Name: generation of genetically modified mice through the microinjection of oocytes
Uploaded: 2017-06-15T13:00:00
Description: Watch this Scientific Journal Video about Generation of Genetically Modified Mice through the Microinjection of Oocytes at JoVE.com

Forfatterne takker personalet på dyreanlegget (BRC) for deres løpende støtte. Dette arbeidet ble finansiert av National Health and Medical Research Council og Australian Research Council.

Alle prosedyrer er godkjent av University of New South Wales Dyrpleie og etikkutvalg. 1. Fremstilling av transgenet (tilfeldig integrasjon) <ol><li> Analytisk agarosegelelektroforese. <ol><li> Fordel plasmidet for å aksessere transgenet ved bruk av passende enzymer (1 time inkubering) eller rask fordøyelsesenzymer (15 til 30 minutter inkubering) i en termocykler som følger produsentens anbefalinger (se figur 2A og dens legende). </li><li> K...

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Kritiske trinn i protokollen Genereringen av genetisk modifiserte mus er kjent for å være teknisk utfordrende. Protokollen som presenteres her er imidlertid en optimalisert og forenklet metode som gjør at man kan mestre og feilsøke teknikken i rekordtid. Det er to trinn som er nødvendige for en vellykket gjennomføring av teknikken. For det første kan syntesen av lineære DNA-maler (for syntese av sgRNAer) oppnås uten magnesiumklorid (MgCl2). Imidlertid anbefales det å systematisk legge...

Dyremodeller, både hos vertebrater og vertebrater, har vært med på å undersøke patofysiologien av menneskelige tilstander som Alzheimers sykdom 1 , 2 . De er også uvurderlige verktøy for å søke etter sykdomsmodifikatorer og til slutt utvikle nye behandlingsstrategier i håp om å kurere. Selv om hver modell har inneboende begrensninger, er bruk av dyr som hele systemiske modeller avgjørende for biomedisinsk forskning. Dette skyldes at det metabolske og komplekse fysiologiske miljøet ikke helt kan simuleres i vevskultur. Hittil er musen den vanligste pattedyrsarten som brukes til genetisk manipulering fordi den har flere fordeler. De fysiologiske prosesser og gener forbundet med sykdommer er sterkt konserverte mellom mus og mennesker. Musen var det første pattedyret som hadde sitt fulle genom sekvensert (2002), ett år før det menneskelige genoMeg (2003). Bortsett fra denne mengden genetisk informasjon, har musen god avlskapacitet, en rask utviklingssyklus (6 uker fra befruktning til avvenning), og en rimelig størrelse. Alle disse fordelene, kombinert med fysiologiske indikatorer, som forskjellige farger (nødvendig for kryssstrategier), gjorde musen til en attraktiv modell for genetisk manipulasjon. Spesielt, i den tidlige alderen av moderne genetikk, begynte Gregor Mendel å jobbe med mus før han flyttet til planter 3 . Gene overføringsteknikker resulterte i generasjonen av den første transgene musen over tre tiår siden 4 , opprinnelig opprettet ved hjelp av viral levering. Imidlertid innså forskere snart at en av hovedutfordringene ved mus transgenese var manglende evne til å kontrollere skjebnen til det eksogene DNA. Fordi den virale tilførsel av transgener i musoocytter resulterte i flere kopier integrert tilfeldig inn i genomet, kunne mulighetenY for å etablere etterfølgende transgene linjer var begrenset. En slik begrensning ble overvunnet da Gordon et al. Genererte den første transgene muselinjen ved mikroinjeksjon 5 , 6 . Dette begynte epoken med rekombinant DNA-teknologi, og parametrene som påvirker utfallet av en mikroinjeksjonssesjon har blitt studert mye 7 . Selv om mikroinjeksjon ikke tillater kontroll over integrasjonsstedet for transgenet (som til slutt resulterer i spesifikke uttrykksnivåer for hver grunnmus), forblir den viktigste fordelen av pronuklear mikroinjeksjon dannelsen av konkatemere ( dvs. arrays av flere kopier av transgenet, Koblet i serie) før genomisk integrasjon 5 . Denne egenskapen har blitt brukt over årene for å etablere tusenvis av transgene muselinjer som overexpresser et gen av interesse. Siden da har transgenese, aRasjonell modifikasjon av et organismers genom, har blitt mye brukt til å identifisere rollen som enkeltgener i forekomsten av sykdommer. En ytterligere nøkkeloppnåelse i manipulering av musegenometet ble nådd da Mario Capecchi forstyrret et enkelt gen i musen, og åpnet epoken med genmålretting 8 . Imidlertid oppstod store ulemper raskt fra ES-cellebasert genmålretting, inkludert utfordringene ved å dyrke ES-celler, den noe variable graden av chimerisme og lengden av prosessen ( dvs. 12-18 måneder, minimum, for å oppnå musen) . Nylig har fremskritt i ny teknologi, slik som konstruerte endonukleaser ( f.eks. Sinkfingernukleaser (ZFN), transkripsjonsaktivator-lignende effektorukleaser (TALEN) og klyngede regelmessige interspaced korte palindromiske gjentakelser (CRISPR / Cas9)) oppstått som alternative metoder for å Akselerere prosessen med genmålretting i mikrofonE 9 , 10 . Disse endonukleaser kan lett injiseres i musoocytter ved mikroinjeksjon, noe som tillater generering av genmålrettede mus på så lite som 6 uker. Siden den første rapporten om bruk av CRISPR for genomredigering 11 , har dette bakterielle adaptive immunsystemet erstattet ZFN og TALEN på grunn av dets mange fordeler, inkludert enkel syntese og evnen til å målrette flere loki på en gang (referert til som &quot;multipleksering &quot;). CRISPR ble først brukt til genmålretting i mus 12 og har siden blitt anvendt på utallige arter, fra planter til mennesker 13 , 14 . Til nå er det ingen rapport om en enkelt art som er resistent mot CRISPR genom redigering. De to hovedbegrensende trinnene i generasjonen av transgene mus er injeksjonen av oocytter og reimplantasjonenAv disse oocytene til pseudo-gravide kvinner. Selv om denne teknikken har blitt beskrevet av oss 15 og andre 16 , har nyere tekniske forbedringer i musembryologi og genoverføringsteknikker revolusjonert prosessen med å generere genetisk modifiserte mus. Disse forbedringene vil bli beskrevet her.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Micropipette 0.1-2.5 ul</td>
			<td style="width:147px;">Eppendorf</td>
			<td style="width:119px;">4920000016</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Micropipette 2-20 ul</td>
			<td style="width:147px;">Eppendorf</td>
			<td>4920000040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Micropipette 20-200 ul</td>
			<td style="width:147px;">Eppendorf</td>
			<td>4920000067</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Micropipette 100-1000 ul</td>
			<td style="width:147px;">Eppendorf</td>
			<td>4920000083</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Molecular weight marker&#160;</td>
			<td>Bioline</td>
			<td>BIO-33025</td>
			<td>HyperLadder 1kb</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Molecular weight marker&#160;</td>
			<td>Bioline</td>
			<td>BIO-33056</td>
			<td>HyperLadder 100 bp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Agarose</td>
			<td>Bioline</td>
			<td>BIO-41025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">EDTA buffer</td>
			<td>Sigma-Aldrich</td>
			<td>93296</td>
			<td>10x - Dilute to 1x</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Ethidium bromide</td>
			<td style="width:147px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15585011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SYBR Safe gel stain</td>
			<td>Invitrogen</td>
			<td>S33102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Gel extraction kit</td>
			<td style="width:147px;">Qiagen</td>
			<td style="width:119px;">28706</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">PCR purification kit (Qiaquick)</td>
			<td style="width:147px;">Qiagen</td>
			<td style="width:119px;">28106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Vacuum system (Manifold)</td>
			<td style="width:147px;">Promega</td>
			<td style="width:119px;">A7231</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nuclease-free microinjection buffer&#160;</td>
			<td>Millipore</td>
			<td>MR-095-10F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrafree-MC microcentrifuge filter&#160;</td>
			<td>Millipore</td>
			<td>UFC30GV00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Cas9 mRNA</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td style="width:119px;">CAS9MRNA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">CRISPR expressing plasmid (px330)</td>
			<td style="width:147px;">Addgene</td>
			<td style="width:119px;">42230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Nuclease free water</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td>W4502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Phusion polymerase</td>
			<td style="width:147px;">New England Biolabs</td>
			<td>M0530L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T7 Quick High Yield RNA kit</td>
			<td style="width:147px;">New England Biolabs</td>
			<td>E2050S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">RNA purification spin columns (NucAway)</td>
			<td style="width:147px;">Thermo Fisher Scientific</td>
			<td>AM10070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">ssOligos</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td>OLIGO STANDARD</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Donor plasmid</td>
			<td style="width:147px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">GeneArt</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Hyaluronidase</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">KSOMaa embryo culture medium</td>
			<td>Zenith Biotech&#160;</td>
			<td>ZEKS-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Mineral oil</td>
			<td>Zenith Biotech&#160;</td>
			<td>ZSCO-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">M2 Medium</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td>M7167</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Cytochalasin B</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td>C6762</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Mouthpiece</td>
			<td style="width:147px;">Sigma-Aldrich</td>
			<td style="width:119px;">A5177</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Glass microcapillaries</td>
			<td style="width:147px;">Sutter Instrument</td>
			<td style="width:119px;">BF100-78-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Proteinase K</td>
			<td>Applichem</td>
			<td>A3830.0100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Dumont #5 forceps</td>
			<td>Fine Science Tools&#160;</td>
			<td style="width:119px;">91150-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Iris scissors</td>
			<td>Fine Science Tools&#160;</td>
			<td style="width:119px;">91460-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Vessel clamp</td>
			<td>Fine Science Tools&#160;</td>
			<td style="width:119px;">18374-43</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Wound clips&#160;</td>
			<td>Fine Science Tools&#160;</td>
			<td style="width:119px;">12040-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Clips applier&#160;</td>
			<td>Fine Science Tools&#160;</td>
			<td style="width:119px;">12018-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-scissors&#160;</td>
			<td>Fine Science Tools&#160;</td>
			<td>15000-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cauterizer</td>
			<td>Fine Science Tools&#160;</td>
			<td>18000-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Non-absorbable surgical sutures (Ethilon 3-0)</td>
			<td>Ethicon</td>
			<td>1691H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">5% CO2 incubator</td>
			<td style="width:147px;">MG Scientific</td>
			<td style="width:119px;">Galaxy 14S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Spectrophotometer</td>
			<td style="width:147px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">Nanodrop 2000c</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Thermocycler</td>
			<td style="width:147px;">Eppendorf</td>
			<td style="width:119px;">6321 000.515</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Electrophoresis set up</td>
			<td style="width:147px;">BioRad</td>
			<td style="width:119px;">1640300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">UV Transilluminator</td>
			<td style="width:147px;">BioRad</td>
			<td style="width:119px;">1708110EDU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Thermocycler</td>
			<td style="width:147px;">Eppendorf</td>
			<td>6334000069</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereoscopic microscope</td>
			<td>Olympus</td>
			<td>SZX7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Inverted microscope</td>
			<td>Olympus</td>
			<td style="width:119px;">IX71</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">2x Micromanipulators</td>
			<td style="width:147px;">Eppendorf</td>
			<td style="width:119px;">5188000.012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Oocytes manipulator</td>
			<td style="width:147px;">Eppendorf</td>
			<td style="width:119px;">5176000.025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Microinjector (Femtojet)</td>
			<td style="width:147px;">Eppendorf</td>
			<td style="width:119px;">5247000.013</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Mice C57BL/6J strain</td>
			<td style="width:147px;">Australian BioResources</td>
			<td>C57BL/6JAusb&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of genetically modified mice through the microinjection of oocytes

Bruken av genetisk modifiserte mus har vesentlig bidratt til studier på både fysiologiske og patologiske in vivo prosesser. Den pronukleære injeksjonen av DNA-ekspresjonskonstruksjoner i befruktede oocytter forblir den mest brukte teknikken for å generere transgene mus for overekspresjon. Med introduksjonen av CRISPR-teknologi for genmålretting, har pronukleær injeksjon i befruktede oocytter blitt utvidet til generering av både knockout- og knock-mus. Dette arbeidet beskriver fremstillingen av DNA til injeksjon og generering av CRISPR-guider for genmålretting, med særlig vekt på kvalitetskontroll. Genotypingsprosedyrene som kreves for identifisering av potensielle grunnleggere er kritiske. Innovative genotyping strategier som utnytter &quot;Multiplexing&quot; evner CRISPR er presentert her. Kirurgiske prosedyrer er også skissert. Sammen vil trinnene i protokollen tillate generering av genEtisk modifiserte mus og for påfølgende etablering av musekolonier for en overflod av forskningsfelt, inkludert immunologi, nevrovitenskap, kreft, fysiologi, utvikling og andre.

Nedenfor er arbeidsflytene for mikroinjeksjon i tilfelle av tilfeldig integrering og CRISPR-mediert genmålretting beskrevet ( figur 1 ). <img alt="Figur 1" class="xfigimg" src="/files/ftp_upload/55765/55765fig1v2.jpg" /> Figur 1: Typisk arbeidsflyt for generering av genetisk modifiserte mus. For tilfeldig integrasjon injiseres det rensede transgenet i pronucleus av befrukted...

Watch this Scientific Journal Video about Generation of Genetically Modified Mice through the Microinjection of Oocytes at JoVE.com

Generation of Genetically Modified Mice through the Microinjection of Oocytes

Mikroinjeksjonen av mus-oocytter blir ofte brukt for både klassisk transgenese ( dvs. tilfeldig integrasjon av transgene) og CRISPR-mediert genmålretting. Denne protokollen vurderer de siste utviklingene i mikroinjeksjon, med særlig vekt på kvalitetskontroll og genotyping-strategier.

Generering av genetisk modifiserte mus gjennom mikroinjeksjon av oocytter

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

Research

JoVE Journal

Developmental Biology

Generation of Human Adipose Stem Cells through Dedifferentiation of Mature Adipocytes in Ceiling Cultures

Generation of Human adipose stamceller gjennom Dedifferentiation av Mature Adipocytter i Ceiling kulturer

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Generering av genmodifiserte organotypiske Skin kulturer Bruke devitalized Menneskelig Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Technique to Target Microinjection to the Developing Xenopus Kidney

Technique to Target Microinjection to the Developing Xenopus Kidney

Teknikken til Target Mikroinjeksjon til utviklings<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 ...

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

Embryo Mikroinjeksjon og Electroporation i chordate<em&gt; Ciona intestinalis</em

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

Generasjon 3D huden Organoid fra ledningen blod-avledet indusert Pluripotent stamceller

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

Generation of Organoids from Mouse Extrahepatic Bile Ducts

Generasjon av Organoids fra mus Extrahepatic galle kanaler

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

Generasjon av en human iPSC-basert blod-brain barriere chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

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 ...

Generation and Expansion of Human Cardiomyocytes from Patient Peripheral Blood Mononuclear Cells

Generering og utvidelse av humane kardiomyocytter fra pasientens perifere blodmonnukleære celler

Generating patient-specific cardiomyocytes from a single blood draw has attracted tremendous interest in precision medicine on cardiovascular disease. ...