Name: crispr-mediated loss of function analysis in cerebellar granule cells using in utero electroporation-based gene transfer
Uploaded: 2018-06-09T13:30:00
Description: Watch this Scientific Journal Video about CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer at JoVE.com

Vi sætter pris på Laura Sieber, Anna Neuerburg, Yassin Harim og Petra Schroeter for teknisk bistand. Vi takker også Drs. K. Reifenberg, K. Dell og P. Prückl for nyttige støtte til dyreforsøg på DKFZ; den Imaging Core faciliteter af DKFZ og Carl Zeiss Imaging Center i DKFZ for konfokalmikroskopi billeddannelse. Dette arbejde blev støttet af Deutsche Forschungsgemeinschaft, KA 4472/1-1 (til D.K.).

Alle dyreforsøg blev gennemført efter dyrevelfærd forordninger og er blevet godkendt af de ansvarlige myndigheder (Regierungspräsidium Karlsruhe, godkendelsesnumre: G90/13, G176/13, G32/14, G48/14 og G133/14).
1. generere pU6-sgRNA-CVH-Cre plasmider
<ol><li>Design sgRNA til at målrette en gen-af-interesse efter tidligere udgivne protokol14. Bemærk: I dette eksperiment, to sgRNAs er designet til at målrette mus Top2b gen og en ikke-målrettet kontrol...

<ol>
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Brug exo utero elektroporation, har vi tidligere rapporteret, siRNA-baseret i vivo funktionelle analyser af Atoh1 på et tidligt stadium af cerebellare granula celle differentiering8. SiRNA fortynding/nedbrydning og eksponering af embryoner uden for livmodervæggen var fænotypiske analyser af electroporated granula celler begrænset til vorden. Men den nuværende metode aktiveret analyse af Fænotypen af postnatal dyr.
Vores tidligere undersøgelse vi...

Hjernesygdomme er en af de mest forfærdelige dødelige sygdomme. De skyldes ofte genetiske mutationer og efterfølgende dysregulering. For at forstå molekylære mekanismer af hjernesygdomme, har nogensinde langvarige bestræbelser på at dechifrere genomer af menneskelige patienter opdaget en række potentielle sygdomsfremkaldende gener. Hidtil, har været udnyttet kønscelleoverførsel genetisk manipuleret dyremodeller for in vivo gevinst-af-funktion (GOF) og tab af funktion (LOF) analyser af sådanne kandidat gener. På grund af den fremskyndede udviklingen af funktionelle valideringsundersøgelser, en mere realistisk og fleksibel i vivo gen assay system for at studere genfunktion er ønskeligt.
Anvendelse af en in vivo elektroporation-baserede gen overførsel systemet at udvikle mus hjernen er velegnet til dette formål. Faktisk har flere undersøgelser, i utero elektroporation vist deres potentiale til at gennemføre funktionelle analyser i udviklingslandene hjernen1,2,3. Faktisk, flere regioner i mus hjernen, såsom cerebral cortex4, nethinden5, diencephalon6, baghjernen7, lillehjernen8og rygmarven9 er blevet angrebet af somatiske gen levering tilgange, så langt.
Faktisk, forbigående genekspression af i vivo elektroporation på embryonale mus hjerner har længe været anvendt til GOF analyse. Seneste transposon-baserede genomisk integration teknologier yderligere aktiveret langsigtet og/eller betingede udtryk af gener af interesse10,11, hvilket er en fordel at dissekere genfunktion i en rumlig og tidsmæssig måde under udvikling. I modsætning til GOF analyse, har LOF analyse været mere udfordrende. Mens forbigående Transfektion af siRNAs og shRNA-regnskabsmæssige plasmider blev udført, er langsigtede virkninger af LOF af gener ikke garanteret på grund af eventuel forringelse af eksogent indførte nukleinsyrer, såsom plasmider og dsRNAs. CRISPR/Cas-teknologien giver imidlertid et gennembrud i LOF analyser. Gener kodning fluorescerende proteiner (f.eks.normal god landbrugspraksis) eller en bioluminescerende proteiner (fxfirefly luciferase) har været co transfekteret med CRISPR-Cas9 og sgRNAs til at mærke de celler udsat for CRISPR-Cas9-medieret somatiske mutationer. Ikke desto mindre, denne tilgang kan have begrænsninger i funktionelle studier om prolifererende celler, da eksogene markørgener er udvandet og forringet efter langsigtede spredning. Mens de transfected celler og deres datter celler gennemgå CRISPR-induceret mutationer i deres genomer, kan deres fodspor få tabt over tid. Genetiske mærkning tilgange vil således egnet til at overvinde dette problem.
Vi har for nylig udviklet en CRISPR-baseret LOF metode i cerebellare granula celler, der undergår langsigtede spredning under deres differentiering12. At genetisk mærke de transfected celler, vi bygget et plasmid, bærer en sgRNA sammen med Cre og indført plasmidet i cerebella af Rosa26-CAG-LSL-Cas9-P2A-EGFP mus13 bruger i utero elektroporation. I modsætning til regelmæssig plasmid vektorer kodning EGFP, denne fremgangsmåde med succes mærket transfekteret granula neuron prækursorer (BNI) og deres datter celler. Denne metode giver stor støtte i forståelse in vivo funktion af gener af interesse i prolifererende celler i normal hjernens udvikling og en tumor-tilbøjelige baggrund.

<table>
	<tbody>
		<tr>
			<td>Alexa 488 Goat anti-Chicken</td>
			<td>ThermoFisher</td>
			<td>A11039</td>
			<td>1:400 dilution</td>
		</tr>
		<tr>
			<td>Alexa 568 Donkey anti-Mouse</td>
			<td>Life Technologies</td>
			<td>A-10037</td>
			<td>1:400 dilution</td>
		</tr>
		<tr>
			<td>Alexa 594 Donkey anti-Rabbit</td>
			<td>ThermoFisher</td>
			<td>A21207</td>
			<td>1:400 dilution</td>
		</tr>
		<tr>
			<td>Alexa 647 Donkey anti-Rabbit</td>
			<td>Life Technologies</td>
			<td>A31573</td>
			<td>1:400 dilution</td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase (FastAP)</td>
			<td>ThermoFisher</td>
			<td>EF0654</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave band</td>
			<td>Kisker Biotech</td>
			<td>150262</td>
			<td></td>
		</tr>
		<tr>
			<td>BamHI (HF)</td>
			<td>NEB</td>
			<td>R3136S</td>
			<td></td>
		</tr>
		<tr>
			<td>BbsI (FastDigest)</td>
			<td>ThermoFisher</td>
			<td>FD1014</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellulose Filter Paper (Whatman)</td>
			<td>Sigma-Aldrich</td>
			<td>WHA10347525</td>
			<td></td>
		</tr>
		<tr>
			<td>Cloth</td>
			<td>Tork</td>
			<td>530378</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Zeiss</td>
			<td>LSM800</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Luciferin</td>
			<td>biovision</td>
			<td>7903-1</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Disposable plastic molds (Tissue-Tek Cyromold)</td>
			<td>VWR</td>
			<td>4566</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM Glutamax</td>
			<td>ThermoFisher</td>
			<td>31966047</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Sigma-Aldrich</td>
			<td>D9663</td>
			<td></td>
		</tr>
		<tr>
			<td>EcoRI (HF)</td>
			<td>NEB</td>
			<td>R3101S</td>
			<td></td>
		</tr>
		<tr>
			<td>Electro Square Porator</td>
			<td>BTX</td>
			<td>ECM830</td>
			<td></td>
		</tr>
		<tr>
			<td>Endofree Maxi Kit</td>
			<td>Qiagen</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck</td>
			<td>107017</td>
			<td></td>
		</tr>
		<tr>
			<td>Eye ointment (Bepanthen)</td>
			<td>Bayer</td>
			<td>81552983</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast Green</td>
			<td>Merck</td>
			<td>104022</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>ThermoFisher</td>
			<td>10270-016</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter (0.22 &#181;m)</td>
			<td>Merck</td>
			<td>F8148</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent cell imager (ZOE)</td>
			<td>Biorad</td>
			<td>1450031</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps straight</td>
			<td>Fine Science Tools</td>
			<td>91150-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze (X100 ES-pads 8f 10 x 10 cm)</td>
			<td>Fisher Scientific</td>
			<td>15387311</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP antibody</td>
			<td>Abcam</td>
			<td>ab13970</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr>
			<td>Gibson Assembly Master Mix</td>
			<td>NEB</td>
			<td>E2611S</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Capillary with Filament</td>
			<td>Narishige</td>
			<td>GD1-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating Pad</td>
			<td>ThermoLux</td>
			<td>463265 / -67</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Processing software (ImageJ and Fiji)</td>
			<td>NIH</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe (B. Braun OMNICAN U-100)</td>
			<td>Carl Roth</td>
			<td>AKP0.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Zoetis</td>
			<td>TU061219</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Lumina LT Series III Caliper</td>
			<td>Perkin Elmer</td>
			<td>CLS136331</td>
			<td></td>
		</tr>
		<tr>
			<td>Kalt Suture Needles</td>
			<td>Fine Science Tools</td>
			<td>12050-02</td>
			<td></td>
		</tr>
		<tr>
			<td>KAPA HIFI HOTSTART READY mix</td>
			<td>Kapa Biosystems</td>
			<td>KK2601</td>
			<td></td>
		</tr>
		<tr>
			<td>Ki67 antibody</td>
			<td>Abcam</td>
			<td>ab15580</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Light Pointer</td>
			<td>Photonic</td>
			<td>PL3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid blocker pen</td>
			<td>Kisker Biotech</td>
			<td>MKP-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Metamizol</td>
			<td>WDT</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Microgrinder</td>
			<td>Narishige</td>
			<td>EG-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjector</td>
			<td>Narishige</td>
			<td>IM300</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>Sutter Instrument Co.</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope software ZEN</td>
			<td>Zeiss</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-sterile Silk Suture Thread (0.12 mm)</td>
			<td>Fine Science Tools</td>
			<td>18020-50</td>
			<td></td>
		</tr>
		<tr>
			<td>O.C.T. Compound (Tissue-Tek)</td>
			<td>VWR</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>p27 antibody</td>
			<td>BD bioscience</td>
			<td>610241</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Roth</td>
			<td>335.3</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (1x)</td>
			<td>Life Technologies</td>
			<td>14190169</td>
			<td></td>
		</tr>
		<tr>
			<td>pCAG-EGxxFP</td>
			<td>Addgene</td>
			<td>50716</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine</td>
			<td>Sigma-Aldrich</td>
			<td>408727</td>
			<td></td>
		</tr>
		<tr>
			<td>pX330 plasmid</td>
			<td>Addgene</td>
			<td>42230</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick Ligation Kit</td>
			<td>NEB</td>
			<td>M2200S</td>
			<td></td>
		</tr>
		<tr>
			<td>Ring Forceps</td>
			<td>Fine Science Tools</td>
			<td>11103-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Slides (SuperFrost)</td>
			<td>ThermoFisher</td>
			<td>10417002</td>
			<td></td>
		</tr>
		<tr>
			<td>Software for biostatistics (Prism 7)</td>
			<td>GraphPad Software, Inc</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Spitacid</td>
			<td>EcoLab</td>
			<td>3003840</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>C-PS</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S5016</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Fine Science Tools</td>
			<td>91460-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors with blunt tip</td>
			<td>Fine Science Tools</td>
			<td>14072-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Suture (Supramid schwarz DS 16, 1.5 (4/0))</td>
			<td>SMI</td>
			<td>220340</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligation Buffer</td>
			<td>NEB</td>
			<td>B0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 PNK</td>
			<td>NEB</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue scissors Blunt (11.5 cm)</td>
			<td>Fine Science Tools</td>
			<td>14072-10</td>
			<td></td>
		</tr>
		<tr>
			<td>TOP2B antibody</td>
			<td>Santa Cruz</td>
			<td>sc13059</td>
			<td>1:200 dilution</td>
		</tr>
		<tr>
			<td>Trypsin (2.5 %)</td>
			<td>ThermoFisher</td>
			<td>15090046</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers w/5mm &#216; disk electrodes Platinum</td>
			<td>Xceltis GmbH</td>
			<td>CUY650P5</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaporizer</td>
			<td>Dr&#228;gerwerk AG</td>
			<td>GS186</td>
			<td></td>
		</tr>
	</tbody>
</table>

crispr-mediated loss of function analysis in cerebellar granule cells using in utero electroporation-based gene transfer

Hjernen misdannelser er ofte forårsaget af genetiske mutationer. Decifrere mutationer i patient-afledte væv har identificeret potentielle årsagsfaktorer af sygdomme. For at validere bidrag af en dysfunktion af de muterede gener til udvikling af sygdom, er generation af dyremodeller transporterer mutationerne en indlysende fremgangsmåde. Mens kønscelleoverførsel genetisk manipuleret musemodeller (GEMMs) er populære biologiske værktøjer og udstille reproducerbare resultater, det er begrænset af tid og omkostninger. I mellemtiden, ikke-genom GEMMs ofte aktiverer udforske genfunktion i en mere realistisk måde. Siden nogle hjerne sygdomme (fx, hjernetumorer) synes at være resultatet af somatiske men ikke kønscelleoverførsel mutationer, ikke-kønscelleoverførsel kimære musemodeller, hvor normale og unormale celler sameksistere, kunne være nyttigt for sygdom-relevant analyse. I denne undersøgelse rapport vi en metode til induktion af CRISPR-medieret somatiske mutationer i lillehjernen. Specifikt, vi udnyttede betinget knock-i mus, hvor Cas9 og normal god landbrugspraksis er kronisk aktiveres af CAG (CMV forstærker/kylling ß-actin) promotor efter Cre-medieret rekombination af genomet. Den selvstændige designet enkelt-guide RNA'er (sgRNAs) og Cre recombinase sekvens, både kodet i et enkelt plasmid konstruktion, blev leveret til cerebellare stilk/stamceller på en embryonale stadie benytter i utero elektroporation. Derfor var transfekteret celler og deres datter celler mærket med grøn fluorescerende proteiner (NGL), således at lette yderligere fænotypiske analyser. Derfor, denne metode ikke kun viser elektroporation-baserede gen levering i cerebellare Embryoceller men også foreslå en roman kvantitative tilgang for at vurdere CRISPR-medieret tab af funktion fænotyper.

I vivo funktionel analyse er det kritisk at identificere de celler, som er indsat om eksogene molekylærgenetisk. Samtidig udtryk for en markør, som normal god landbrugspraksis i ikke-prolifererende celler kan blive fulgt op i en lang periode, bliver signalet sekventielt tabt i prolifererende celler. En illustration af denne effekt er vist i figur 1. For at omgå mister fodaftryk af transfekteret celler i LOF analyser, udviklet vi en ny metode ved a...

Watch this Scientific Journal Video about CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer at JoVE.com

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Konventionelle tab af funktion undersøgelser af gener ved hjælp af knockout dyr har ofte været dyrt og tidskrævende. Elektroporation-baseret CRISPR-medieret somatiske mutagenese er et kraftfuldt værktøj til at forstå genet fungerer in vivo. Her rapporterer vi en metode til at analysere knockout fænotyper i prolifererende celler af lillehjernen.

CRISPR-medieret tab af funktion analyse i cerebellare granula celler ved hjælp af In Utero elektroporation-baseret overførsel af gener

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

Research

JoVE Journal

Developmental Biology

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

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 ...

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

Ex Vivo Kultur Chick cerebellare Skiver og Rumligt Målrettet Elektroporation af granulat celleforstadier

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Underskudsgivende og Gain-of-funktion tilgang til Undersøge Tidlig Cell Fate Determinanter i præimplantationsperioden Mouse Embryoner

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Gene Transfer til kylling Auditory Organ med<em&gt; I Ovo</em&gt; Micro-elektroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

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 ...

Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM

Cell Lineage Analyser og genfunktion undersøgelser under anvendelse af Twin-spot MARCM

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological ...

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Struktur og funktion Studier i embryonale stamceller fra mus Brug rekombinase-medieret Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Identifikation af skeletmuskulatur satellit celler ved immunfluorescens med Pax7 og Laminin antistoffer

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

Visualisere Node og Notochordal plade i Gastrulating mus embryoner ved hjælp af Scanning elektronmikroskopi og hele Mount immunfluorescens

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...