Name: a simple and efficient method for in vivo cardiac-specific gene manipulation by intramyocardial injection in mice
Uploaded: 2018-04-16T13:30:00
Description: Watch this Scientific Journal Video about A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice at JoVE.com

Dette arbejde blev støttet af National Science Fond for fremtrædende unge forskere (81625002), National Natural Science Foundation of China (81470389, 81270282, 81601238), programmet for Shanghai akademisk Forskningsleder (18XD1402400), Shanghai Municipal Uddannelse Kommissionen Gaofeng klinisk medicin tilskud (20152209), Shanghai Shenkang Hospital Development Center (16CR3034A), Shanghai Jiao Tong University (YG2013MS42), Shanghai Jiao Tong University School of Medicine (15ZH1003 og 14XJ10019), Shanghai sejlads Program (18YF1413000), og Postgraduate Innovation Program af Bengbu Medical College (Byycx1722). Vi takker Dr. Erhe Gao for hans tidligere hjælp i vores laboratorium.

Alle dyreforsøg blev udført ifølge den nationale institutter sundhed retningslinjer for brugen af forsøgsdyr, og blev godkendt af instituttets dyr etisk komité. Mandlige C57BL/6J mus (i alderen 8-10 uger) blev brugt til alle eksperimenterne. Mus har været opstaldet under patogenfrie betingelser på 24 ° C ± 4 ° C, under en 12-h lys/mørke cyklus, med fri adgang til vand og mad.
1. forberedelse af Adenovirus løsning
<ol><li>Ved ankomsten af renset adenovirus løsningen, skal du gemm...

<ol>
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Den aktuelle betænkning viser en modificeret teknik for intramyocardial injektion af virale vektorer for kardiale genmanipulation, som blev ændret fra en metode for myokardieinfarkt induktion af Gao et al. 13 i øjeblikket, i vivo karakterisering af bestemte gen funktioner oftest involverer generation af knockout eller Transgene mus3,14,15,16,</s...

Hjertesygdom er den hyppigste årsag til sygelighed og dødelighed på verdensplan1,2. Manglen på effektive terapeutiske strategier for livstruende hjertesygdomme myokardieinfarkt og hjertesvigt tiltrækker intensiv efterforskning af underliggende patomekanismer og identifikation af nye terapeutiske muligheder3. For disse videnskabelige udforskninger er hjerte-specifikke genmanipulation udbredte4,5. Hjerte genmanipulation kan opfyldes af genom-redigering ved hjælp af kraftfulde transkription aktivere-lignende effektor nukleasen (TALEN) og grupperet regelmæssigt interspaced korte palindromiske gentagelser (CRISPR) / CRISPR forbundet protein 9 (Cas9) værktøjer, eller ved levering af ektopiske genetiske materiale (fx, virus vektorer bærer gener kodning proteiner af interesse)6. Selvom genom-redigering giver præcise og spatiotemporelle genom ændringer i levende mus, er det stadig en tidskrævende og arbejdskrævende praksis6. Alternativt, hjerte-specifikke genmanipulation af virus vektor eller små blande RNA (siRNA) komplekse levering er rutinemæssigt udføres6.
Virus vektor levering til voksen mus hjertet opnås ved omtrent to strategier: systemiske eller lokale injektion. Systemisk injektion af cardiotropic serotype af AAVs såsom AAV9 er noninvasive for hjerte bestemt gen manipulation7. Men denne metode kræver en forholdsvis stor mængde af vektor nødvendigt for effektiv transduktion og genekspression, og kan resultere i betydelige transduktion af nontarget organer såsom muskel og lever7. Lokale virus injektion er opnået ved intramyocardial injektion eller Intrakoronar levering7. Intrakoronar levering fører til en mere jævn fordeling af virus i hjertet sammenlignet med intramyocardial injektion. Men ulemperne ved denne teknik er den hurtig vask ud af virale vektorer til systemisk cirkulation og transduktion i nontarget organer8, og dets krav om udstyr til trykmåling under operationen. Derimod vektor intramyocardial injektion giver bedre virus fastholdelse i myokardiet samt stedsspecifik levering, men det mislykkes at jævnt distribuere viral7. For små dyr er Intrakoronar levering teknisk vanskeligt at udføre, mens systemiske AAV9 injektion og intramyocardial injektion er mere almindeligt praktiseres4,5,7. Selvom systemisk injektion er nem at udføre, konventionelle intramyocardial injektion kræver mekanisk ventilation og torakotomi, forårsager omfattende vævsskader, og er tidskrævende.
I denne betænkning beskrevet vi en let, tidsbesparende og yderst effektiv metode til intramyocardial injektion. Adenovirus kodning luciferase og VDR eller shRNA målretning VDR, blev sprøjtet for at manipulere hjerte genekspression. Når først beherskes, kan denne metode bruges til genmanipulation, som indsprøjtning af celler eller andre materialer i mus hjertet.

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			<td colspan="4" height="40" style="height:40px;width:916px;">Equipments</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Laminar flow sterile hood</td>
			<td style="width:267px;">Fengshi Animal Experimental&#160; Equipment Techonology Co., Ltd. (Soochow, China)</td>
			<td style="width:143px;">FS-CJ-2F</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Centrifuge</td>
			<td style="width:267px;">Thermo Scientific (Waltham, USA)</td>
			<td style="width:143px;">75005282</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Tissue grinding machine</td>
			<td style="width:267px;">Scientz Biotechnology Co., Ltd. (Ningbo, China)</td>
			<td style="width:143px;">Scientz-48</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">High temperature/high pressure sterilizer</td>
			<td style="width:267px;">Hirayama (Saitama, Japan)</td>
			<td style="width:143px;">HVE-50</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Isoflurane vaporizer&#160;</td>
			<td style="width:267px;">Matrix (Orchard Park, USA)</td>
			<td style="width:143px;">VIP3000</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">IVIS&#160; Lumina III imaging system</td>
			<td style="width:267px;">PerkinElmer (Waltham, USA)</td>
			<td style="width:143px;">CLS136334</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Precision&#160;balance</td>
			<td style="width:267px;">Sartorius (G&#246;ttingen, Germany)</td>
			<td style="width:143px;">28091873</td>
			<td style="width:156px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Instruments&#160;</td>
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			<td style="width:143px;"></td>
			<td style="width:156px;"></td>
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			<td height="40" style="height:40px;width:351px;">Eppendorf pipette (100 &#181;L)</td>
			<td style="width:267px;">Eppendorf (Westbury, USA)</td>
			<td style="width:143px;">&#160;4920000059</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Eppendorf pipette (10 &#181;L)</td>
			<td style="width:267px;">Eppendorf (Westbury, USA)</td>
			<td style="width:143px;">&#160;4920000113</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Forceps</td>
			<td style="width:267px;">Shanghai Medical Instruments (Group) Ltd., Corp.&#160;</td>
			<td style="width:143px;">JD4020</td>
			<td style="width:156px;">Curved tip</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Hamilton syringe</td>
			<td style="width:267px;">Hamilton (Nevada, USA)</td>
			<td style="width:143px;">80501</td>
			<td style="width:156px;">Volume 50 &#956;L</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Micro-mosquito hemostat</td>
			<td style="width:267px;">F.S.T (Foster City, USA)</td>
			<td style="width:143px;">13011-12</td>
			<td style="width:156px;">Curved, tip width 1.3mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Needle holder&#160;</td>
			<td style="width:267px;">Shanghai Medical Instruments (Group) Ltd., Corp. (Shanghai, China)</td>
			<td style="width:143px;">J32110</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Surgical scissors</td>
			<td style="width:267px;">F.S.T (Foster City, USA)</td>
			<td style="width:143px;">14002-12</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">1-mL Syringe</td>
			<td style="width:267px;">WeiGao Group Medical Polymer Co.,Ltd. (ShangDong, China)</td>
			<td style="width:143px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Materials and reagents</td>
			<td style="width:267px;"></td>
			<td style="width:143px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Anti-GAPDH antibody</td>
			<td style="width:267px;">CST (Danvers,&#160; USA)</td>
			<td style="width:143px;">#2118</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Anti-Luciferase antibody</td>
			<td style="width:267px;">Abcam (Cambridge, UK)</td>
			<td style="width:143px;">ab187340</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Anti-rabbit IgG</td>
			<td style="width:267px;">CST (Danvers,&#160; USA)</td>
			<td style="width:143px;">&#160;#7074</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Anti-VDR antibody</td>
			<td style="width:267px;">Abcam (Cambridge, UK)</td>
			<td style="width:143px;">&#160;ab109234</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Buprenorphine</td>
			<td style="width:267px;">Thermo Scientific (Waltham, USA)</td>
			<td style="width:143px;">PA175056</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Chloralic hydras</td>
			<td style="width:267px;">LingFeng Chemical (ShangHai, China)</td>
			<td style="width:143px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Cryogenic Vials</td>
			<td style="width:267px;">Thermo Scientific (Waltham, USA)</td>
			<td style="width:143px;">375418</td>
			<td style="width:156px;">1.8 mL&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Depilatory cream</td>
			<td style="width:267px;">Veet (Shanghai, China)</td>
			<td style="width:143px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Dulbecco&#39;s phosphate buffered saline&#160;</td>
			<td style="width:267px;">Gibco (Grand Island,&#160; USA)</td>
			<td style="width:143px;">14040133</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Entoiodine</td>
			<td style="width:267px;">LiKang (Shanghai, China)</td>
			<td style="width:143px;">310132</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">EP tube</td>
			<td style="width:267px;">Sarstedt (Newton, USA)</td>
			<td style="width:143px;">PCR001</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Filter</td>
			<td style="width:267px;">Millipore (Bedford, USA)</td>
			<td style="width:143px;"></td>
			<td style="width:156px;">Pore size 0.2 &#181;m&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Isoflurane</td>
			<td style="width:267px;">Yipin Pharmaceutical Company (Hebei, China)</td>
			<td style="width:143px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Luciferin</td>
			<td style="width:267px;">Promega (Madison, USA)</td>
			<td style="width:143px;">P1041</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Lysis buffer for western&#160; blot</td>
			<td style="width:267px;">Beyotime (Shanghai, China)</td>
			<td style="width:143px;">P0013J</td>
			<td style="width:156px;">Without inhibitors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Ophthalmic cream</td>
			<td style="width:267px;">Apex Laboratories ( Melbourne, Australia))</td>
			<td style="width:143px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">PBS</td>
			<td style="width:267px;">Gibco (Grand Island,&#160; USA)</td>
			<td style="width:143px;">10010023</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Protease inhibitor cocktail</td>
			<td style="width:267px;">Thermo Scientific (Waltham, USA)</td>
			<td style="width:143px;">78438</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">5-0 silk suture</td>
			<td style="width:267px;">Shanghai Medical Instruments (Group) Ltd., Corp. (Shanghai, China)</td>
			<td style="width:143px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Steel ball</td>
			<td style="width:267px;">Scientz Biotechnology Co., Ltd. (Ningbo, China)</td>
			<td style="width:143px;"></td>
			<td style="width:156px;">Width 1.5 mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Syringe needle</td>
			<td style="width:267px;">Kindly Medical Devices Co., Ltd. (Zhejiang, China)</td>
			<td style="width:143px;"></td>
			<td style="width:156px;">30 gauge&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Warm mat</td>
			<td style="width:267px;">Warmtact Electrical Heating Technology Co., Ltd. (Guangdong, China )</td>
			<td style="width:143px;">NF-GNCW</td>
			<td style="width:156px;"></td>
		</tr>
	</tbody>
</table>

a simple and efficient method for in vivo cardiac-specific gene manipulation by intramyocardial injection in mice

Genmanipulation specifikt i hjertet forstærke betydeligt undersøgelse af hjertesygdom patomekanismer og deres terapeutiske potentiale. In vivo hjerte-specifikke gen levering er almindeligt opnås ved enten systemiske eller lokale levering. Systemisk injektion via hale vene er nemt og effektivt i manipulere hjerte genekspression ved hjælp af rekombinante adeno-associeret virus 9 (AAV9). Men denne metode kræver en forholdsvis stor mængde af vektor for effektiv transduktion, og kan resultere i nontarget orgel gen transduktion. Her, beskriver vi en enkel, effektiv og tidsbesparende metode af intramyocardial injektion for i vivo hjerte-specifikke genmanipulation i mus. Under anæstesi (uden ventilation), større og mindre brystmuskulaturen var ligefremt dissekeret, og musen hjertet var hurtigt udsat af manuel udlægning gennem et lille snit på den fjerde interkostale rum. Efterfølgende, blev adenovirus kodning luciferase (Luc) og vitamin D-receptoren (VDR) eller korte hårnål RNA (shRNA) rettet mod VDR, sprøjtet med en Hamilton sprøjte ind i myokardiet. Efterfølgende i vivo billeddannelse demonstreret at luciferase var med held overexpressed specielt i hjertet. Endvidere, vestlige skamplet analyse bekræftet den succesfulde overekspression eller hæmning af VDR midt mus. Når først beherskes, kan denne teknik bruges til genmanipulation, som indsprøjtning af celler eller andre materialer som nanogels midt mus.

Eksperiment-protokollen, og nogle af de vigtigste skridt for den rapporterede metode er vist i figur 1. 5 dage efter intramyocardial injektion af adenovirus kodning luciferase (Adv-luc) angivet i vivo billeddannelse i adv-luc injiceres mus robust overekspression af luciferase specifikt i hjertet (figur 2A, B), som blev bekræftet af Western blot analyse (figur 2 c), hvilket ...

Watch this Scientific Journal Video about A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice at JoVE.com

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Her præsenterer vi en protokol for hjerte-specifikke genmanipulation i mus. Under anæstesi, var musen hjerter externalized gennem den fjerde interkostale rum. Efterfølgende, blot adenovirus kodning specifikke gener blev sprøjtet med en sprøjte ind i myokardiet, efterfulgt af protein udtryk måling via i vivo billeddannelse og Western analyse.

En enkel og effektiv metode til In Vivo hjerte-specifikke genmanipulation af Intramyocardial injektion i mus

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

Research

JoVE Journal

Biology

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

Isolering af ribo Bundet Gryende polypeptider<em&gt; In vitro</em&gt; Til at identificere Translationelle Pause steder langs mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Brug SecM Arrest Sequence som et redskab til at isolere ribo bundne polypeptider

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Simpel og effektiv teknik til Udarbejdelse af Testikel blodlegemesuspensioner

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

In Vivo siRNA transfektion og Gene Knockdown i Spinal Cord via Rapid Noninvasiv Lumbar Intrathekal Injektioner i mus

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional sporing af hæmatopoietisk stamcelletransplantation og stamcelle i Voksen Mouse calvarie knoglemarv

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Intravenous Injections in Neonatal Mice

Intravenøse injektioner i neonatale mus

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

Filtrering Isolering af nukleinsyrer: en enkel og hurtig DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

En simpel metode til dyrkning af sojabønner Protoplasts og ansøgning til forbigående gen Expression analyser

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. ...

Simple Detection of Primary Cilia by Immunofluorescence

Simpel påvisning af primær cilier ved immunofluorescens

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to ...