Name: genetic manipulation of the mouse developing hypothalamus through in utero electroporation
Uploaded: 2013-07-24T12:00:00
Description: Watch this Scientific Journal Video about Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation at JoVE.com

Dette arbejde blev finansieret af den tyske Research Foundation (Deutsche Forschungsgemeinschaft).

1.. Forberedelse af DNA og glas Mikropipetter Injection <ol><li> God kvalitet glas mikropipetter er afgørende for at reducere indledende høje aborter skyldes tab af fostervand. Proceduren til at trække glas mikropipetter har været veldokumenteret 13,18,25. Brug 1,2 mm diameter kapillærer trukket i en konventionel Sutter P-97-enhed med indstillingerne P = 500, Heat = 300; Pull = 40, Velocity = 50, Time = 50. Monter aftrækker med 3 mm &quot;trough&quot; filamenter (Sutter Instrument FT330B). De 2 mm st?...

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Om anæstesi: Da i livmoderen elektroporation i hypothalamus kan være teknisk vanskelig og kræve længere bedøvelsestilstand gange, vi foretrækker at inducere og vedligeholde anæstesi gennem administration af en blanding af oxygen og isofluran. Det er vores erfaring, kan dyrene forbliver passende bedøvet på denne måde i perioder på op til en time i det mindste, inddrivelse af moderen er meget hurtigt, og embryo overlevelse forbedres. Andre tilgange til anæstesi er også tilgængelige. Den mest enkle f...

Genetisk manipulation af embryonale muse hjerne er en foretrukken metode til at lære om udviklingsmæssige regulering. Generering af mutante muselinjer dog er langsomt og dyrt. En kraftfuld metode til at indføre særlige genetiske ændringer i udviklingen neuroner i pattedyrs hjerne er i livmoderen elektroporation. Dybest set Teknikken består i at transficere DNA i embryonale hjerne neuroepithelium ved hjælp af elektriske impulser, derefter lade embryonet til at overleve en vis periode, indsamle hjernen og undersøge dem for mulig roman, informative fænotyper. På denne måde, kan forsøgslederen teste hypoteser næsten øjeblikkeligt uden lange ventetider er nødvendige for produktionen af ​​muse-mutanter. Transfektion af DNA i fostre startede med in ovo elektroporation på kyllingefostre 1.. Den væsentlige proof-of-concept for musen blev udført i kultur <sup&gt; 2.. Dette blev hurtigt efterfulgt af de første beskrivelser af teknikken på musen i livmoderen 3,4. Det vigtigste problem er at transficere hjernen af embryoner udvikler i livmoderen uden at dræbe dem eller moderen. At lære at udføre den nødvendige kirurgi (laparotomi, injektion, elektroporation) kræver en lang uddannelsesperiode. Når operationen er blevet mestret til det punkt, hvor embryo overlevelse forholdet er acceptabel, er det næste centrale spørgsmål er: hvilke hjernens strukturer er tilgængelige? Ikke overraskende, de første offentliggjorte papirer viser resultater opnået med in utero elektroporation fokus på kortikal udvikling 5-9. Dette er stadig sandt for de fleste af de publikationer ved hjælp af denne teknik, da regionen udviklingslandene musehjerne mest tilgængelige for kirurgiske procedurer er cortex (figur 1). Proceduren for in utero elektroporation i cortex er blevet beskrevetpå tryk 10 og i video 11-14. En modifikation af teknikken kan anvendes til at målrette en ventral del af telencephalon, basalganglierne 15.. Ud telencephalon, er diencephalon (klassisk opdelt i thalamus og hypothalamus) en region i forhjernen mere vanskeligt at nå. Et lille antal papirer rapporter målrettet sin ryg og mest tilgængelige del, thalamus 16-19. Hypothalamus er den mest ventrale del af forhjernen, derfor den ene lokaliserede mest dybt fra den dorsale overflade (cortex) (figur 1). Denne region er fortsat en vanskelig udfordring for forskerne forpligtet til genetisk manipulere musehjernen i livmoderen. Til vores viden, melder kun meget få artikler om resultaterne af i livmoderen transfektion i musen hypothalamus 20,21. Men den funktionelle betydning af hypothalamus cannot overvurderes, da det regulerer adfærd som at spise og drikke, parring, avl og forældreskab 22.. Desuden ændringer i hypothalamus udvikling bidrager til at stamme senere i livsbetingelser som fedme, forhøjet blodtryk, diabetes og tidlig pubertet 23.. At være i stand til at ændre genetisk hypothalamus under udvikling vil give et meget kraftfuldt værktøj til at forstå det. Den grundlæggende kirurgiske protokol for laparotomi af gravide mus, som vi bruger her, er magen til den, der anvendes i de andre protokoller 11,13,14,24. Vi vil beskrive dem her kort for fuldstændighedens. Nøglen til vores procedure på den anden side er typen af ​​anæstesi, stedet for indsprøjtning, typen af ​​elektroder og indsættelse og placering af den positive elektrode med hensyn til fosterets hoved. Vi foretrækker at inducere og vedligeholde anæstesi gennem gas indånding løbet enkel intraperitoneal anæstesi, idet førstnævnte giver mulighed forlidt længere narkose kræves for en vanskelig operation. Isofluran inhalation medfører hurtigere helbredelse fra anæstesi, da regel moderen demonstrerer normal adfærd allerede få minutter efter operationen. Den nemmeste punktet af indsprøjtning af DNA opløsningen med glasmikropipette er den laterale ventrikel, som dog er helt uegnet til hypothalamus elektroporation. Injektion direkte i den tredje ventrikel er helt afgørende at målrette dybe diencephalic strukturer. Det er muligt at transfektere hypothalamus fra E12.0 eller E12.5 med standard, off-the-shelf elektroder. Vi har fundet nogle af elektroderne fremstillet af Nepa Gene (Chiba, Japan) særligt velegnet til dette formål. Med vores procedure opnår vi transfektion af hele hypothalamisk neuroepithelium eller delvis, regional transfektion afhængigt elektrode orientering. Her har vi demonstrere teknikken ved transfektion af mammillary kroppen, velsagtensden dybeste og mest forsænket fremmest hypothalamus kerner. Derudover viser vi detaljeret histologisk analyse af de transficerede celler ned til celleniveau af opløsning. En sammenligning af in utero elektroporation med andre tilgange til transfektion af mus hjernens udvikling i livmoderen kan findes i Discussion afsnit.

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	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>REAGENTS</td>
		</tr>
		<tr>
			<td>Acepromazine</td>
			<td>Sanofi GmbH</td>
			<td>&nbsp;</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>HDG9623</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Ketamin</td>
			<td>Pharma GmbH</td>
			<td>&nbsp;</td>
			<td>anesthetic</td>
		</tr>
		<tr>
			<td>Fast Green</td>
			<td>Fluka</td>
			<td>44715</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Rimadyl</td>
			<td>Pfizer</td>
			<td>&nbsp;</td>
			<td>non-steroidal anti-inflammatory</td>
		</tr>
		<tr>
			<td>Braunoderm</td>
			<td>Braun</td>
			<td>3887138</td>
			<td>povidone-iodine</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline PBS</td>
			<td>Gibco</td>
			<td>14190</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Temgesic (buprenorphine)</td>
			<td>Essex Pharma</td>
			<td>&nbsp;</td>
			<td>opioid analgesic</td>
		</tr>
		<tr>
			<td>Eye Ointment</td>
			<td>Pan-Ophtal</td>
			<td>7136926</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Anaesthetic Device Komesaroff Mark-5</td>
			<td>Medical Developments Australia</td>
			<td>ACN 004 903 682</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Capillary puller P-97</td>
			<td>Sutter Instrument Co.</td>
			<td>P-97</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Compresstome</td>
			<td>Precisionary Instr.</td>
			<td>VF-300</td>
			<td>Vibratome-type device</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Zeiss</td>
			<td>LSM700</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM3050S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>Nepa Gene Co. Ltd.</td>
			<td>CUY21EDIT</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electrode 1</td>
			<td>Nepa Gene Co. Ltd.</td>
			<td>CUY550-10</td>
			<td>Stainless Steel Needle Electrode, 10 mm-Tip, 0. 5 mm diam.</td>
		</tr>
		<tr>
			<td>Electrode 2</td>
			<td>Nepa Gene Co. Ltd.</td>
			<td>CUY700P4L</td>
			<td>Cover Round Platinum Plate 4 mm diameter</td>
		</tr>
		<tr>
			<td>Fiberoptic cold light source</td>
			<td>Leica</td>
			<td>KL2500 LCD</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Glass capillaries</td>
			<td>Harvard Apparatus</td>
			<td>GC120T-15</td>
			<td>1. 2 mm O.D. x 0. 94 mm I.D.</td>
		</tr>
		<tr>
			<td>Glass bead sterilizer</td>
			<td>Fine Science Tools</td>
			<td>FST250</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Harvard Apparatus</td>
			<td>py872-5272</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Injection device</td>
			<td>World Precision Instruments</td>
			<td>Pneumatic Pico Pump PV820</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Suture Thread Coated Vicryl</td>
			<td>Ethicon</td>
			<td>V4914</td>
			<td>Peritoneal Suture</td>
		</tr>
		<tr>
			<td>Suture Thr. Supramid</td>
			<td>Serag Wiessner</td>
			<td>TO07171L</td>
			<td>Skin Suture</td>
		</tr>
	</tbody>
</table>

genetic manipulation of the mouse developing hypothalamus through in utero electroporation

Genetisk modifikation af specifikke regioner i udviklingslandene pattedyrs hjerne er en meget kraftfuld eksperimenterende tilgang. Imidlertid genererer nye mus mutanter er ofte frustrerende langsomme. Det har vist sig, at adgangen til musehjernen udvikler i livmoderen med rimelig post-operatory overlevelse er mulig. Alligevel har resultater med denne procedure blevet rapporteret næsten udelukkende til det mest overfladiske og let tilgængelige del af hjernens udvikling, dvs cortex. Thalamus, en smallere og mere mediale region, har vist sig sværere at målet. Transfektion i dybere kerner, især dem i hypothalamus, er måske den mest udfordrende, og derfor meget få resultater er blevet rapporteret. Her viser vi en procedure for at målrette hele hypothalamus neuroepithelium eller en del af det (hypothalamus regioner) til transfektion via elektroporation. Nøglerne til vores tilgang er længere narkose tider, injektion i tHird ventrikel, og passende form og placering af elektroderne. Derudover viser vi resultaterne af målretning og efterfølgende histologisk analyse af de mest forsænket hypothalamus kerne, det mammillary kroppen.

De fleste hypothalamus neuroner er født mellem E11.5 til E15.2 ifølge fødsel-dating analyse rotte 26 oversat til noget kortere musen udvikling 27,28. Toppen af hypothalamus neurogenese nås ved E12.5 29-31. Følgelig, ved transfektion alder valgt til denne undersøgelse (E12.5), kan en stor del af hypothalamus neuroner mærkes på et givent Rostro-caudale niveau. Analyse ved E18.5 på tykke vibratome-typen sektioner (fig. 3A og 3B)...

Watch this Scientific Journal Video about Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation at JoVE.com

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Trods den funktionelle og medicinske betydning af hypothalamus,<em&gt; In utero</em&gt; Genmanipulation af sin udvikling er sjældent blevet forsøgt. Vi viser en detaljeret procedure for<em&gt; In utero</em&gt; Elektroporation i musen hypothalamus og viser repræsentative resultater af total og delvis (regional) hypothalamus transfektion.

Genetisk Manipulation af musen Udvikling Hypothalamus gennem<em&gt; In Utero</em&gt; Elektroporering

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

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Neuroscience

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

In vivo Electroporation of Developing Mouse Retina

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

Genoverførsel til udviklingslandene Mouse indre øre af<em&gt; In Vivo</em&gt; Elektroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

Visualisering og genetisk manipulering af dendritter og udløberne i mus cerebrale cortex og hippocampus under anvendelse<em&gt; I utero</em&gt; Elektroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Genetisk manipulation af Cerebellær granuleneuroner<em&gt; In Vitro</em&gt; Og<em&gt; In Vivo</em&gt; At studere Neuronal morfologi og Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral septal Nucleus- og striatum-specifikke<em&gt; In Utero</em&gt; Elektroporation i C57BL / 6 mus

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

I Utero Elektroporation Approaches to Undersøgelse ophidselse af neuronale subpopulationer og Single-celle Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

Time-lapse konfokal billeddannelse af migrerende neuroner i organotypisk skive kultur af embryonale mus hjernen ved hjælp af<em&gt; I Utero</em&gt; Elektroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...