High Resolution Hele Mount<em> In Situ</em> Hybridisering inden Zebrafisk embryoner at studere genekspression og funktion
Summary
Den zebrafisk, en lille tropisk fisk, er blevet en populær model for at studere genfunktion under hvirveldyr udvikling og sygdom. Det tidsmæssige og rumlige ekspression af målgener kan bestemmes ved<em> In situ</em> Hybridisering. Vores forbedrede protokol giver mulighed for påvisning af lave rigelige transkripter med lav ikke-specifik baggrund signal.
Abstract
Denne artikel fokuserer på hel-mount in situ hybridisering (WISH) i zebrafisk embryoner. Den WISH teknologi letter vurderingen af genekspression både vævsfordeling og udviklingsstadiet. Protokollerne beskrevet for brugen af WISH af zebrafisk embryoner med antisense RNA-prober mærket med digoxigenin. Prober genereres ved at inkorporere digoxigenin-linked nukleotider gennem in vitro-transkription af genet skabeloner, der er blevet klonet og lineariseret. De chorions af embryoner høstet ved definerede udviklingsstadier fjernes før inkubation med specifikke prober. Efter en vask procedure for at fjerne overskydende probe, er embryoner inkuberet med anti-digoxigenin-antistof konjugeret med alkalisk phosphatase. Ved at anvende et kromogent substrat for alkalisk phosphatase, kan specifik genekspression vurderes. Afhængig af størrelsen af genekspression hele proceduren kan afsluttes inden for 2-3 dage.
Introduction
Den zebrafisk (Danio rerio) er dukket op som en stærk dyremodel for studiet af hvirveldyr udvikling, sygdom, adfærd og in-medikamentscreeningsanalyse 1-3. Zebrafisk embryoner kan opnås i stort tal fra et enkelt passage. Befrugtning og udvikling sker ex utero og optisk klare embryoner udvikle sig hurtigt. Kritiske udviklingsmæssige hændelser forekommer i de første 48 timer efter befrugtning (HPF). Dette omfatter fremkomsten af orgel primordier og påbegyndelsen af cytodifferentiation. Knockdown eller over-ekspression af proteiner kan opnås gennem mikroinjektion af fostre med antisense morpholino (MO) oligonucleotider eller mRNA henholdsvis den ene eller to celle stadiet (0,75 HPF).
Ønsket om zebrafisk embryoner letter undersøgelse af spatio-temporale ekspression af specifikke gener af interesse. Anvendelse af WISH teknikken efter mikroinjektion af fostre med mRNA eller MO at over-udtrykess eller knockdown specifikke protein niveauer afslører differentieret regulering af andre gener.
Ændringer i genekspression mønstre kan korreleres med fænotypiske ændringer og afslører funktionen af målgener i den tidlige organogenese. Da prober kan fremstilles og lagres på forhånd, kan ISH teknik anvendes til at afsløre genekspressionsmønstre i mindst 20 gener ad gangen med seks-brønds plader og specialfremstillede kurve.
Protocol
Representative Results
Discussion
Vi har udviklet forbedrede metoder til at visualisere RNA med høj opløsning. Den in situ hybridisering procedurer gennemføres ved hjælp af simple skræddersyede kurve med porøse bunde (Figur 1). Embryoer behandlet ved hjælp af RNase-fri løsninger i plader med 6 brønde ved stuetemperatur under sterile betingelser.
At adskille embryoner kurve er fremstillet af forskellige farvede Eppendorf-rør. Kurve med eller uden en fælg bruges til nem orientering og til a…
Disclosures
The authors have nothing to disclose.
Materials
| Taq Polymerase | Invitrogen | 10342053 | |
| TopoTA Cloning Kit- Dual Promoter pCRII-TOPO | Invitrogen | K4650-01 | |
| HQ Mini Plasmid Purification Kit | Invitrogen | K2100-01 | |
| Agarose | Roche | 11 685 660 001 | |
| Not1 and HindIII Restriction Enzymes | Invitrogen | 15441-025, 15207-012 | |
| Buffer Saturated Phenol, ultrapure | Invitrogen | 15513039 | Store at 4 °C. Eyes, skin, and respiratory tract irritant and suspected carcinogen. Care should be taken while handling. |
| Chloroform | Fisher | C607-1 | Toxic and suspected carcinogen. Work under fume hood. |
| RNase-free DNAse I | Roche | 4716728001 | Prepare small aliquots and store at -20°C. |
| DIG-RNA Labeling Mix with Sp6, T7 and T3 RNA Polymerase | Roche | 11277073910 | |
| RNase Inhibitor | Roche | 10777-019 | |
| 3.0 M Sodium Acetate (pH 5.5) | Fisher | 50-751-7355 | |
| 100% Ethanol | Commercial alcohols | ||
| Diethyl Pyrocarbonate (DEPC) | Sigma | 40718-25ML | DEPC is an eye, skin, and respiratory irritant. Avoid contact with skin and eyes. Use safety glasses, gloves, and mask. |
| NaOH | Fisher | SS255-1 | |
| EDTA 0.5 M (pH 8.0) | Fisher | 50-751-7404 | |
| Instant Ocean Salt | Aquarium Systems | N/A | |
| Phosphate Buffered Saline (PBS) | Fisher | FL-03-0900 | |
| Paraformaldehyde (PFA) | Sigma | P6148-1KG | Prepare and store at -20°C as 40 ml aliquots for later use. PFA is toxic. Use safety glasses, gloves, and dust mask. |
| Phenylthiocarbomide (PTC) | Sigma | P7629-25G | PTC is highly toxic. Use safety glasses, gloves, and dust mask. |
| Methanol | Fisher | A947-4 | |
| Dumont (Watchmaker’s) Forceps pattern no. 5 | Fine Science Tools | ||
| Six-well Cell Culture Cluster | Sarstedt | 83.1839 | |
| Baskets are made of nylon mesh and Eppendorf tubes | In-house preparation | To make the baskets, cut Eppendorf tubes (1.5 ml) with or without rims to remove the conical end. Cut nylon mesh into small pieces corresponding to the size of the cut ends of the Eppendorf tubes. Place tubes with the nylon mesh covering the cut end on an electrical hot plate until both the tube and nylon mesh stick together (carry out in fume hood). Cut off the excess mesh from the baskets and store them in 100% methanol until used. | |
| Polyoxyethylenesorbitan Monolaurate (Tween 20) | Sigma | P1379-500ML | |
| Proteinase K | Fermentas | EO0491 | |
| Acetic Anhydride | Sigma | 242845 | |
| Triethanolamine | Sigma | 90279-100ML | |
| Formamide, high purity grade | Sigma | F9037 | |
| AG501-X8 Resin | Bio Rad | 142-6424 | |
| Citric Acid monohydrate | Sigma | C0706-500G | |
| Heparin Sodium Salt | Sigma | H3393-25KU | |
| Saline-sodium Citrate Buffer (SSC) | Sigma | S6639 | |
| tRNA from bakers yeast type X | Sigma | R5636-1ML | |
| NaCl | Fisher | S640-500 | |
| Tris-HCl | Fisher | BP153-1 | |
| MgCl2 | Fisher | S25403 | |
| Levamisole Hydrochloride | Sigma | 31742 | |
| N,N-Dimethylformamide anhydrous (DMF) |
Sigma | 227056 | Irritant, toxic, combustible, and suspected teratogen. Handle with proper safety attire including gloves and goggles. |
| 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) | Sigma | N6639 | Protect this solution from light. |
| Nitroblue tetrazolium (NBT) | Sigma | 840W | Protect this solution from light. |
| Albumin from Bovine Serum, purified fraction V (BSA) | Sigma | A8806 | |
| Sheep Anti-digoxigenin-AP Fab Fragments | Roche | 1093274910 | |
| Glycerol | Sigma | G5516-500ML | |
| 96-well cell culture plates | Sarstedt | 83.1835 | |
| Tg(mnx1:GFP)ml2/Tg(hb9:GFP)ml2 | ZIRC | Tg(mnx1:GFP)ml2 | |
| Instant Ocean | Aquarium Systems | N/A |
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