Name: induction of protein deletion through in utero electroporation to define deficits in neuronal migration in transgenic models
Uploaded: 2015-01-12T12:30:00
Description: Watch this Scientific Journal Video about Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models at JoVE.com

We are indebted to Pierre Mattar and Freda Miller for their assistance with the in utero electroporation and to Noel Ghanem and Bensun Fong for creating diagrams. We thank Linda Jui and Jason G. MacLaurin for excellent technical assistance. Equipment was supported by the Centre for Stroke Recovery.
This work was supported by a scholarship from HSFO and OGS to D.S.S. and a CIHR grant to R.S.S.

BEMÆRK: Alle forsøg blev godkendt af University of Ottawa Animal Care etiske udvalg, der tilslutter sig retningslinjerne fra den canadiske Rådet om Animal Care. 1. Pre-kirurgiske Forberedelse og materialer <ol><li> Plasmid Forberedelse: <ol><li> Forbered plasmider under anvendelse af Qiagen Megaprep ifølge producentens protokol. Inokulering mindst 400 ml LB-bouillon med transformerede bakterier at sikre et højt DNA udbytte og inkuberes natten over. </li><li> Resuspender plasmid i 100-...

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To af de primære udfordringer i at udføre i livmoderen elektroporeringer er 1) at forebygge embryo dødelighed og 2) faldende variabilitet mellem elektroporeringer. En af de vigtigste faktorer i at forebygge dødelighed er nål kvalitet, som stumpe nåle påfører mere skade på fosteret. Ved skæring af nålen, holder spidsen så længe som muligt, mens det stadig tillader en synlig dråbe af 0,1-0,2 pi væske til at blive vist efter en pumpe af foden padle (Figur 1J). Hvis nålen er for ked...

Radial migration er en central proces til tidlig kortikale udvikling. Det afhænger af en række celle autonome faktorer, såsom korrekt mitotisk udgang og neuronal differentiering, cellepolaritet, regulering af cytoskeletale dynamik og ekspression af transmembrane receptorer, såvel som ikke-celle autonome faktorer, såsom dannelse af den radiale glial stillads og sekretion af vandrende vejledning molekyler 1-3. Da afbrydelse af nogen af disse mekanismer kan forringe neuronal migration i en transgen musemodel 4-8, bestemme den underliggende årsag til en defekt i migration kan være en kompliceret og vanskelig proces. I livmoderen elektroporation (IUE) kan bruges til at supplere og forenkle fortolkning af fænotypen af en genetisk knock-out model og derved belyse vigtige mekanismer, der kræves til radial migration 7,9-11. I livmoderen elektroporation (IUE) er den proces, hvorved et plasmid carryi<html> ng både et gen af interesse og et reporter indsprøjtes i ventrikel i hjernen hos en mus eller rotte foster og derefter trukket ind i celler, der beklæder ventrikel ved anvendelse af en elektrisk strøm 12-14. Dette tillader investigator at analysere effekten af ​​op eller nedregulering af et gen af ​​interesse i udvikling eller funktion af elektroporerede neuroner. Efter IUE kan hjerner behandles til immunhistokemi 7,9-11, elektrofysiologi 15 eller cellekultur 16,17. Den største fordel ved IUE er, at der tillader meget specifik manipulation af genekspression. Endvidere kan IUE anvendes til at målrette en specifik region af hjernens udvikling gennem en rettet strøm (figur 2). IUE kan også anvendes til at målrette en specifik celletype ved injektion af plasmider, der bærer gener under kontrol af forskellige promotorer eller plasmid aktiveringssystemer (tetracyclin induceret genekspression er et eksempel) 10,18-21. jove_content &quot;&gt; IUE kan anvendes i forbindelse med Cre-Lox-medieret gen excision systemet 7-9,11,22. kan et plasmid indeholdende et gen, der koder meddelelsen for Cre protein elektroporeret ind et foster homozygot for floxed allel ( hvor genet eller specifik DNA-regioner er flankeret af to loxP-sites for Cre-medieret DNA-rekombination). Cre vil derefter inducere rekombination af genet af interesse specifikt i elektroporerede neurale forstadier, generering af et knock-out af Floxed allele.The virkning protein knockdown på neural migration og udvikling inden for individuelle neuroner kan derefter undersøgt. Elektroporation af Cre inducerer rekombination i kun en lille population af påvirkede celler, der forlader den understøttende miljø intakt. I modsætning, vævsspecifik ekspression af Cre under kontrol af en celletype specifik promotor, forekommer i hele væv så både migrerer neuroblaster og det omgivende miljø kan blive påvirket. Således Juxtaposition af disse to tilgange kan afgøre, om en given migration defekt skyldes celle autonome eller celle ikke-selvstændige mekanismer. Defekt migration i både eksperimentelle systemer tyder på, at den observerede fænotype skyldes en celle autonom mekanisme; normal migration efter elektroporering af Cre med defekt migration på en vævsspecifik Cre model indikerer, at genet af interesse virker gennem en ikke-celleautonom mekanisme. IUE kan også anvendes til at udføre redning eksperimenter ved elektroporering potentielle interagerende gener i knock out dyr 7,9,10. For eksempel kunne en investigator forsøge at redde en migration fænotype i en transgen model ved elektroporere en downstream mål og bestemme, om migrationen defekt korrigeres i elektroporerede neuroner. Dette har den yderligere fordel, at en vellykket redning indikerer, at normal migration kan genoprettes ved at manipulere proteinekspression i en specifikke neuron, selvom det omgivende miljø stadig mangelfuld for målgenet. Igen, denne tilgang er mere tid og omkostningseffektiv end at krydse eller generere transgene linier, med den ekstra fordel af at fastslå, om den defekte mekanisme er celle autonom. IUE kan bruges til at spore migrerende neuroner gennem elektroporering af et reporterplasmid i knock out embryoner (figur 4C). Da kun neuronerne foring ventriklen på tidspunktet for kirurgi er elektroporeret 17 kan IUE bruges til at følge de neuroner, der er født på et bestemt tidspunkt med den fordel visualisering af deres morfologi in vivo. Endelig er det muligt at anvende IUE at målrette hjerneregioner som den mediale eller dorsale telencephalon, hippocampus eller ganglioniske eminence (figur 2). Det giver forskerne beføjelse til at undersøge genfunktion i et bestemt område uafhængig af komplikationer resultating fra protein knockdown i tilstødende bygninger. Retinoblastoma protein (pRb), p107 og p130 omfatter lommen protein familie og er veletablerede regulatorer af cellecyklus exit. Men der er stigende tegn på, at disse proteiner også regulere cellecyklus uafhængige aspekter af neural udvikling. Som vi tidligere har vist, pRb og p107 spiller en afgørende rolle i både tangential 4,23 og radial migration 6. Her viser vi rollen af lommen protein familiemedlemmer pRb og p107 på neurale migration 6 at eksemplificere anvendelsen af in utero elektroporering af Cre i en transgen model. Sammenfattende IUE giver en kraftfuld måde at analysere celleautonom virkninger af gendeletion (eller overekspression). Når det kombineres med vævsspecifik knock out modeller kan IUE give yderligere oplysninger om de mekanismer, der styrer neurale migration.

<table border="0" cellpadding="0" cellspacing="0" width="538">
	<tbody>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;width:233px;">Table 1: Equipment and Materials</td>
			<td style="width:64px;"></td>
			<td style="width:64px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:119px;">Product Name</td>
			<td style="width:115px;">Company</td>
			<td colspan="2" style="width:128px;">Catalog Number</td>
			<td style="width:177px;">Description</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:119px;">ECM 830 Square Wave Electroporator</td>
			<td style="width:115px;">BTX Harvard Apparatus</td>
			<td colspan="2" style="width:128px;">45-0052</td>
			<td style="width:177px;">Electroporator only &#8211; buy cables separately (45-0204)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">1250FS Footswitch for ECM 830</td>
			<td style="width:115px;">BTX Harvard Apparatus</td>
			<td colspan="2" style="width:128px;">45-0211</td>
			<td style="width:177px;">Foot paddle is necessary for hands-free electroporation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Tweezertrodes</td>
			<td style="width:115px;">BTX Harvard Apparatus</td>
			<td colspan="2" style="width:128px;">45-0489 (5mm)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 45-0488 (7mm)</td>
			<td style="width:177px;">Platinum forcep style electrodes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Femtojet</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">920010504</td>
			<td style="width:177px;">Microinjector</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Griphead 0</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">920007414</td>
			<td style="width:177px;">Holds the pulled needle</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Universal Capillary Holder</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">920007392</td>
			<td style="width:177px;">Connects griphead to tube</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Injection Tube</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">920007431</td>
			<td style="width:177px;">Connects capillary holder to Femtojet</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Foot Control</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">920005098</td>
			<td style="width:177px;">Foot paddle for hands-free injection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Microloader Tips</td>
			<td style="width:115px;">Eppendorf</td>
			<td colspan="2" style="width:128px;">930001007</td>
			<td style="width:177px;">Tips for loading the pulled capillary tubes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Plasmid Mega Kit</td>
			<td style="width:115px;">Qiagen</td>
			<td colspan="2" style="width:128px;">12181</td>
			<td style="width:177px;">5 QIAGEN-tip 2500, Reagents, Buffers</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Fast Green FCF</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td colspan="2" style="width:128px;">F7252-5G</td>
			<td style="width:177px;">Non-toxic dye</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Extra Fine Bonn Scissors</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td colspan="2" style="width:128px;">14084-08</td>
			<td style="width:177px;">For trimming the needles</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Bonn Strabismus Scissors</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td colspan="2" style="width:128px;">14084-09</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Graefe Forceps</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td colspan="2" style="width:128px;">11050-10</td>
			<td style="width:177px;">Straight</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Colibri Retractors</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td colspan="2" style="width:128px;">17000-03</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Stapler</td>
			<td style="width:115px;">Fine Science Tools</td>
			<td colspan="2" style="width:128px;">12031-07</td>
			<td style="width:177px;">For 7mm clips</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Castroviejo Needle Holder</td>
			<td style="width:115px;">Medical Tools</td>
			<td colspan="2" style="width:128px;">SNH-6737</td>
			<td style="width:177px;">Straight, with lock</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Needle Puller</td>
			<td style="width:115px;">Narishige</td>
			<td colspan="2" style="width:128px;">PC-10</td>
			<td style="width:177px;">For pulling microcappillaries</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:119px;">Microcapillaries</td>
			<td style="width:115px;">Drummond</td>
			<td colspan="2" style="width:128px;">1-000-800</td>
			<td style="width:177px;">0.4 mm inner diameter glass capillary tubes</td>
		</tr>
	</tbody>
</table>

induction of protein deletion through in utero electroporation to define deficits in neuronal migration in transgenic models

Genetisk sletning ved hjælp af Cre-Lox-system i transgene mus linjer er et kraftfuldt værktøj, der anvendes til at studere protein funktion. Men bortset fra i meget specifikke Cre-modeller, sletning af et protein gennem et væv eller celle population fører ofte til komplekse fænotyper som følge af flere interagerende mekanismer. Vurdering af, om en fænotype resultater fra afbrydelse af en celle autonom mekanisme, som er uløseligt forbundet med den pågældende celle, eller fra en ikke-celle autonom mekanisme, der ville følge af nedskrivning af denne celle miljø, kan være svært at få øje på. At få indsigt i proteinfunktion i en in vivo kontekst, utero elektroporation (IUE) gør gendeletion i en lille delmængde af celler i cortex udvikling eller en anden udvalgt område af hjernen. IUE kan bruges til at målrette specifikke områder i hjernen, herunder dorsale telencephalon, mediale telencephalon, hippocampus, eller ganglie eminence. Dette letter observation of konsekvenserne af celleautonom deletion gen i forbindelse med et sundt miljø. Målet med denne protokol er at vise, hvordan IUE kan bruges til at analysere en defekt i radial migration i en floxed transgene mus linje, med vægt på at skelne mellem celle autonome og ikke-celle selvstændige effekter af sletning protein. Ved at sammenligne den fænotype, der hidrører fra gen deletion i hele cortex versus IUE-medieret deletion gen i en begrænset cellepopulation, større indsigt i proteinfunktion i hjernens udvikling kan opnås end ved anvendelse af enten teknik i isolation.

Different regions of the brain can be targeted for electroporation by varying the orientation of the paddle electrodes over the embryo&#8217;s head (Figure 2). For all parts of the forebrain, the plasmid is injected into the lateral ventricle (the hind brain can be targeted by injecting into the fourth ventricle). In general, place the electrodes so that the straight line between them passes through the region of interest, with DNA being drawn toward the positive electrode. To target the lateral cortex (...

Watch this Scientific Journal Video about Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models at JoVE.com

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

Induktion af protein Sletning Gennem<em&gt; In Utero</em&gt; Elektroporering Definer Underskud i neuronal migration i transgene modeller

Neuron migration is regulated by numerous cell autonomous and non-cell autonomous factors. This protocol shows how in utero electroporation can be used to determine whether a phenotype in a transgenic mouse is due to disruption of a cell intrinsic mechanism or impairment of interaction between the neuron and its environment.

Genetic deletion using the Cre-Lox system in transgenic mouse lines is a powerful tool used to study protein function. However, except in very specific ...

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JoVE Journal

Developmental Biology

Induction of Protein Deletion Through In Utero Electroporation to Define Deficits in Neuronal Migration in Transgenic Models

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

En optogenetic fremgangsmåde for vurdering af Dannelse af neuronal forbindelser i en co-kultur System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Ex Utero Elektroporering og organotypisk Slice kultur Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

Analyse af zebrafisk Larver Skeletmuskel Integritet med Evans blå farvestof

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Technique to Target Microinjection to the Developing Xenopus Kidney

Technique to Target Microinjection to the Developing Xenopus Kidney

Teknik Target Mikroinjektion til udviklingslandene<em&gt; Xenopus</em&gt; Nyre

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

Rumlig og tidsmæssig analyse af Active ERK i<em&gt; C. elegans</em&gt; Kimcellelinje

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

Instrueret differentiering af Primitive og endelige hæmatopoietisk ophav fra humane pluripotente stamceller

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

Generering af genetisk modificerede mus gennem mikroinjektion af oocytter

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissektion og immunfluorescent farvning af champignon krop og fotoreceptor neuroner i voksen Drosophila melanogaster hjerner

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Anvendelse af RNAi og Heat-shock-induceret transskription faktor udtryk til at omprogrammere kønsceller til neuroner i C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...