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 ønsker å takke våre av &quot;Animalerie Centrale de Gif-sur-Yvette&quot; for dyrehold. Dette arbeidet ble støttet av midler fra ANR (ANR-09-BLAN-0262-02 og ANR-11-JSV2-002-01) til Michael Schubert, av EU FP6 stipend &quot;Embryomics&quot; og av ANR stipend &quot;ANR- 10-BLAN-121801 Dev-Process &quot;til Jean-François Nicolas og Nadine Peyriéras. João Emanuel Carvalho er finansiert av en FCT doc (SFRH / BD / 86878/2012). Forespørsler om vektorene er beskrevet her kan rettes direkte til forfatterne.

1. Klargjøring av instrumenter og reagenser <ol style="margin-left: 40px;"><li> Overføre Pasteur pipetter <ol><li> Generere en rekke overføring Pasteur pipetter med ulike tips diametre ved å trekke 230 mm lange Pasteur pipetter over en flamme på ulike hastigheter. Sørg for at taper er så lenge som mulig for jevn og fin kontroll av aspirasjon. </li><li> Med en diamantspiss, riper i pipetten langs en linje vinkelrett på lengden av pipetten. Med begge hender, trekk pipette parallelt med sin lengde for å generere...

<ol>
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I denne artikkelen presenterer vi, for første gang, en detaljert og reproduserbar protokoll for injeksjon av B. lanceolatum eggceller, som etter B. floridae 13,14 og B. belcheri 15, er således den tredje amphioxus art, hvor en slik teknikk er beskrevet. Viktigere protokollen beskrevet her også omfatter beskrivelsen av vektorsystemer er egnet for produksjon av fluorescerende proteiner i B. lanceolatum fra injisert mRNA produsert in vitro (beskrevet ned...

Under utviklingen gir en enkelt celle opphav til en hel organisme i en meget kompleks prosess som involverer både celledelinger og bevegelser. For bedre å forstå de biologiske prinsippene dynamikken i celle atferd, har utviklingsbiologer begynt å bruke fluorescens-basert in vivo imaging teknikker. Spesifikke avdelinger av celler, for eksempel cellemembraner, kan enten bli merket ved behandling med fluorescerende fargestoffer, en tilnærming hemmet av mangel på spesifisitet og vev penetrasjon 1, eller ved den spesifikke innføring i embryo av eksogene mRNA som koder for fluorescerende proteiner 2. Forskjellige teknikker kan brukes for effektiv levering av eksogene forbindelser, slik som mRNA. Disse inkluderer, men er ikke begrenset til, mikroinjeksjon, elektroporering, bombardement med mikropartikler, lipofeksjon, og transduksjon 3,4. Selv om alle disse fremgangsmåter kan brukes for å innføre eksogene forbindelser i enutvikling av embryo, tillater bare mikroinjeksjon anvendelsen av forhåndsdefinerte og presise mengder inn i hver celle 3. Mikroinjeksjon teknikker har blitt beskrevet for alle de store utviklingsmodellsystemer 4 (for eksempel fruktfluer, nematodeormer, sebrafisk, frosker, mus) samt for noen alternative modeller 4, inkludert de som brukes for sammenlignende studier med sikte på å forstå utviklingen av utviklingsmessige mekanismer (f.eks, sjøanemoner, annelid ormer, kråkeboller, ascidian kappedyr, den Lansettfisker amphioxus). Cephalochordates, som sammen med kappedyr og virveldyr etablere chordate phylum, er spesielt godt egnet modeller for å studere utviklingen av chordatar og spredning av virveldyr fra en virvelløse stamfar 5-8. Den Lansettfisker avstamning avvek veldig tidlig i løpet chordate evolusjon; og bevarte cephalochordates, som er inndelt i tre slekter (Branchiostoma, Asymmetron og Epigonichthys), ligner virveldyr både i form av generelle anatomi og genom arkitektur 5-8. Av de ca 30 arter av cephalochordates som har blitt beskrevet så langt, fem er tilgjengelig for embryologiske og utviklingsstudier 6,9: Asymmetron lucayanum (Bahama Lansettfisker), Branchiostoma floridae (Florida amphioxus), Branchiostoma lanceolatum (den europeiske amphioxus), Branchiostoma belcheri (den kinesiske amphioxus) og Branchiostoma japonicum (den japanske amphioxus). Modne voksne i tre av disse artene (B. lanceolatum, B. belcheri og B. japonicum) kan induseres å gyte on-demand i hekkesesongen 10,11. I tillegg, i det minste for B. lanceolatum, kan effektiv gyting også bli indusert i kunstig sjøvann 12, og dermed gjør denne spesielle Lansettfisker arter tilgjengelig for laboratoriumtallet som ikke har tilgang til naturlig sjøvann. Kombinasjonen, i B. lanceolatum, av en praktisk og pålitelig tilgang til embryoer med en effektiv leveringsmetode, for eksempel mikroinjeksjon, så langt det eneste levering teknikk utviklet i amphioxus (i både B. floridae og B. belcheri) 13-15, vil muliggjøre utvikling av en roman suite av manipulerende teknikker, inkludert avstamning tracing- og dynamisk celleatferdsbaserte tilnærminger. En protokoll for effektiv mikroinjeksjon av mRNA for å uttrykke fluorescerende proteiner i B. lanceolatum embryo ble derfor utviklet. Videre, for å gi en grunnleggende verktøykasse for levende avbildning av B. lanceolatum embryoer ble vektorsystemer utviklet som tillater membran-forbundet og kjernefysisk uttrykk for fluorescerende proteiner. For membran målretting, ble forbedret grønt fluorescerende protein (EGFP) smeltet til den menneskelige HRAS CAAX boksen og kjernefysiske lokalisering av mCherry og EGFP varoppnås ved fusjon til sebrafisk histone 2B (H2B) ekson (Figur 1, Supplerende Fil 1). Videre, med det mål å optimalisere protein oversettelse, de Kozak sekvenser og kodonene av konstruksjonene har blitt modifisert og tilpasset bruk i B. lanceolatum. Til sammen vil de injeksjon metode og ekspresjonsvektorene presenteres her tjene som grunnlag for generering av nye eksperimentelle tilnærminger for cephalochordates, særlig analyser bruker den nyeste fluorescens-basert 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 gir grunnlag for mikroinjeksjon av B. lanceolatum eggceller og dermed for innføring i å utvikle B. lanceolatum embryoer av mRNA som koder for fluorescerende proteiner for in vivo avbilding. Selv om teknikken er absolutt robust og pålitelig, forblir hastigheten av vellykkede injeksjoner ved hjelp av denne protokoll variabel (tabell 1). Den meget sannsynlig forklaring på dette faktum interessant er den ekstreme variabilitet av oocytten clutcher:...

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 robust og effektiv uttrykk for fluorescerende proteiner etter mRNA injeksjon i ubefruktede eggceller av Branchiostoma lanceolatum. Utviklingen av mikroinjeksjon teknikk i denne basal chordate vil bane vei for vidtrekkende tekniske nyvinninger i denne nye modellen systemet, inkludert in vivo avbildning og genspesifikke manipulasjoner.

Uttrykk for Fluorescent Proteiner i<em&gt; Branchiostoma lanceolatum</em&gt; Av mRNA Injeksjon i ubefruktet 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

Manipulasjon og<em&gt; In Vitro</em&gt; Modning av<em&gt; Xenopus laevis</em&gt; Oocytter, etterfulgt av Intracytoplasmatisk Sperm Injection, for å studere embryoutvikling

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

Negl Perler inn Utvikling kyllingembryo Limbs å identifisere signaltransduksjonsveier 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 inn i Chicken hørbar Organ av<em&gt; I Ovo</em&gt; Micro-electroporation

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

Induksjon av akutt Skeletal Muscle Regenerering av 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 merking av piskehale som henger og Centrosomal proteiner i Ex Vivo tarmen Tissue og 3D In Vitro Intestinal 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

Rask og effektiv uttrykk for flere proteiner i avian embryo bruke mRNA electroporation

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

Hemogenic omprogrammering av Human fibroblaster av tvungen uttrykk for transkripsjon faktorer

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 immunlokalisering av arabinogalaktane proteiner og pektiner i celleveggen av plantevev

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 Volum Vitrifisering av umodne Feline Oocytes

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

Separasjon av follikulære celler og oocytter i eggstokksekkene av sebrafisk

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 ...