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

Bu eser Ulusal Bilim Fonu tarafından desteklenen ayırt edici genç akademisyenler (81625002), Ulusal Doğa Bilimleri Vakfı Çin (81470389, 81270282, 81601238), Program Shanghai akademik araştırma, lider (18XD1402400), Şangay belediye için Eğitim Komisyonu Gaofeng klinik tıp hibe desteği (20152209), Shanghai Shenkang hastane Geliştirme Merkezi (16CR3034A), Shanghai Jiao Tong Üniversitesi (YG2013MS42), Shanghai Jiao Tong Üniversitesi Tıp (15ZH1003 ve 14XJ10019), Shanghai yelken programı (18YF1413000) ve Bengbu Tıp Fakültesi (Byycx1722) yüksek lisans yenilik programı. Dr. Erhe Gao bizim laboratuarımızda onun önceki yardım için teşekkür ediyoruz.

Tüm hayvan deneyleri Ulusal Sağlık kurallarına Enstitüleri kullanım laboratuvar hayvanları üzerinde göre yapılmıştır ve Enstitü nün hayvan Etik Komitesi tarafından kabul edildi. Erkek C57BL/6J fareler (8-10 hafta yaşındaki) tüm deneyler için kullanılmıştır. Fareler 24 ° C ± 4 ° C, bir 12-h ışık/karanlık döngü, su ve gıda ücretsiz erişim altında patojen-Alerjik koşullarda muhafaza.
1. Adenovirus çözüm hazırlanması
<ol><li>Arıtılmış adenovirus ç?...

<ol>
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Geçerli raporu miyokard infarktüsü indüksiyon bir yönteminden Gao vd tarafından güncellenmiştir kardiyak gen manipülasyon için viral vektörler intramyocardial enjeksiyon için değiştirilmiş bir teknik gösterir 13 şu anda, karakterizasyonu vivo içinde belirli bir gen fonksiyonları en sık dahil nakavt veya transgenik fareler3,14,15,16<s...

Kalp hastalığı morbidite ve mortalite dünya çapında1,2önde gelen nedenidir. Miyokard İnfarktüsü ve kalp yetmezliği gibi ölümcül kalp sorunları için etkili tedavi stratejileri eksikliği temel pathomechanisms yoğun keşfi ve kimliği roman tedavi olanakları3çekiyor. Bu bilimsel keşifler için yaygın olarak kullanılan4,5kalp özel gen manipülasyon olduğunu. Kardiyak gen manipülasyon güçlü transkripsiyon harekete geçirmek gibi efektör nükleaz (TALEN) kullanarak genom düzenleyerek elde edilebilir ve düzenli olarak interspaced kısa palindromik yineler (CRISPR) kümelenmiş / CRISPR ilişkili protein 9 (Cas9) araçları, ya da Ektopik genetik malzemelerin (örneğin, virüs vektörel çizimler proteinler ilgi kodlama genleri taşıyan) teslim edilmesi6. Genom düzenleme kesin ve kronolojik zamanmekansal genom değişiklikler yaşayan fareler sağlar rağmen hala bir zaman alıcı ve emek yoğun pratik6olduğunu. Alternatif olarak,6kalp özel gen manipülasyon virüs vektör veya küçük müdahale RNA (siRNA) karmaşık teslim rutin tarafından gerçekleştirilen.
Virüs vektör teslim yetişkin fare kalp için kabaca iki stratejileri ile elde edilir: sistemik veya yerel enjeksiyon. AAVs AAV9 gibi cardiotropic serotip sistemik enjeksiyon noninvaziv kardiyak belirli bir gen manipülasyon7için. Ancak, bu yöntem vektör verimli iletim ve gen ekspresyonu için gerekli nispeten yüksek miktarda gerektirir ve kas ve karaciğer7gibi nontarget organların önemli iletim neden olabilir. Yerel virüs enjeksiyon intramyocardial enjeksiyon veya intrakoroner teslim7tarafından sağlanır. İntrakoroner teslim virüs intramyocardial enjeksiyon için karşılaştırıldığında kalp içinde daha eşit dağılımı yol açar. Ancak, bu teknik dezavantajları sistemik dolaşım ve iletim nontarget organları8ve onun gereksinimi için basınç ölçüm işlemi sırasında aygıt için viral vektörler dışarı hızlı yıkama vardır. Buna karşılık, viral olarak dağıtabilmenizi daha iyi virüs saklama Miyokardiyum yanı sıra site belirli teslim, ama başarısız intramyocardial enjeksiyon sağlar7vektör. Küçük hayvanlar için intrakoroner teslimat sistemik AAV9 enjeksiyon ve intramyocardial enjeksiyon daha yaygın olarak uygulanan4,5,7iken yapmak teknik olarak zor olur. Sistemik enjeksiyon gerçekleştirmek kolay olsa da, geleneksel intramyocardial enjeksiyon mekanik havalandırma ve Torakotomi gerektiren, geniş doku hasarına neden olur ve zaman alır.
Bu raporda, intramyocardial enjeksiyon için bir kolay, zaman tasarrufu ve yüksek verimli yöntemi açıklanmıştır. Luciferase ve VDR veya VDR, hedefleme shRNA kodlama adenovirus kardiyak gen ekspresyonu işlemek için enjekte ettiler. Hakim sonra gene manipülasyon, aynı zamanda hücreleri ve fare kalp diğer maddeleri enjeksiyonu için bu yöntem kullanılabilir.

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			<td colspan="4" height="40" style="height:40px;width:916px;">Equipments</td>
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			<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>
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			<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>
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			<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>
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			<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>
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			<td height="40" style="height:40px;width:351px;">Isoflurane vaporizer&#160;</td>
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			<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>
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			<td height="40" style="height:40px;width:351px;">Precision&#160;balance</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>
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			<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>
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			<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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			<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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			<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>
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		<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>
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			<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>
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			<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>
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			<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>
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			<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

Gene manipülasyon özellikle kalbinde önemli ölçüde soruşturma kalp hastalığı pathomechanisms ve tedavi potansiyellerini artırmak. Vivo kalp özel gen teslim sık sistemik veya yerel teslim tarafından elde edilir. Kuyruk ven yoluyla sistemik enjeksiyon kolay ve verimli kardiyak gen ekspresyonu rekombinant adeno ilişkili virüs 9 (AAV9) kullanarak işleme. Ancak, bu yöntem için verimli iletim vektör nispeten yüksek miktarda gerektirir ve nontarget organ gen iletim neden olabilir. Burada, intramyocardial enjeksiyon vivo içinde kalp özel gen manipülasyon farelerde için bir basit, etkili ve zaman kazandıran yöntemi açıklanmaktadır. Anestezi (havalandırma) olmadan altında göğüs birincil ve ikincil kasları açık açık disseke ve fare kalp hızlı bir şekilde dördüncü interkostal alan küçük bir kesi yoluyla el ile dışa tarafından maruz kalmış. Daha sonra luciferase (Luc) ve vitamin D reseptör (VDR) veya VDR, hedefleme kısa saç tokası RNA (shRNA) kodlama adenovirus Hamilton şırınga Miyokardiyum enjekte ettiler. Sonraki vivo içinde görüntüleme bu luciferase özellikle kalbinden başarılı bir şekilde overexpressed gösterdi. Ayrıca, Western blot analizi başarılı overexpression veya fare kalbinde VDR susturmak doğruladı. Kez hakim, bu teknik gen manipülasyon yanı sıra enjeksiyon hücreleri veya fare kalbinde nanogels gibi diğer malzemeler için kullanılabilir.

Deney Protokolü ve bazı önemli adımlar bildirilen yöntemi için şekil 1' de gösterilmiştir. İntramyocardial enjeksiyon adenovirus kodlama luciferase (Adv-luc) sonra 5 gün, adv-luc enjekte farelerde Imaging vivo içinde kalbinde olan özellikle (şekil 2A, B), luciferase sağlam overexpression belirtti. nontarget organ iletim yokluğu düşündüren Western blot analizi ile (<strong class="xfig"...

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

Burada fareler bir protokol kalp özel gen manipülasyon için mevcut. Anestezi altında fare kalpler dördüncü interkostal uzayda externalized. Daha sonra kodlama belirli genler bir şırınga vivo içinde görüntüleme ve Batı ile protein ifade ölçü tarafından takip Miyokardiyum içine enjekte edildi adenovirüse bakın analiz leke.

Vivo içinde kalp özel gen manipülasyon farelerde Intramyocardial enjeksiyonla için basit ve etkili bir yöntem

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

Pyrosequencing: Doğru Genotiplendirme için Basit Bir Yöntem

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

Ribozom Bağlı Doğan Polipeptitler İzolasyonu<em&gt; In vitro</em&gt; MRNA boyunca Translasyonel Duraklat Siteleri Belirleme

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

Ribozom Ciltli Polipeptitler izole bir aracı olarak SECM Tutuklanma sırası kullanılarak

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

Testis Hücre Cezalı Hazırlanması için basit ve Verimli Tekniği

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

Farelerde Hızlı noninvaziv Lomber İntratekal enjeksiyon yoluyla Vivo siRNA&#39;nın Transfaksiyon ve Omurilik Gen demonte olarak

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

Yetişkin Fare Kalvaryal Kemik İliği içinde Hematopoietik kök hücrelerin Vivo 4-Boyutlu Takip

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

Intravenous Injections in Neonatal Mice

Yenidoğan Farelerde intravenöz enjeksiyonlar

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

Nükleik Asitlerin süzülmesi İzolasyonu: basit ve hızlı DNA izolasyonu,

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

Soya önceki ve geçici gen ifade analiz için uygulama yalıtım için basit bir yöntem

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

İmmünfluoresan siyon ile Primer Silya'nın Basit Tespiti

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