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DNA。 </li><li>使用无内毒素的塑料制品和处理的DNA与照顾，以确保内毒素是不会重新引入的DNA样品中去除步骤之后。 </li><li>通过使用MICR确定其260nm处的吸光度测量的无内毒素的质粒DNA的浓度如下所述邻体积紫外分光光度计或标准，比色皿型分光光度计。 <ol><li>清洁微型分光光度计样品保留系统的下部和上部的光学表面如下。吸取1μL的干净的去离子水到较低的光学系统。关闭杠杆臂，并点击了几次，洗澡上的光学系统，然后打开杆并用纸巾擦拭干净。 </li><li>打开微分光光度计的软件，并选择核酸模块。 </li><li>将降低光学系统1微升干净的去离子水，降低杠杆臂，然后从程序软件"初始化"。一旦初始化一个组织完整的，既清洁光学表面.. </li><li>和从程序软件选择"空白"，通过加载1微升的无内毒素的TE缓冲液（1mM EDTA的10mM Tris-CL，pH 8.0）中进行空白测定。一旦空白测量完成后，既清洁光学表面与组织。 </li><li>从程序软件加载1微升的内毒素的DNA样本，并选择"测量"进行样品测量。记录DNA浓度。通过记录吸光度比值260/280 nm的屏幕上会出现评估的DNA纯度。因为对于HGD使用纯DNA（260/280比值为1.8）是非常可取的，避免使用DNA样品与260/280的比例在1.7以下。一次测量完成后，用纸巾清洁两个光学表面。 </li></ol></li></ol> 2，称量小鼠<ol><li>马克小鼠的方式适当的应变。注:尾部的标记，不建议这个程序。 <ol><li>有白苔（ 如瑞士韦伯斯特）在他们的后面有一黑色标记标记鼠种。随着时间的推移重新标记作为必要的。 </li><li>有黑色皮草（ 如 C57BL / 6）由耳标鼠种冲孔数天前。 </li></ol></li><li>称取小鼠individuaLLY轻轻放置在动物秤盘或桶放在数字实验室天平。记录实验中所用各小鼠的体重，在实验当天。 </li></ol> 3，准备注射器<ol><li>注射混合制备<ol><li>计算所需要的假设5的DNA的量 - 50微克无内毒素的质粒DNA用于注入每只小鼠。 <ol><li>实验方法确定质粒DNA的最佳量。 </li><li>当确定的DNA所需的时间，假定注入每组的一个额外的鼠标，由于注射器的正确装载将需要一些额外的注射组合。为了帮助计算，考虑下面的例子:注射4实验和4个对照组，每个重25克，每只小鼠50微克质粒DNA。以确定需要在这种情况下的DNA的量，计算出以每组5只小鼠:需要为每个组5×50微克= 250微克的DNA。 </li></ol></li><li>确定所需的假设体重10％为注射每只小鼠的盐水量（0.9％氯化钠）。 <ol><li>根据上面的例子中，于2.5ml盐水（2.5克液体）与50微克质粒DNA注入每个25克鼠标。 </li><li>当确定需要对小鼠的整个组生理盐水的量，假设注入每组附加鼠标，以允许注射器的正确装载。考虑给定的实验中，计算所需的5只小鼠的各组中的盐水的量:5×2.5毫升= 12.5 ml生理盐水为每个组（或25毫升总数）。注意:如果在同一组内的老鼠是不一样的体重（体重超过10％的差异），准备注射液分别混合。 </li></ol></li><li>使用已批准供人类使用防腐剂和无内毒素，无菌生理盐水，10毫升，多用小瓶。 </li><li>使用连接到20ml的注射器的鲁尔锁头20号针头（0.9毫米×25毫米），从生理盐水瓶中抽出液体，并清空注射器到50毫升锥形管中。为了帮助DNA的盐水注入混合物的制备过程中盐的精确移液，收集比所需的注射所有的小鼠在实验的更多的盐水。对于上面的实验中，从3撤回盐水，将10毫升小瓶（总约30毫升）中，即使盐水的实验所需的计算量仅为25毫升</li><li>吸取DNA的计算量为不同的50ml锥形管中。小心不要触摸与移液管主体管的一侧，以避免引进的内毒素。根据本示例中，吸取250微克实验的质粒DNA为单独的锥形管的控制和250微克。 </li><li>添加所需的盐水的量来使用血清吸液管将DNA。考虑到样本实验，吸管12.5毫升生理盐水各母液（实验组和对照组）的。 </li><li>让所有的解决方案，以达到室温下在注射之前。 </li></ol></li><li>注射器的制备<ol><li>对于每个小鼠，填充无菌的3毫升，鲁尔锁注射器用无菌20号针头（0.9毫米×25毫米）的DNA的盐水混合。注意避免气泡。避免使用更高容量的注射器注射的流速不能被控制得很好，而且也难以用一只手操纵较大的注射器。弹出任何多余的DNA的盐水混合放回锥形管，因为这可能是重新使用。 </li><li>针切换到一个无菌的27号针头。液体填充针完全没有引入气泡。调整计算基于鼠标的重量将DNA-盐水混合物的体积。 </li><li>不要让不封顶的针头接触任何非无菌表面。放置在一个平面上的盖，插入针没有握住瓶盖用另一只手，按上限针:如果有必要，针头可使用单手技术重新皑皑对一个公司的对象来保护帽将针头。注射器现在已准备好尾静脉注射。 </li></ol></li></ol> 4，水动力DNA传递<ol><li>需要注意的是麻醉的小鼠可能会​​降低活力。避免在一个房间里太冷执行HGD，因为这也将降低生存能力。如果房间有20°C的环境温度下，或者使用麻醉，将小鼠一个加热垫设置在37℃后程序，以避免任何动物的损失。如果使用麻醉，确保鼠标的嘴和鼻子都在热垫通畅的同时，观察鼠标，直到它完全恢复。不要隐瞒的食物或水从小鼠前HGD。 </li><li>暖鼠来扩张血管。 <ol><li>将鼠标笼（床上用品包括在内）在5分钟加热垫，使被褥的温度大约为38-39°C。的温度应足够低，以保持小鼠在热垫为一延长期限。 </li><li>或者，将鼠标变成背的访问限制器以最小的克制。轻轻地用一块纱布浸湿温水中保持温暖鼠标1分钟的尾巴。让鼠标重新定位，如果必要的。擦拭尾部用纸巾擦干。 </li><li>另外，保暖鼠标放置在热灯下笼不超过2分钟。如果使用烤灯，谨慎操作，避免鼠标过热。 </li></ol></li><li>大容积尾静脉注射<ol><li>通过实验室胶带固定背侧访问阻挡在一个平面上。 </li><li>的尾巴持有，请将鼠标轻轻放入阻挡并插入插头。使用尽可能少的约束作为必要保持尾固定。确保鼠标自由地呼吸。 </li><li>如果鼠标是在严重的困扰任意点的过程中，从阻挡立即移开鼠标，并允许它休息。 </li><li>将鼠标等等横向尾静脉中的一个是可见的。红线在尾部的中间运行是动脉，而在尾部两侧的蓝线是横向尾静脉:直视下在小鼠背部定位的横向尾静脉。 </li><li>擦拭鼠标的尾部用酒精棉签彻底。 </li><li>使用非注射手，紧紧握住尾巴的食指和中指之间，以使尾部穿过食指下，在中间和第三手指和下小指。让拇指保持自由移动。 （ 图1A） </li><li>用另一只手，擦拭被用酒精棉签再注入的区域。允许区域干燥。 </li><li>拿起装入注射器与注射手和用拇指和其它四个数字之间的枪管握住。不要守住柱塞。 </li><li>定位与针指向鼠标的主体平行于尾部的注射器和朝上针的斜面（倾斜边缘）。 </li><li>将针头插入尾静脉。继续进行注射仅当静脉正确定位，这是显而易见的，从针无阻力滑动。需要注意的是进针的深度是个人喜好的问题然而，完全插入时不因静脉剪切的风险增加建议。 避免移动针头。 </li><li>用尾巴保持手的拇指，关闭上针，然后按集线器反对尾巴的位置保持针的枢纽。不要施加强压，不守住轴针，因为这些都会阻碍液体流入静脉。持用食指尾部松弛地在这一点上，以便不妨碍喷射（ 图1A）。 </li><li>重新定位注射器夹持手，使得食指和中指握住凸缘和拇指是在柱塞的端部（FIGURË1A）。 </li><li>向下按柱塞于一体，连续运动，在6-8秒注入液体的最大音量。 </li><li>从静脉取出针和通过应用温和的压力，尾部用纸巾止血。 </li><li>从限制器和地点鼠标移除鼠标进入恢复笼放置在加热垫的顶部（床上用品应该是37-38°C）。如果注射室是冷的，这一步是为鼠标生存的关键。守住鼠标的尾部，直到出血完全停止。 </li><li>观察小鼠1小时以下的水动力DNA传递。气喘吁吁，不动的初期是正常而引起的HGD临时心律失常，但要确保鼠标显示出复苏的迹象，约5分钟。如果鼠标的呼吸变得非常浅，轻轻按摩鼠标促进呼吸的腹部。注意恢复的速度可以由所使用的小鼠品系略有影响。 </li><li>返回鼠标住房笼，确保鼠标有食物和水供应充足。 </li></ol></li><li>使用替代手的位置大容量尾静脉注射<ol><li>把鼠标放到阻挡和注射制备由下列步骤4.3.1至4.3.5中，如前所述。 </li><li>用四个手指在一个90度的角度弯曲，休息的非注射的手放在一平面上。手指（除了拇指）应让彼此的顶端，创造了一个平台。容纳拇指和指向手指（ 图1B）之间的小鼠的尾部。 </li><li>用另一只手，擦拭被用酒精棉签再注入的区域。允许区域干燥。 </li><li>抓住注射器与注射手和通过凸缘与柱塞，准备用于注射的结束握住。 </li><li>通过从步骤4.3.9至步骤4.3.17下面这个协议中，如前面所述完成该过程。 </li></ol></li></ol>

<ol>
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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. J., 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=Development+of+catheter-based+procedures+for+transducing+the+isolated+rabbit+liver+with+plasmid+DNA.">Development of catheter-based procedures for transducing the isolated rabbit liver with plasmid DNA.</a> Hum Gene Ther. 13, 2065-2077 (2002).</li><li>Yoshino, H., Hashizume, K., Kobayashi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naked+plasmid+DNA+transfer+to+the+porcine+liver+using+rapid+injection+with+large+volume.">Naked plasmid DNA transfer to the porcine liver using rapid injection with large volume.</a> Gene Ther. 13, 1696-1702 (2006).</li><li>Vanden Bossche, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+general+aspects+of+the+distribution+and+epidemiology+of+bovine+trypanosomosis+in+southern+Africa.">Some general aspects of the distribution and epidemiology of bovine trypanosomosis in southern Africa.</a> Int J Parasitol. 31, 592-598 (2001).</li><li>Fevre, E. M., Picozzi, K., Jannin, J., Welburn, S. C., Maudlin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+African+trypanosomiasis:+Epidemiology+and+control.">Human African trypanosomiasis: Epidemiology and control.</a> Adv Parasitol. 61, 167-221 (2006).</li><li>Lugli, E. B., Pouliot, M., Portela Mdel, P., Loomis, M. R., Raper, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+primate+trypanosome+lytic+factors.">Characterization of primate trypanosome lytic factors.</a> Mol Biochem Parasitol. 138, 9-20 (2004).</li><li>Pays, E., 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=The+trypanolytic+factor+of+human+serum.">The trypanolytic factor of human serum.</a> Nat Rev Microbiol. 4, 477-486 (2006).</li><li>Raper, J., Fung, R., Ghiso, J., Nussenzweig, V., Tomlinson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+novel+trypanosome+lytic+factor+from+human+serum.">Characterization of a novel trypanosome lytic factor from human serum.</a> Infect Immun. 67, 1910-1916 (1999).</li><li>Vanhamme, L., 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=Apolipoprotein+L-I+is+the+trypanosome+lytic+factor+of+human+serum.">Apolipoprotein L-I is the trypanosome lytic factor of human serum.</a> Nature. 422, 83-87 (2003).</li><li>Nielsen, M. J., 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=Haptoglobin-related+protein+is+a+high-affinity+hemoglobin-binding+plasma+protein.">Haptoglobin-related protein is a high-affinity hemoglobin-binding plasma protein.</a> Blood. 108, 2846-2849 (2006).</li><li>Molina-Portela Mdel, P., Lugli, P., Recio-Pinto, E., Raper, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypanosome+lytic+factor,+a+subclass+of+high-density+lipoprotein,+forms+cation-selective+pores+in+membranes.">Trypanosome lytic factor, a subclass of high-density lipoprotein, forms cation-selective pores in membranes.</a> Mol Biochem Parasitol. 144, 218-226 (2005).</li><li>Perez-Morga, D., 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=Apolipoprotein+L-I+promotes+trypanosome+lysis+by+forming+pores+in+lysosomal+membranes.">Apolipoprotein L-I promotes trypanosome lysis by forming pores in lysosomal membranes.</a> Science. 309, 469-472 (2005).</li><li>Molina-Portela, M. P., Samanovic, M., Raper, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+roles+of+apolipoprotein+components+within+the+trypanosome+lytic+factor+complex+revealed+in+a+novel+transgenic+mouse+model.">Distinct roles of apolipoprotein components within the trypanosome lytic factor complex revealed in a novel transgenic mouse model.</a> J Exp Med. 205, 1721-1728 (2008).</li><li>Oli, M. W., Cotlin, L. F., Shiflett, A. M., Hajduk, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum+resistance-associated+protein+blocks+lysosomal+targeting+of+trypanosome+lytic+factor+in+Trypanosoma+brucei.">Serum resistance-associated protein blocks lysosomal targeting of trypanosome lytic factor in Trypanosoma brucei.</a> Eukaryot Cell. 5, 132-139 (2006).</li><li>Thomson, R., Molina-Portela, P., Mott, H., Carrington, M., Raper, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+gene+delivery+of+baboon+trypanosome+lytic+factor+eliminates+both+animal+and+human-infective+African+trypanosomes.">Hydrodynamic gene delivery of baboon trypanosome lytic factor eliminates both animal and human-infective African trypanosomes.</a> Proc Natl Acad Sci U S A. 106, 19509-19514 (2009).</li><li>Genovese, 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=Association+of+trypanolytic+ApoL1+variants+with+kidney+disease+in+African+Americans.">Association of trypanolytic ApoL1 variants with kidney disease in African Americans.</a> Science. 329, 841-845 (2010).</li><li>Tzur, S., 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=Missense+mutations+in+the+APOL1+gene+are+highly+associated+with+end+stage+kidney+disease+risk+previously+attributed+to+the+MYH9+gene.">Missense mutations in the APOL1 gene are highly associated with end stage kidney disease risk previously attributed to the MYH9 gene.</a> Hum Genet. 128, 345-350 (2010).</li><li>Suda, T., Gao, X., Stolz, D. 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>

作者宣称，他们有没有竞争的财务权益。

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被注入的...

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>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Alcohol Prep Wipe</td>
			<td style="width:113px;">Webcol</td>
			<td align="right" style="width:119px;">6818</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AST kit, Amplite Colorimetric</td>
			<td style="width:113px;">AAT Bioquest</td>
			<td align="right" style="width:119px;">13801</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conical Tube, 50 ml</td>
			<td style="width:113px;">BD Falcon</td>
			<td align="right" style="width:119px;">352070</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EndoFree Plasmid Maxi Kit</td>
			<td style="width:113px;">Qiagen</td>
			<td align="right" style="width:119px;">12362</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KimWipes</td>
			<td style="width:113px;">KIMTECH</td>
			<td align="right" style="width:119px;">34120</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Mouse Tail Illuminator</td>
			<td style="width:113px;">Braintree Scientific</td>
			<td style="width:119px;">MTI RST</td>
			<td style="width:167px;">Or equivalent</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Needle, 20 Gx 1 (0.9mmx25mm)</td>
			<td style="width:113px;">BD</td>
			<td align="right" style="width:119px;">305175</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Needle, 27 Gx 1/2 (0.9mmx25mm)</td>
			<td style="width:113px;">BD</td>
			<td align="right" style="width:119px;">305109</td>
			<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>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Sodium Chloride, 0.9% INJ USP 10 ml, Hospira&#160;</td>
			<td style="width:113px;">Fisher</td>
			<td style="width:119px;">NC9054335</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Syringe, 3ml, Luer-Lok Tip</td>
			<td style="width:113px;">BD</td>
			<td align="right" style="width:119px;">309657</td>
			<td style="width:167px;">Luer Lock necessary to withstand pressure from HGD</td>
		</tr>
	</tbody>
</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

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。

蛋白通过流体动力基因传递在小鼠体内瞬时表达

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

transient-expression-of-proteins-by-hydrodynamic-gene-delivery-in-mice

Research

JoVE Journal

Biology

28.3K 次观看.  Hunter College, CUNY.  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。 

视频: Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

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

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

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

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

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

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

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

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 to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

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 ...

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

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

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

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

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 ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

代谢率的测量<em&gt;果蝇</em&gt;使用呼吸运动

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

重组人蛋白与HEK293悬浮细胞的瞬时转染高产表达

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

We describe a method to knock down gene expression in a growing population of E. coli cells using sequence-targeted sRNA expression cassettes delivered by an M13 phagemid vector.