Microsoft Word doc

Size: px

Start display at page:

Download "Microsoft Word doc"

签庞
6 years ago
Views:

1 HEREDITAS (Beijing) , 35(4): ISSN 实验指南 DOI: /SP.J 斑马鱼高分辨率整胚原位杂交实验方法与流程张春霞, 刘峰,, 整胚原位杂交技术是利用反义 RNA 探针检测体内 mrna 表达的一项技术, 在利用模式动物研究基因时空表达方面有着重要的应用如何使用该技术得到特异高敏感度的表达结果, 对每一个使用该技术的实验室来说都很重要本实验室参照常规的实验方法, 对该技术加以改进, 使之更加灵敏, 结果更加特异文章主要以斑马鱼为例, 介绍了整胚原位杂交技术的发展历史, 并重点介绍了本实验室所用的整胚原位杂交实验流程, 同时还分析了实验结果不理想的原因及其解决方法斑马鱼 ; 整胚原位杂交 A brief protocol for high-resolution whole mount in situ hybridization in zebrafish ZHANG Chun-Xia, LIU Feng State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing , China Abstract: Whole mount in situ hybridization (WISH) is a popular technique that is used to study temporal-spatial gene expression at mrna level in model systems. Sensitive and high-resolution WISH is important for every laboratory that uses this technique to study expression pattern of genes in wildtype and perturbed embryos. Based on the commonly used protocols in fish community, our lab optimizes them to get more sensitive and specific results. Here, we briefly summarize the history of this technology using zebrafish as an example, describe a detailed experimental protocol, and provide troubleshooting for imperfect results and their solution. Keywords: zebrafish; whole mount in situ hybridization RNA mrna, 1,, RNA mrna,,,, 收稿日期 : ; 修回日期 : 作者简介 :,, Tel: ; zhangchxa@163.com 通讯作者 :,, liuf@ioz.ac.cn 网络出版时间 : :14:36 URL:

2 4 : 523, (1), ; (2), 1969 Gall Pardue DNA [1],,, Westerfield [2], Nusslein-Volhard [3] Cox RNA, RNA [4], 2008, Bernard Thisse,, 2009,,, [6] The Zebrafish Book [7] Bernard Thisse [5],,, T7RNA polymerase(promega, P2075); SP6 RNA 图 1 斑马鱼整胚原位杂交技术原理 RNA mrna,,, 14 hpf myod

3 524 HEREDITAS (Beijing) polymerase(promega, P1085); DNase I(NEB, M0303S); Heparin sodium salt (Sigma, H4784); trna(rna from torlua yeast, Type VI, Sigma, R6225); Pronase (Sigma, P6911); Protease K(Amresco, 0706); Blocking Reagent(Roche, ); anti-digoxigenin-ap Fab Fragments(Roche, ); BM Purple(Roche, ), PFA(Paraformaldehyde, Merck, ); 1.2 LF-, ZF-A4 (, ) PBS (1L, ) 80 g NaCl, 2 g KCl, g Na 2 HPO 4 12H 2 O, 2.4 g KH 2 PO 4, 1 L 1 PBST(1 L, ) 100 ml 10 PBS, 1 ml Tween 20, 1 L 4% PFA(500 ml, 4 ) 20 g PFA, 500 ml 1 PBS PFA,, SSC (ph7.0, 1 L, ) g NaCl, g C 6 H 5 Na 3 O 7 2H 2 O, 1 L 2 SSC(200 ml, 65 ) 20 ml 20 SSC, 200 μl Tween-20, 200 ml 0.2 SSC(500 ml, 65 ) 5 ml 20 SSC, 500 μl Tween-20, 500 ml Pronase(Pronase, 30 mg/ml, 20 ) 390 mg Pronase, 13 ml, 37 1 h,, 20, 1 PBS 1 mg/ml, 2~3 (100 ml) g Tris, 58.5 g NaCl, 100 ml Protease K( 20 ) Protease K 10 mg/ml,, 1: PBST, 10 μg/ml Hybe-(1 L,, 65 ) 500 ml, 250 ml 20 SSC, 9.2 ml 1 mol/l, 1 ml Tween-20, 1 L Hybe+(4, 65 ) Hybe- trna, 500 μg/ml 50 μg/ml trna (50 mg/ml, 4 ) 5 g trna, 100 ml trna, (100 mg/ml, 20 ) 100 mg, 1 ml MAB (1 L, ) 11.6 g, 8.8 g NaCl, 950 ml, 8 g NaOH ph 7.5, 1 L MABT (1 L, ) 1 L MAB 1 ml Tween %(v/v) (MAB Block, 1 L, 20 ) 20 g Blocking Reagent, 1 L MAB, 2%(w/v), 65,,, 20, ( ) anti-digoxigenin-ap Fab Fragments (BCL Buffer, 200 ml, ) 1 mol/l Tris-HCl(pH9.5)20 ml, 5 mol/l NaCl 4 ml, 0.5 mol/l MgCl 2 10 ml, 20% Tween-20 1 ml, 50 ml RNA DNA RNA, DNA, pgem-t pgem-t easy,, T7 SP6 RNA PCR RNA,,,, RNA,, 3, PCR 800~1 200 bp, T7, T7,, SP6, 1 μl, 1 μl DNase min,

4 4 : 525 RNase Free ddh 2 O 100 μl, 2 (200 μl)rnase Free 1/10 (10 μl)rnase Free (3 mol/l),, min RNA, 50~80 μl( )RNase Free ddh 2 O RNA, 30~100 ng/μl Hybe+ 1 10, 80, Hybe 表 1 体外转录反应体系 DNA template 5 Buffer DDT DIG-Labeling Mix RNase inhibitor SP6 RNA Polymerase μl 4 μl 2 μl 2 μl 0.5 μl 1 μl 37, 1.5 h 1 d, 2 d 3 d ZF-A4 ( ) 整胚原位杂交准备工作 1. (24 hpf 0.003% PTU ), Pronase, 4% PFA, % PFA,, 20 2 h 第 1 d 探针杂交 1. ( 30 ), PBST 75% methanol + 25% PBST, 5 min; 50% methanol + 50% PBST, 5 min; 25% methanol + 75% PBST, 5 min; 100% PBST, 5 min, 4 ; μg/ml Protease K,, 24 hpf, 24 hpf 8~9 min, 36 hpf 13 min, 4 dpf 40 min; 3. PBST 5 min, 2 ; 4. 4% PFA 20 min; 5. PBST 5 min, 5, ; 6. 50% PBST +50% Hybe- 5 min; 7. Hybe-, 65 (LF- ) 1h; ~200 μl Hybe RNA (200 μl 30~100 ng RNA), 65, 4 10 ml 5.67 ml H 2 O, 3.33 ml H 2 O 2, 1 ml(50% formamide+5 SSC),, EP, (, ), 4 d 8~10 min,, EP, 第 2 d 抗体孵育, 1., 20 ( 4~6 ); % Hybe-, 10 min; 75% Hybe-+ 25% 2 SSC, 10 min; 50% Hybe- + 50% 2 SSC, 10 min; 25% Hybe-+ 75% 2 SSC, 10 min; 2 SSC, 10 min; 0.2 SSC, 15 min, 4 ; 3. 75% 0.2 SSC + 25% MABT, 5 min; 50% 0.2 SSC +50% MABT, 5 min; 25% 0.2 SSC +75% MABT, 5 min; 100% MABT, 5 min; 4. ( MAB Block), 1 h, ; 5. MAB Block, (Roche), 1 h, 4, 1. 1, 500 μl 65 Hybe-,, 65 Hybe- ;

526 HEREDITAS (Beijing) 2013 35 2., 2 d, 2 d, 2, ; 3.

5 526 HEREDITAS (Beijing) , 2 d, 2 d, 2, ; % MABT,, ( MAB Block, 20 ), 1 h, ; 4. MAB Block, (Roche), 1 h, 第 3 d 染色, 1. 1 h, ( 2 ); 2. MABT 15 min, 8 ; 3. BCL Buffer 5 min, 3 ; 4. BCL Buffer BM Purple 1 1, ; 5., 4% PFA 20 min; 6. PBST 5 min, 3 ; 7. PBST, 90% glycerol, 4,,, 4 图 2 斑马鱼整胚原位杂交自动程序 A: ZF-A4 ; B 2 d 3 d

6 4 : 527, 1. 1,, 3 d, 2, 2., EP, BCL Buffer BM Purple 1 1,, ; 3., 5~ 图像采集 90%glycerol, 3A,, Nikon SMZ1500 Nikon DIGITAL SIGHT DS-Ri B 3C,,,,,,,,,,,,,,, ; 图 3 斑马鱼整胚原位杂交结果拍照及范例 A, 90% glycerol, ; B C 10 hpf shh 4 dpf rag1

7 528 HEREDITAS (Beijing) 表 2 整胚原位杂交技术常见问题及解决方案 K K 4%PFA,, RNA-RNA 65, K K RT-PCR PFA 4%PFA, K,, ;,,, ( 100 ng), RNA - -BM Purple,, NBT/BCIP BM Purple, 参考文献 (References): [1] Gall JG, Pardue ML. Formation and detection of RNA- DNA hybrid molecules in cytological preparations. Proc Natl Acad Sci USA, 1969, 63(2): DOI [2] Ekker M, Akimenko MA, Bremiller R, Westerfield M. Regional expression of three homeobox transcripts in the inner ear of zebrafish embryos. Neuron, 1992, 9(1): DOI [3] Cox KH, DeLeon DV, Angerer LM, Angerer RC. Detection of mrnas in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev Biol, 1984, 101(2): DOI [4] Schulte-Merker S, Ho RK, Herrmann BG, Nüsslein- Volhard C. The protein product of the zebrafish homologue of the mouse T gene is expressed in nuclei of the germ ring and the notochord of the early embryo. Development, 1992, 116(4): DOI [5] Thisse C, ThisseB. High-resolution in situ hybridization to whole-mount zebrafish embryos. Nat Protoc, 2008, 3(1): DOI [6] Wang L, Zhang PP, Wei YL, Gao Y, Patient R, Liu F. A blood flow-dependent klf2a-no signaling cascade is required for stabilization of hematopoietic stem cell programming in zebrafish embryos. Blood, 2011, 118(15): DOI [7] WesterfieldM. The zebrafish book: A guide for the laboratory use of zebrafish Danio ' (Brachydanio) rerio. 4th ed. Eugene: University of Oregon Press, DOI

討論

MTT cell viability assay 1 [45] succinate dehydrogenase MTT Kreb s cycle 2 MTT 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide DMSO 570 nm 15 3 MTT solution MTT Sigma Cat. M-2128 ( PBS ) 5