Name: in vivo microinjection and electroporation of mouse testis
Uploaded: 2014-08-23T15:00:00
Description: Watch this Scientific Journal Video about In Vivo Microinjection and Electroporation of Mouse Testis at JoVE.com

Vi takker Birgit Westernstroeer af Center for Reproductive Medicine og Andrologi ved universitetet i Muenster til undervisning testikel mikroinjektion. Desuden er vi taknemmelige for Ursula Antkewitz og Petra Reckling til teknisk bistand. Vi takker den tyske Research Foundation (DFG) til at støtte dette arbejde (WE2458 / 10-1). &#160;

Alle udførte dyreforsøg er blevet godkendt af den lokale etiske komité (Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei, Mecklenburg-Vorpommern, Tyskland). 1. Plasmid Fremstilling <ol><li> Til plasmidpræparat bruge plasmid oprensningskit (se Materialer tabellen) eller lignende metoder med endotoksin fjernelse puffer, således at immunreaktioner af dyret kan undgås. Følg instruktionerne i manualen. Ansæt Hedeselskabet 2 O for at fortynde plasmidet løsn...

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Forskning inden for reproduktiv biologi, især på området for mandlig fertilitet og spermatogenesen uundgåeligt afhængig levende organismer. For at undersøge testikelfunktionen, er blevet etableret nogen passende cellekultur / in vitro-system, der kan afspejle alle de vigtige morfologiske ændringer fra en diploid spermatogonium til en haploide moden sædcelle 1,2. Således generation af genmodificerede dyr er ofte en nødvendig og som sådan et værdifuldt værktøj i mandlige ...

Pattedyrs spermatogenese anses for at være en sofistikeret proces selvfornyende stamceller successivt undergår mitose, meiose og differentiering med henblik på at udvikle sig til modne haploide spermatozoer. Disse morfologiske ændringer er orkestreret af forskellige celletyper og trods dybtgående forsøg, er det stadig umuligt at efterligne disse processer i cellekulturer 1,2. Derfor forskning i spermatogenesen indtil nu er afhængig af levende organismer, som in vivo modeller. I almindelighed gen-funktionsundersøgelser normalt baseret på transgene dyr. Men generere og opretholde denne type dyremodel er tidskrævende, omkostningskrævende og ganske omfattende. Dette tilskrives den krævede lange avl proces til frembringelse og opretholdelse af transgenet gennem generationer. Derudover genmanipulation af hele organismen af ​​transgene eller knockout fremgangsmåde er tilbøjelig til at give fysiologiske svækkelser når målretning gener med væsentlige funktioner i flere områder, f.eks uden testiklerne eller systemisk. Endvidere er visse forbigående transfektionsfremgangsmåder forbundet med nogle vigtige ulemper. For eksempel typiske ulemper ved virus-medieret genoverførsel er de mulige provokation af immunreaktioner og yderligere sikkerhedsbestemmelser, mens lipofektion 3 og mikropartikelbombardement 4 kan beskadige vævet og er begrænset til en bestemt celle dybde tilstrækkelig transfektionseffektivitet. I modsætning hertil, elektroporation (EP) som en anden almindelig måde transient transfektion, synes at udgøre en lovende teknik til at muliggøre in vivo-transfektion og konsistent in vivo-analyse. I almindelighed EP benævnt et dynamisk fænomen, der afhænger af lokal transmembrane spænding med følgelig medierede porer i nanosekunder. Disse huller kan opretholdes i millisekunder, tilstrækkelig to give adgang til DNA, RNA eller små molekyler 5. Når den anvendte spænding er for høj, er det sædvanligvis forbigående karakter EP modvirkes på grund af varme produktion og induktion af for omfattende permeabilisering med deraf irreversibel ødelæggelse af cellen 5. Her viser vi, at elektroporering er en effektiv og økonomisk transfektion, som er i stand til at blive udnyttet til genetisk testis transformation for at belyse testikulære gen egenskaber in vivo. Denne artikel omhandler plasmidpræparat, mikroinjektion via efferente kanal og den efterfølgende elektroporering af muse testis. Denne procedure kan være midlet til valg til at opnå hurtig, konkret og effektiv transfektion af sædkanalerne af muse testikler in vivo for at undersøge processer spermatogenesen.

<table border="0" cellpadding="0" cellspacing="3">
	<tbody>
		<tr>
			<td>
			centrifuge
			</td>
			<td>Sigma</td>
			<td>1-15 PK</td>
			<td></td>
		</tr>
		<tr>
			<td>spectrophotometer</td>
			<td>Nanodrop</td>
			<td>ND-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>micropipette puller</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td>vertical capillary puller</td>
		</tr>
		<tr>
			<td>microinjection capillaries</td>
			<td>Clark</td>
			<td>GC100-10</td>
			<td>borosilicate standard wall</td>
		</tr>
		<tr>
			<td>micropipette beveler</td>
			<td>Bachofer</td>
			<td>Typ 462</td>
			<td>rotating disk beveler</td>
		</tr>
		<tr>
			<td>electroporator</td>
			<td>Nepagene</td>
			<td>CUY 21EDIT</td>
			<td>square wave electroporator</td>
		</tr>
		<tr>
			<td>tweezer electrodes</td>
			<td>Nepagene</td>
			<td>CUY 650P5</td>
			<td>5 mm &#216; disk electrodes</td>
		</tr>
		<tr>
			<td>stereo microscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000-C</td>
			<td></td>
		</tr>
		<tr>
			<td>cold light source</td>
			<td>Zeiss</td>
			<td>KL 1500LCD</td>
			<td></td>
		</tr>
		<tr>
			<td>microinjection pump</td>
			<td>Eppendorf</td>
			<td>Femtojet</td>
			<td></td>
		</tr>
		<tr>
			<td>micromanipulator</td>
			<td>homemade</td>
			<td></td>
			<td>XYZ cross table</td>
		</tr>
		<tr>
			<td>surgical instruments</td>
			<td>FST</td>
			<td></td>
			<td>scissors, forceps, 
			needle holder</td>
		</tr>
		<tr>
			<td>fine forceps</td>
			<td>FST</td>
			<td>Dumont #5, #7</td>
			<td>efferent ducts preparation</td>
		</tr>
		<tr>
			<td>Michel clip applying stapler</td>
			<td>FST</td>
			<td>Michel, 12029-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Michel suture clips</td>
			<td>FST</td>
			<td>Michel, 12040-01</td>
			<td>7.5 x 1.75 mm</td>
		</tr>
		<tr>
			<td>surgical suture</td>
			<td>Catgut 00</td>
			<td>Catgut 
			Markenkirchen</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe, with 30G needle</td>
			<td>B. Braun</td>
			<td>Omnican 40</td>
			<td>to load micropipette and 
			for anesthesia</td>
		</tr>
		<tr>
			<td>plasmid isolation kit</td>
			<td>Promega</td>
			<td>Cat. # A2495</td>
			<td>plasmid Midiprep</td>
		</tr>
		<tr>
			<td>plasmid pEGFP-C1</td>
			<td>Clontech</td>
			<td>Cat. #6084-1</td>
			<td>CMV-promoter + EGFP</td>
		</tr>
		<tr>
			<td>plasmid pDsRed2-N1</td>
			<td>Clontech</td>
			<td>Cat. #6084-1</td>
			<td>CMV-promoter + DsRed2</td>
		</tr>
		<tr>
			<td>Fast Green dye</td>
			<td>Sigma</td>
			<td>F7258-25G</td>
			<td>for dilution in ddH2O</td>
		</tr>
		<tr>
			<td>10x PBS, pH 7.4</td>
			<td>Carl Roth</td>
			<td>3957.1</td>
			<td>NaCl [1.37 M]</td>
		</tr>
		<tr>
			<td>6781.1</td>
			<td>KCl [27 mM]</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T106.2</td>
			<td>Na2HPO4+2H2O [100 mM]</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3904.1</td>
			<td>KH2PO4 [18 mM]</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10% Ketamin</td>
			<td>Serum Werk 
			Bernburg</td>
			<td>Urotamin</td>
			<td>mix in a rate 1:1</td>
		</tr>
		<tr>
			<td>2% Xylazin</td>
			<td>Serum Werk 
			Bernburg</td>
			<td>Xylazin</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilium</td>
			<td>Bode Chemie</td>
			<td>Sterillium</td>
			<td>disinfection</td>
		</tr>
		<tr>
			<td>vet ointment</td>
			<td>S&#38;K Pharma 
			GmbH</td>
			<td>Kerato Biciron 5% 
			Augensalbe</td>
			<td>or similar to prevent 
			dryness of eyes</td>
		</tr>
		<tr>
			<td>To-Pro-3 iodide</td>
			<td>Invitrogen</td>
			<td>T3605</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo microinjection and electroporation of mouse testis

Denne video og artiklen bidrag giver en omfattende beskrivelse af mikroinjektion og elektroporation af muse testikler in vivo. Denne særlige transfektion teknik til testikulære museceller tillader undersøgelse af unikke processer i spermatogenese. Følgende protokol fokuserer på transfektion af testikulære museceller med plasmidkonstruktioner. Specifikt har vi brugt reporter vektoren pEGFP-C1, som udtrykker forbedret grønt fluorescerende protein (eGFP) samt pDsRed2-N1 vektor, der udtrykker rødt fluorescerende protein (DsRed2). Begge kodede reportergener var under kontrol af den humane cytomegalovirus immediate-early-promotoren (CMV). For at kunne udføre genoverførsel til musetestiklerne, er reporter plasmidkonstruktioner injiceret i testiklerne af levende mus. Til dette formål er testiklerne af en bedøvet dyr eksponeret og stedet for mikroinjektion er forberedt. Vores foretrukne stedinjektion er den efferente kanalen, med den i sidste ende tilsluttede rete testis som anatomiske transportvej af spermatozoer mellem testiklerne og epididymis. På denne måde er fyldningen af ​​sædkanalerne efter mikroinjektion glimrende styres og kontrolleres på grund af anvendelsen af ​​farvede DNA-opløsninger. Efter at have observeret en tilstrækkelig fyldning af testis ved sin farvede tubulus struktur, er organet elektroporeres. Dette muliggør overførsel af DNA-opløsningen i de testikulære celler. Efter 3 dages inkubation testikler fjernes og undersøges under mikroskop for grøn eller rød fluorescens, der illustrerer transfektion succes. Generelt kan denne protokol anvendes til at levere DNA- eller RNA- konstruktioner i levende mus testikel for at (over) udtrykker eller vælte gener, letter in vivo-funktion analyse. Desuden er det velegnet til at studere reporterkonstruktioner eller formodede gen regulatory elementer. Således er de vigtigste fordele ved elektroporationsteknik er hurtige resultater i kombination med lav indsats samt den moderate teknisk udstyr og færdigheder, der kræves i forhold til alternative teknikker.

Den eksperimentelle indstilling til udførelse mikroinjektion og elektroporation af muse testis in vivo, som det anvendes i overensstemmelse med den protokol er vist i figur 1. Selv om det er muligt at erhverve industrielt fremstillede mikropipetter, foretrak vi at generere vores egne pipetter ved at trække (figur 1A ) og beveling (Figur 1B) glaskapillarer så de monteret vores behov. Udstyret til mikroinjektion og elektroporation er illustreret i figu...

Watch this Scientific Journal Video about In Vivo Microinjection and Electroporation of Mouse Testis at JoVE.com

In Vivo Microinjection and Electroporation of Mouse Testis

In vivo Mikroinjektion og Elektroporation af Mouse Testis

Denne artikel beskriver mikroinjektion og elektroporation af muse testis in vivo som en transfektion teknik til testikulære museceller at studere unikke processer spermatogenesen. Den præsenterede protokol involverer trin glaskapillar forberedelse, mikroinjektion via efferente kanalen og transfektion ved elektroporation.

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...

Research

JoVE Journal

Biology

In Vivo Microinjection and Electroporation of Mouse Testis

In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos

In-utero in-vivo injection and electroporation of the embryonic mouse neocortex provides a powerful tool for the manipulation of individual progenitors ...

Single Cell Electroporation in vivo within the Intact Developing Brain

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Germ Cell Transplantation og testikel væv Xenografting i mus

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

In vivo Elektroporation af Morpholinos i den voksne zebrafisk Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

In vivo elektroporation af Morpholinos i Regenerating Adult zebrafisk Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...