Name: expression of fluorescent proteins in branchiostoma lanceolatum by mrna injection into unfertilized oocytes
Uploaded: 2015-01-12T12:45:00
Description: Watch this Scientific Journal Video about Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes at JoVE.com

Forfatterne vil gerne anerkende den støtte fra &quot;Animalerie Centrale de Gif-sur-Yvette&quot; for dyrehold. Dette arbejde blev støttet af midler fra ANR (ANR-09-Blan-0262-02 og ANR-11-JSV2-002-01) til Michael Schubert, som Den Europæiske Union FP6 tilskud &quot;Embryomics&quot; og af ANR tilskud &quot;ANR- 10-Blan-121801 Dev-processen &quot;til Jean-François Nicolas og Nadine Peyriéras. João Emanuel Carvalho er finansieret af en FCT ph.d. stipendium (SFRH / BD / 86878/2012). Anmodning om vektorerne er beskrevet her, kan rettes direkte til forfatterne.

1. Fremstilling af instrumenter og reagenser <ol style="margin-left: 40px;"><li> Overførsel Pasteur-pipetter <ol><li> Generer en række overførsel Pasteur-pipetter med forskellige tip diameter ved at trække over en flamme ved forskellige hastigheder 230 mm lange Pasteur-pipetter. Sørg for, at tilspidsning er så længe som muligt for glat og fin kontrol med aspiration. </li><li> Med en diamant Skriveren ridse pipetten langs en linie vinkelret på længden af ​​pipetten. Med begge hænder, træk pipetten parall...

<ol>
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I denne artikel præsenterer vi for første gang, en detaljeret og reproducerbar protokol til injektion af B. lanceolatum oocytter, som efter B. floridae 13,14 og B. belcheri 15, er således den tredje amphioxus arter, for hvilke der er beskrevet en sådan teknik. Vigtigere er det, protokollen beskrevet her, omfatter også beskrivelsen af vektorsystemer egnede til fremstilling af fluorescerende proteiner i B. lanceolatum fra injicerede mRNA fremstillet in vit...

Under udvikling, en enkelt celle giver anledning til en hel organisme i en meget kompleks proces, der involverer både celledelinger og bevægelser. For bedre at forstå de biologiske principper dynamikken i celle adfærd, har udviklingsmæssige biologer begyndt at bruge fluorescens-baserede in vivo imaging teknikker. Specifikke rum i celler, såsom cellemembraner, kan enten mærkes ved behandlinger med fluorescerende farvestoffer, en fremgangsmåde hæmmes af en mangel på specificitet og vævspenetration 1, eller af den specifikke indførelse i embryo af exogene mRNA&#39;er koder fluorescerende proteiner 2. Forskellige teknikker kan anvendes til effektiv levering af exogene forbindelser, såsom mRNA&#39;er. Disse indbefatter, men er ikke begrænset til, mikroinjektion, elektroporation, bombardement med mikropartikler, lipofektion og transduktion 3,4. Selv om alle disse fremgangsmåder kan anvendes til at indføre eksogene forbindelser i enudviklende embryo, kun mikroinjektion tillader anvendelse af foruddefinerede og nøjagtige mængder i hver celle 3. Mikroinjektionsteknikker er blevet beskrevet for alle større udviklingsmæssige modelsystemer 4 (fx bananfluer, nematoder orme, zebrafisk, frøer, mus) samt for nogle alternative modeller 4, herunder dem, der anvendes til komparative undersøgelser med henblik på at forstå udviklingen i udviklingsmæssige mekanismer (fx søanemoner, Ledorme orme, søpindsvin, søpung sækdyr, den cephalochordate amphioxus). Cephalochordates, som sammen med sækdyr og hvirveldyr etablere chordate phylum, er særligt velegnede modeller til at studere udviklingen af chordater og diversificering af hvirveldyr fra en hvirvelløse forfader 5-8. Den cephalochordate slægt afveg meget tidligt under chordate evolution; og bevarede cephalochordates, som er opdelt i tre slægter (Branchiostoma, Asymmetron og Epigonichthys), ligner hvirveldyr både med hensyn til den samlede anatomi og genom arkitektur 5-8. Af de omkring 30 arter af cephalochordates der er blevet beskrevet indtil nu, fem er til rådighed for embryologiske og udviklingsmæssige undersøgelser 6,9: Asymmetron lucayanum (Bahamas lancelet), Branchiostoma floridae (Florida amphioxus), Branchiostoma lanceolatum (den europæiske amphioxus) Branchiostoma belcheri (kinesisk amphioxus) og Branchiostoma japonicum (japansk amphioxus). Modne voksne tre af disse arter (B. lanceolatum, B. belcheri og B. japonicum) kan induceres til at gyde on-demand i ynglesæsonen 10,11. Desuden, i det mindste for B. lanceolatum kan effektiv gydning også induceres i kunstig havvand 12 og derved gøre denne særlige cephalochordate art tilgængelige for LABORATORerne, der ikke har adgang til naturligt havvand. Kombinationen, i B. lanceolatum, af en praktisk og pålidelig adgang til fostre med en effektiv levering metode, såsom mikroinjektion, indtil videre den eneste levering teknik udviklet i amphioxus (i både B. floridae og B. belcheri) 13-15, vil gøre det muligt at udvikle en roman suite af manipulerende teknikker, herunder afstamning tracing- og dynamisk celleopførsel tilgange. En protokol for effektiv mikroinjektion af mRNA&#39;er til at udtrykke fluorescerende proteiner i B. lanceolatum embryo blev derfor udviklet. Desuden at give en grundlæggende værktøjskasse til levende billeddannelse af B. lanceolatum embryoner blev vektorsystemer udviklet, der tillader membran-associeret og nuklear ekspression af fluorescerende proteiner. For målretning membran blev forstærket grønt fluorescerende protein (eGFP) fusioneret til den humane HRAS CAAX kasse og kernelokalisering af mCherry og eGFP varopnået ved fusion til zebrafisk histon 2B (H2B) exon (figur 1, Supplerende fil 1). Desuden, med det mål at optimere protein translation, Kozak-sekvenser og kodoner af konstruktionerne er blevet modificeret og tilpasset til brug i B. lanceolatum. Tilsammen vil de injektion metode og ekspressionsvektorer præsenteret her tjene som grundlag for generering af nye eksperimentelle tilgange til cephalochordates, bl.a. analyser ved hjælp af den nyeste fluorescens-baserede in vivo imaging teknikker.

<table border="0" cellpadding="0" cellspacing="0" width="1009">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Name of Material/ Equipment</td>
			<td style="width:156px;">Company</td>
			<td style="width:143px;">Catalog Number</td>
			<td style="width:413px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Consumables</td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">35 mm Petri dishes</td>
			<td>Falcon</td>
			<td>353001</td>
			<td>culture-treated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Filtration unit (Stericup 1L)</td>
			<td style="width:156px;">Fisher</td>
			<td style="width:143px;">W21719</td>
			<td>0.22 micron filtration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Spin-X tubes</td>
			<td style="width:156px;">Costar</td>
			<td style="width:143px;">8160</td>
			<td>0.22 micron filtration tube</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">Needle storage jar for 1.2 mm diameter capillaries</td>
			<td style="width:156px;">WPI</td>
			<td style="width:143px;">E212</td>
			<td style="width:413px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Pasteur Pipettes</td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td>230 mm long</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Aspiration tube</td>
			<td style="width:156px;">Dutscher</td>
			<td style="width:143px;">75056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Capillaries for injection needles</td>
			<td style="width:156px;">Sutter</td>
			<td style="width:143px;">BF 120-94-10</td>
			<td>Borosilicate glass with filament, OD 1.20 mm, ID 0.94 mm, length 10 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;"></td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Reagents</td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Low-melting agarose</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">A9414</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Phenol Red</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">114537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Glycerol</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">G2025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Poly-L-Lysine hydrobromide</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">P9155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">H2O Dnase, Rnase-free</td>
			<td style="width:156px;">Gibco</td>
			<td style="width:143px;">10977-035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">mMessage mMachine SP6 Transcription kit</td>
			<td style="width:156px;">Ambion</td>
			<td style="width:143px;">AM1340</td>
			<td>mRNA synthesis kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Phenol pH8</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">P4557</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">24:1 chloroform:isoamylic alcohol</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">C0549</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">5:1 phenol pH4.7:chloroform</td>
			<td style="width:156px;">SIGMA</td>
			<td style="width:143px;">P1944</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Reef Crystal salts (200 kg)</td>
			<td style="width:156px;">Europrix&#160;</td>
			<td style="width:143px;"></td>
			<td>Commercial salts</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;"></td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Equipment</td>
			<td style="width:156px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">Fluorescent dissecting scope with 200x magnification&#160;</td>
			<td style="width:156px;">Leica</td>
			<td style="width:143px;">MZ16F</td>
			<td>25x oculars, DSR and GFP2 filters</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Micromanipulator</td>
			<td style="width:156px;">Marzhauzer</td>
			<td style="width:143px;">M-33</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Injector</td>
			<td style="width:156px;">Picospritzer</td>
			<td style="width:143px;">model II or III</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Needle puller</td>
			<td style="width:156px;">Sutter</td>
			<td style="width:143px;">P97</td>
			<td>heating-filament needle puller</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:297px;">Fine forceps</td>
			<td>FINE SCIENCE TOOLS GMBH</td>
			<td>11252-30</td>
			<td>Dumont #5</td>
		</tr>
	</tbody>
</table>

expression of fluorescent proteins in branchiostoma lanceolatum by mrna injection into unfertilized oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Protokollen beskrevet ovenfor giver grundlag for mikroinjektion af B. lanceolatum oocytter og dermed for indslæbning udvikle B. lanceolatum embryoner af mRNA, der koder for fluorescerende proteiner til in vivo billeddannelse. Selvom teknikken er helt sikkert robust og pålidelig, satsen for succesfulde injektioner ved hjælp af denne protokol forbliver variabel (tabel 1). Den meget sandsynlige forklaring på dette spændende kendsgerning er den ekstreme variation i oocyt kobl...

Watch this Scientific Journal Video about Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes at JoVE.com

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

Vi rapporterer her den robuste og effektiv ekspression af fluorescerende proteiner efter mRNA injektion i ubefrugtede oocytter fra Branchiostoma lanceolatum. Udviklingen af mikroinjektion teknik i denne basale chordate vil bane vejen for vidtgående tekniske innovationer i denne nye model, herunder in vivo imaging og gen-specifikke manipulationer.

Ekspression af fluorescerende proteiner i<em&gt; Branchiostoma lanceolatum</em&gt; Af mRNA Injektion i befrugtede oocytter

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

Research

JoVE Journal

Developmental Biology

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Manipulation og<em&gt; In vitro</em&gt; Modning af<em&gt; Xenopus laevis</em&gt; Oocytter, efterfulgt af ICSI, for at studere fosterudviklingen

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

Podning af perler i at udvikle Chicken Embryo lemmer at identificere signaltransduktionsveje påvirker Gene Expression

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Gene Transfer til kylling Auditory Organ med<em&gt; I Ovo</em&gt; Micro-elektroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Induktion af akut Skeletal Muscle Regeneration af cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

Immuno-fluorescerende mærkning af mikrotubuli og Centrosomal proteiner i Ex Vivo tarmens væv og 3D In Vitro tarm Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

Fast and Efficient Expression of Multiple Proteins in Avian Embryos Using mRNA Electroporation

Hurtig og effektiv ekspression af flere proteiner i aviær embryoner ved hjælp af mRNA-elektroporation

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

Hemogen omprogrammering af humane fibroblaster ved tvungen ekspression af transkriptionsfaktorer

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...

Fluorescent Immunolocalization of Arabinogalactan Proteins and Pectins in the Cell Wall of Plant Tissues

Fluorescerende immunolocalisering af Arabinogalactan proteiner og pektiner i cellevæggen af plantevæv

Plant development involves constant adjustments of the cell wall composition and structure in response to both internal and external stimuli. Cell walls ...

Minimum Volume Vitrification of Immature Feline Oocytes

Minimum volumen vitrifikation af umodne Feline Oocytter

In wild animals&#8217; conservation programs, gamete banking is crucial to safeguard genetic resources of valuable individuals and rare species and to ...

Separation of Follicular Cells and Oocytes in Ovarian Follicles of Zebrafish

Adskillelse af follikulære celler og oocytter i æggestokkene Follikler af zebrafisk

Zebrafish has become an ideal model to study the ovarian development of vertebrates. The follicle is the basic unit of the ovary, which consists of ...