啟動子驅動YFP和EGFP的表達,在全身的神經元中特異表達標記基因。elavl3基因別名huc,最早在果蠅中發現,該基因缺失會導致視覺神經系統缺陷伴隨神經系統發育不良,嚴重致死。剛過原腸期,Elavl3基因開始在分布在中線兩側的神經元中表達,神經系統二次發育開始不久,Elavl3基因表達急劇上升。elavl3在斑馬魚早期神經發育過程中發揮了重要的作用,是神經系統發育的重要標記基因(Lyons et al., 2003; Zhao et al., 2006; Wu et al., 2018)。

CZ413, tsu33Tg, Tg1(elavl3:YFP)( Wu et al., 2018)

CZ413, tsu33Tg, Tg1(elavl3:YFP)

CZ160, knu3Tg, Tg(elavl3:EGFP)
CZ154(vu19Tg, Tg(olig2:DsRed2))轉基因利用紅色熒光蛋白DsRed2標記運動神經元和頭部少突膠質細胞群。在神經系統發育過程中,放射狀膠質細胞既是神經元的前身,也充當著新生神經元遷移的支架。Notch信號通路在放射狀膠質細胞的維持中發揮了重要的作用,同時抑制其形成運動神經元,促進其向少突膠質細胞分化(Kim et al., 2008)。放射狀膠質細胞在發育中的中樞神經系統中居要角,扮演引導神經元遷移的角色。缺少膠質細胞的胚胎中,運動神經元會偏離預定遷移軌跡,向脊髓外遷移,形成異常的軸突投射(Kucenas et al., 2008)。

CZ154, vu19Tg, Tg(olig2:DsRed2)
CZ224(nju1Tg, Tg(cyp26a1:EYFP))轉基因品系由cyp26a1基因一段2.5kb的啟動子驅動EYFP標記基因的表達,表達部位主要分布在視網膜、嗅球、背側脊髓前部、后腸、脊索后部和咽弓等組織中(Hu et al., 2008)。cyp26a1基因是細胞色素P450家族成員,相對應的產物為Cyp26氧化酶,可以和視黃醛脫氫酶RALDHs(aldh1a2基因產物)共同協調維持視黃酸(RA)的均衡狀態。視黃酸參與了細胞增殖和凋亡、細胞分化和組織形態等多個重要的生理功能中,對機體的視覺、皮膚、免疫、生殖和神經系統等都有一定的影響。

CZ224, nju1Tg, Tg(cyp26a1:EYFP)
對特異單一神經相關組織感興趣的朋友可以關注CZ25(Tg(OTM:dGFP) ihb27Tg,標記中腦組織),CZ31(Tg(-1.0pomca:GFP) zf44Tg,標記腦垂體),CZ36(Tg(-2.5tshb:EGFP) ihb5Tg,標記松果體),CZ151(Tg(ompb:tau-eGFP) jt0012Tg,標記嗅球),CZ153(Tg(mbp:EGFP) ck1Tg,標記成髓鞘膠質細胞),CZ353(Tg(atoh1a:dTomato)nns8Tg,標記小腦組織)和CZ415(Tg(crybb1:CFP) tsu24Tg,標記晶狀體)等品系。

標記單一特異神經組織的品系
還有很多有意思的品系在這里就不一一贅述了,有興趣的朋友請點擊品系列表的CZ號就可以進入品系信息頁面了解啦。未來中心的全體員工將繼續努力以為斑馬魚研究團隊提供更好的科研支撐。
CZ ID | Genotype | construct | Note |
CZ25 | ihb27Tg/+ | Tg(OTM:GFP) | Optimal Tcf motif, TOP driving GFP expression, midbrain GFP |
CZ31 | zf44Tg/+ | Tg(-1.0pomca:GFP) | pituitary |
CZ36 | ihb5Tg/+ | Tg(UAS:casp3a-UTR-nanos3,CMV:EGFP) | pituitary/pineal/thyroid |
CZ77 | Tg(atoh7:Gal4); Tg(UAS:mcherry) | Tg(atoh7:Gal4); Tg(UAS:mcherry) | retinal ganglion cells |
CZ78 | cf2Tg/+ | Tg(elavl3:YC2) | neurons, retina |
CZ79 | ml3Tg/+ | Tg(mnx1:mGFP) | Primary motor axons, motoneurons |
CZ81 | rw0Tg/+ | Tg(isl1:GFP) | cranial motor neuron |
CZ82 | zf147Tg/+ | Tg(apoeb:LY-EGFP) | Neuronal |
CZ86 | kca66Tg/+ | Tg(h2afva:h2afva-GFP) | neuromast primordium migration, posterior lateral line nerve development |
CZ150 | ck2Tg/+ | Tg(eef1a1l1:Kaede) | Brain, hatching gland |
CZ151 | jt0012Tg/+ | Tg(ompb:tau-EGFP) | Olfactory bulb, Olfactory placode |
CZ153 | ck1Tg/+ | Tg(mbp:EGFP) | myelinating glia, central nervous system |
CZ154 | vu19Tg/+ | Tg(olig2:DsRed2) | Oligodendrocyte, motor neuron |
CZ159 | Tg(mbp:mGFP) | Tg(mbp:mGFP) | myelinating glia |
CZ160 | knu3Tg/+ | Tg(elavl3:EGFP) | neurons, nervous system |
CZ162 | Tg(ift46:GAL4-VP16;UAS:nfsB-mCherry) | Tg(ift46:GAL4-VP16;UAS:nfsB-mCherry) | eye, pronephric duct, spinal cord |
CZ224 | nju1Tg/+ | Tg(cyp26a1:EYFP) | retina,olfactory vesicle, anterior dorsal spinal cord,proctodeum,caudal notochord,pharyngeal arches |
CZ353 | nns8Tg/+ | Tg(atoh1a:dTomato) | cerebellum |
CZ354 | nl1Tg/+ | TgBAC(neurod1:EGFP) | Cranial ganglia, lateral line nerve cells |
CZ355 | um14Tg/+ | Tg(EPV.Tp1-Mmu.Hbb:EGFP) | Notch-responsive tissues such as the developing CNS, vasculature, liver, intestine and pancreas |
CZ413 | Tg(huc:YFP)/+ | Tg1(elavl3:YFP) | neurons |
CZ415 | Tg(crybb1:CFP)/+ | Tg(crybb1:CFP) | eye lens |
CZ417 | ihb304Tg/+ | Tg(gad1b:mCherry) | olfactory pit, optic tectum, medulla oblong, eye, spinal cord |
神經系統特異表達的轉基因斑馬魚品系(直接點接CZ號即可進入相應品系信息頁面)
參考文獻
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