Name: transient expression of proteins by hydrodynamic gene delivery in mice
Uploaded: 2014-05-05T16:30:00
Description: Watch this Scientific Journal Video about Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice at JoVE.com

我们感谢刘霞（Regeneron公司制药）的最初教我们的HGD技术和罗素汤姆逊博士（医学阿尔伯特·爱因斯坦学院），他在技术的各个方面不断的指导。这项工作是由亨特学院，纽约市立大学启动基金和国家科学基金会颁发的面包IOS-1249166。我们感谢NYULMC病理组织学核心NYUCI中心支援津贴，NIH / NCI 5 P30CA16087-31为组织学上的小鼠肝脏。

这里描述的所有实验均批准了亨特学院的纽约市立大学机构动物护理和使用委员会。 1，制备无内毒素的质粒DNA <ol><li>挑含有在哺乳动物表达载体目的基因从新鲜的划线平板选择性细菌的单菌落。 </li><li>按照市售的无内毒素质粒纯化试剂盒用于种植和收获细菌的手册中找到的建议。 </li><li>通过以下的市售无内毒素的质粒纯化试剂盒的方案纯化从细菌细胞的无内毒素的质粒DN...

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+progress+and+prospects:+hydrodynamic+gene+delivery.">Gene therapy progress and prospects: hydrodynamic gene delivery.</a> Gene Ther. 14, 99-107 (2007).</li><li>Kobayashi, N., Nishikawa, M., Takakura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hydrodynamics-based+procedure+for+controlling+the+pharmacokinetics+of+gene+medicines+at+whole+body,+organ+and+cellular+levels.">The hydrodynamics-based procedure for controlling the pharmacokinetics of gene medicines at whole body, organ and cellular levels.</a> Adv Drug Deliv Rev. 57, 713-731 (2005).</li><li>Al-Dosari, M. S., Knapp, J. E., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+delivery.">Hydrodynamic delivery.</a> Adv Genet. 54, 65-82 (2005).</li><li>Bell, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+delivery+of+the+Sleeping+Beauty+transposon+system+to+livers+of+mice+by+hydrodynamic+injection.">Preferential delivery of the Sleeping Beauty transposon system to livers of mice by hydrodynamic injection.</a> Nat Protoc. 2, 3153-3165 (2007).</li><li>Osorio, F. G., de la Rosa, J., Freije, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luminescence-based+in+vivo+monitoring+of+NF-kappaB+activity+through+a+gene+delivery+approach.">Luminescence-based in vivo monitoring of NF-kappaB activity through a gene delivery approach.</a> Cell Commun Signal. 11, (2013).</li><li>Knapp, J. E., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+delivery+of+DNA.">Hydrodynamic delivery of DNA.</a> Methods Mol Biol. 245, 245-250 (2004).</li><li>Yang, J., Chen, S., Huang, L., Michalopoulos, G. K., Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+expression+of+naked+plasmid+DNA+encoding+hepatocyte+growth+factor+in+mice+promotes+liver+and+overall+body+growth.">Sustained expression of naked plasmid DNA encoding hepatocyte growth factor in mice promotes liver and overall body growth.</a> Hepatology. 33, 848-859 (2001).</li><li>Maruyama, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-level+expression+of+naked+DNA+delivered+to+rat+liver+via+tail+vein+injection.">High-level expression of naked DNA delivered to rat liver via tail vein injection.</a> J Gene Med. 4, 333-341 (2002).</li><li>Hen, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+foreign+genes+in+chicks+by+hydrodynamics-based+naked+plasmid+transfer+in+vivo.">Expression of foreign genes in chicks by hydrodynamics-based naked plasmid transfer in vivo.</a> Domestic animal endocrinology. 30, 135-143 (2006).</li><li>Eastman, S. 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B., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+impact+of+hydrodynamic+injection+on+mouse+liver.">Structural impact of hydrodynamic injection on mouse liver.</a> Gene Ther. 14, 129-137 (2007).</li></ol>

当正确执行，HGD是一个非常安全和有效的转基因运载工具。的关键步骤，以成功HGD是：1）提供的DNA适量在一个大体积的盐水车辆2）插入到小鼠尾静脉3）在小于8秒。 虽然注射过程本身无可否认需要一些手巧，这半年的第二个过程的成功往往在于精心准备实验。必要的，以达到最大的基因表达的质粒DNA的量会有很大的改变取决于所用的质粒，也可以因此表达的基因，DNA被注入?...

自从其首次描述Liu 等人 ，张等人 ，流体力学基因传递（对冲）已经成为一个非常宝贵的工具，在啮齿动物模型系统1，2基因功能研究。该技术涉及的溶液的大量（8-12％体重）的快速注射（5-7秒）到小鼠尾静脉中由靶器官1，2的细胞，以促进质粒DNA的摄取。这些条件导致的肾，脾，肺和心脏，肝脏和较少的基因表达健壮的基因表达。 注射10微克质粒pCMV-LacZ的质粒可以转染高达40％的肝细胞，使得HGD最高效的，非病毒， 体内基因递送方法至今1。不像病毒携带者，质粒DNA是很容易准备，不会引起在啮齿动物宿主3的免疫反应和结尾重组不会对健康构成威胁ogenous病毒。另外，由于通过HGD交付的DNA分子不需要的包装，这种方法适合于细菌人工染色体（BAC）一样大的150 kb的4输送。其它类型的已交付由一个流体动力法分子包括RNA 5-10，吗啉11，蛋白质12，13和其它小分子12,14。 HGD超过其它递送方法的优点和缺点的优良评论已被讨论在文献15-20和许多作者所提供的程序21-23的详细描述。 由HGD引入转基因到小鼠中是安全和有效的1-3，24，并 ​​且该方法已在大鼠可比成功25被使用。有了一定的修改，概念验证的实验已经进行了26只鸡，饶位27和28的猪，虽然在较大的动物的体内应用这一技术仍然是一个挑战。当使用这种方法，另一种常见的限制是，许多现有的哺乳动物表达载体中缺少的组件来实现基因表达的持续性，高水平。使用目标器官的pCMV-吕克质粒，基因表达是明显的，因为早在HGD后十几分钟，然而，最初的高表达水平在注射之后的第一周急剧下降。长期的转基因表达可能依赖于在质粒设计3，24然而，维护高水平的基因表达，往往需要反复注射用的启动子和内含子。出于这个原因，HGD可能会对研究慢性疾病是长期暴露于有害的蛋白质或蛋白质产物的结果不太适合。有了这些限制，HGD是一个非常强大的工具，学习宝基因和它的影响作用潜力在体内 ，以及治疗效果和蛋白质的调控和建立疾病的动物模型突变体（综述见15）。例如，HGD，可以使用由单独引入各种基因构建到小鼠体内已各基因敲除分配功能结构域和蛋白质的氨基酸。此外，该技术可以在任何小鼠品系使用。 本协议描述HGD小鼠，专注于必要的技术方面实现成功转染：正确的针头插入静脉，注射量交付和速度。这种方法的应用是体现在非洲锥虫病，人类的一种致命的疾病和牲畜29，30的小鼠模型。虽然一些物种锥虫引起疾病的家畜，最不能引起疾病，由于先天免疫复合物在B人类载量称为锥虫溶解因子（TLFS）29，31，32。这些孔隙形成，高密度脂蛋白（HDL）包含两个独特的，灵长类特异性蛋白质：HPR，配体，这有利于TLFS的摄取锥虫和APOL-I，裂解组件31，33-38 锥虫布氏罗得西亚能够感染人类由于血清耐药相关蛋白（SRA），其结合并中和人类APOL-I 34，39的表达。狒狒TLF未通过SRA由于其不同的APOL-I蛋白40中和。据此前报道，使用哺乳动物表达载体（pRG977），狒狒TLF成分的转基因小鼠表达赋予的保护，防止人类感染锥虫40。这里提出的有代表性的数据展示如何流体力学基因递送可用于研究蛋白质的治疗效果。

<table border="0" cellpadding="0" cellspacing="0" width="615">
	<tbody>
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			<td height="21" style="height:21px;width:216px;">EndoFree Plasmid Maxi Kit</td>
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			<td align="right" style="width:119px;">12362</td>
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			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">pRG977 (mammalian expression vector)</td>
			<td style="width:113px;">Regeneron Pharmaceuticals</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Material Transfer Agreement required</td>
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			<td height="41" style="height:41px;width:216px;">Sodium Chloride, 0.9% INJ USP 10 ml, Hospira&#160;</td>
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</table>

transient expression of proteins by hydrodynamic gene delivery in mice

体内转基因的有效表达是在基因功能研究和开发治疗疾病至关重要。在过去几年中，水动力基因传递（对冲）已成为交付的转基因老鼠成一个简单，快速，安全，有效的方法。这种技术依赖于通过快速注射大量生理溶液，以增加灌注器官的细胞膜的渗透性的产生的力，从而将DNA传递到细胞内。一个HGD的主要优点是使用裸质粒DNA（pDNA的）引入转基因到哺乳动物细胞的能力。使用质粒导入外源基因是微创费力，高效，违背病毒携带者，非常安全。 HGD最初用于将基因递送到小鼠体内，它现在被用于提供一个范围广泛的物质，包括寡核苷酸，人工染色体，RNA，蛋白质和小分子进入小鼠，大鼠并且，在有限的程度，其他动物。本协议描述HGD小鼠，专注于那些能够成功执行的关键步骤的方法的三个主要方面：针头插入静脉的正确插入，注入的量和交货速度。实施例是为了显示本方法的应用，以2基因编码分泌的，灵长类特异性蛋白的瞬时表达，载脂蛋白李（APOL-I）和触珠蛋白相关蛋白（HPR）。

针的正确定位到小鼠尾静脉中（ 如图1）可以通过流体力学的基于转染成功递送的转基因的先决条件。通常的技术中最具挑战性的部分，但是，尾静脉内针不移动的保持，从而使注射的总体积可在6-8秒钟时间内被传递。在注射过程中的小错误可能导致大大降低转染效率和蛋白表达的影响。因此，必须监测HGD的每个实验的成功。如果感兴趣的转基因蛋白难以通过蛋白印迹分析，由于缺乏...

Watch this Scientific Journal Video about Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice at JoVE.com

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

蛋白通过流体动力基因传递在小鼠体内瞬时表达

在由流体动力基因递送裸DNA的体内转染引入基因到最小的炎症反应的动物的组织中。产生基因产物的足够量，使得基因功能和调节以及蛋白质结构和功能可以被分析。

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

Research

JoVE Journal

Biology

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

科研

生物学

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

无有机溶剂和醋酸，蛋白质与考马斯亮蓝G - 250凝胶染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

In vitro Reconstitution of the Active T. castaneum Telomerase

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

高级别瞬态表达重组蛋白的植物高效的农杆菌

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...