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 dyreanlægets personale (BRC) for deres løbende støtte. Dette arbejde blev finansieret af National Health and Medical Research Council og Australian Research Council.

Alle procedurer er godkendt af University of New South Wales Dyrpleje og Etikudvalg. 1. Fremstilling af transgenet (tilfældig integration) <ol><li> Analytisk agarosegelelektroforese. <ol><li> Fordel plasmidet for at punge transgenet ved hjælp af passende enzymer (1 time inkubation) eller hurtigt fordøjelsesenzymer (15 til 30 minutter inkubation) i en termocykler efter producentens anbefalinger (se figur 2A og dens legende). </li><li> Stiv e...

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Kritiske trin i protokollen Genereringen af ​​genetisk modificerede mus er kendt for at være teknisk udfordrende. Protokollen, der præsenteres her, er imidlertid en optimeret og forenklet metode, der gør det muligt for en at mestre og fejlfinding teknikken i rekordtid. Der er to trin nødvendige for en vellykket gennemførelse af teknikken. For det første kan syntesen af ​​lineære DNA-skabeloner (til syntese af sgRNA&#39;er) opnås uden magnesiumchlorid (MgCl2). Det anbefales dog s...

Dyremodeller, både hos hvirveldyr og hvirvelløse dyr, har været med til at undersøge patofysiologien af ​​humane tilstande såsom Alzheimers sygdom 1 , 2 . De er også uvurderlige værktøjer til at søge efter sygdomsmodifikatorer og til sidst udvikle nye behandlingsstrategier i håb om en kur. Selvom hver model har indre begrænsninger, er anvendelsen af ​​dyr som hele systemiske modeller afgørende for biomedicinsk forskning. Dette skyldes, at det metaboliske og komplekse fysiologiske miljø ikke helt kan simuleres i vævskulturen. Til dato er musen den mest almindelige pattedyrsart, der anvendes til genetisk manipulation, fordi den har flere fordele. De fysiologiske processer og gener forbundet med sygdomme er stærkt konserveret mellem mus og mennesker. Musen var det første pattedyr, der havde sin fulde genom sekventeret (2002), et år før den menneskelige genoMig (2003). Bortset fra denne rigdom af genetisk information har musen god avlskapacitet, en hurtig udviklingscyklus (6 uger fra befrugtning til fravænning) og en rimelig størrelse. Alle disse fordele, kombineret med fysiologiske indikatorer, som f.eks. Klare farver (kræves til krydsstrategier), gjorde musen til en attraktiv model til genetisk manipulation. I den tidlige moderne genetikkens begyndelse begyndte Gregor Mendel at arbejde på mus, før han flyttede til planter 3 . Genoverførselsteknikker resulterede i dannelsen af ​​den første transgene mus over tre årtier siden 4 , der oprindeligt blev oprettet ved hjælp af viral levering. Forskere indså imidlertid snart, at en af ​​de vigtigste udfordringer ved mus transgenese var manglende evne til at kontrollere skæbnen af ​​det eksogene DNA. Fordi den virale afgivelse af transgener i musoocytter resulterede i flere kopier integreret tilfældigt i genomet, kunne mulighedenY for at etablere efterfølgende transgene linjer var begrænset. En sådan begrænsning blev overvundet, når Gordon et al. Dannet den første transgene muselinie ved mikroinjektion 5 , 6 . Dette begyndte æra med rekombinant DNA-teknologi, og parametrene, der påvirker resultatet af en mikroinjektionssession, er blevet studeret bredt 7 . Selv om mikroinjektion ikke tillader kontrol over transgenets integrationssted (som i sidste ende resulterer i specifikke ekspressionsniveauer for hver grundmus), forbliver den primære fordel ved pronuklear mikroinjektion dannelsen af ​​concatemers ( dvs. arrays af flere kopier af transgenet, Forbundet i serie) før genomisk integration 5 . Denne egenskab er blevet brugt gennem årene til at etablere tusindvis af transgene muselinier, der overudtrykker et gen af ​​interesse. Siden da har transgenese, aRtificerende modifikation af et organismer genom har i vid udstrækning været anvendt til at identificere de enkelte geners rolle i forekomsten af ​​sygdomme. En yderligere vigtig præstation i manipulering af musegenomet blev nået, da Mario Capecchi med succes forstyrrede et enkelt gen i musen og åbner æra med genmålretning 8 . Imidlertid opstod der store ulemper hurtigt fra ES-cellebaseret genmålretning, herunder udfordringerne ved dyrkning af ES-celler, den noget variable grad af chimerisme og længden af ​​processen ( dvs. 12-18 måneder minimum for at opnå musen) . For nylig er fremskridt inden for ny teknologi, såsom manipulerede endonukleaser ( fx zinkfingernukleaser (ZFN), transkriptionsaktivatorlignende effektor-nukleaser (TALEN) og klyngede regelmæssigt interspaced korte palindromiske gentagelser (CRISPR / Cas9)) fremkommet som alternative metoder til Fremskynde processen med genmålretning i mikrofonenE 9 , 10 . Disse endonucleaser kan let injiceres i musococytter ved mikroinjektion, hvilket tillader generering af genmålrettede mus i så lidt som 6 uger. Siden den første rapport om brugen af ​​CRISPR til genomredigering 11 har dette bakterielt adaptive immunsystem erstattet ZFN og TALEN på grund af dets mange fordele, herunder den lette syntese og evnen til at målrette flere loci på én gang (kaldet multiplexing &quot;). CRISPR blev først anvendt til genmålretning i mus 12 og er siden blevet anvendt på utallige arter fra planter til mennesker 13 , 14 . Til dato er der ingen rapport om en enkelt art, der er resistent over for CRISPR genom redigering. De to hovedbegrænsende trin i generationen af ​​transgene mus er injektionen af ​​oocytter og reimplantationenAf disse oocytter til pseudo-gravide kvinder. Selv om denne teknik er blevet beskrevet af os 15 og andre 16 , har de seneste tekniske forbedringer i musembryologi og genoverførselsteknikker revolutioneret processen med at generere genetisk modificerede mus. Disse forbedringer vil blive beskrevet heri.

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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

Anvendelsen af ​​genetisk modificerede mus har signifikant bidraget til studier på både fysiologiske og patologiske in vivo- processer. Den prækukleære injektion af DNA-ekspressionskonstruktioner i befrugtede oocytter forbliver den mest almindeligt anvendte teknik til frembringelse af transgene mus til overekspression. Med introduktionen af ​​CRISPR-teknologi til genmålretning er pronukleær injektion i befrugtede oocytter blevet udvidet til dannelsen af ​​både knockout- og knock-mus. Dette arbejde beskriver fremstillingen af ​​DNA til injektion og dannelsen af ​​CRISPR-guider til genmålretning med særlig vægt på kvalitetskontrol. Genotypeprocedurerne til identifikation af potentielle grundlæggere er kritiske. Innovative genotypebestemmelser, der udnytter CRISPR&#39;s &quot;multiplexing&quot; -muligheder, præsenteres her. Kirurgiske procedurer er også beskrevet. Sammen vil protokollens trin tillade generering af genEtisk modificerede mus og til efterfølgende etablering af musekolonier for en overflod af forskningsområder, herunder immunologi, neurovidenskab, cancer, fysiologi, udvikling og andre.

Nedenfor beskrives arbejdsprocesserne for mikroinjektion i tilfælde af tilfældig integration og CRISPR-medieret genmålretning ( Figur 1 ). <img alt="figur 1" class="xfigimg" src="/files/ftp_upload/55765/55765fig1v2.jpg" /> Figur 1: Typisk arbejdsstrøm til generering af genetisk modificerede mus. Til tilfældig integration injiceres det oprensede transgen i pronucleus af b...

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

Mikroinjektionen af ​​musoocytter er almindeligt anvendt til både klassisk transgenese ( dvs. den tilfældige integration af transgene) og CRISPR-medieret genmålretning. Denne protokol gennemgår de seneste udviklinger inden for mikroinjektion, med særlig vægt på kvalitetskontrol og genotyping-strategier.

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 ...

Research

JoVE Journal

Developmental Biology

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

Generation af menneskelige fedt-stamceller gennem dedifferentiering af Ældre Adipocytter i Loft Cultures

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 af genetisk modificerede Organotypiske Skin Cultures Brug devitaliseret 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

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 ...

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

Embryo Mikroinjektion og Elektroporering i Chordatzoologi<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

Generation af 3D hud Organoid fra ledningen blod-afledte induceret pluripotente 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

Generation af Organoids fra mus ekstrahepatisk galdeveje

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

Generation af en menneskelig iPSC-baseret blod-hjerne barrierchip

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 volumen vitrifikation af umodne Feline Oocytter

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

Adskillelse af follikulære celler og oocytter i æggestokkene Follikler af zebrafisk

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 udvidelse af humane kardiomyocytter fra patientens perifere blod mononukleare celler

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