Name: handling of the cotton rat in studies for the pre-clinical evaluation of oncolytic viruses
Uploaded: 2014-11-24T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses at JoVE.com

Breanne Cuddington holds a fellowship from the Canadian Breast Cancer Foundation. This work was sponsored by operating grants from the Cancer Research Society and the Canadian Cancer Society Research Institute (formerly the Canadian Breast Cancer Research Alliance). We thank Ann Tollefson (Saint Louis University School of Medicine) for LCRT cells and Dr. Kathleen Delaney and Marion Corrick for technical assistance with cotton rat housing and sedation.

注:根据加拿大会议上的动物护理准则使用的协议已经批准了我们的机构动物研究伦理委员会麦克马斯特大学。实验是在麦克马斯特大学动物中心进行设施。 1.培养LCRT细胞<ol><li>培养LCRT细胞在Dulbecco改进的Eagle培养基（DMEM），补充有10％胎牛血清（FBS），2mM的L-谷氨酰胺，100U / ml青霉素和100μg/ ml链霉素。维持细胞在T-150细胞培养瓶中，在37℃和5％的CO 2。传代细胞时它们形成90％的汇合单层（每2-3天， 图1）。 </li><li>预暖1X磷酸盐缓冲盐水（PBS），1×胰蛋白酶和介质在37℃的水浴中之前，分裂细胞10分钟。 </li><li>从烧瓶中吸出培养基并冲洗细胞用5ml的1×PBS中。 </li><li>冲洗后，吸PBS和孵化细胞用2ml的1×胰蛋白酶直到细胞分离从烧瓶（〜2分钟）。 </li><li>重悬的细胞在8ml培养基（总共为10ml的细胞悬浮液），并轻轻地吸液管上下打散细胞团块。 </li><li>播种1毫升细胞悬浮于24毫升培养基（总共每T-150 25ml）和岩石烧瓶轻轻地从一边到另一边维持细胞的T-150烧瓶中。维持细胞在37℃和5％的CO 2，直到下一次分裂。 </li></ol> 2.评估病毒复制和细胞毒性LCRT细胞<ol><li>病毒复制 注:表达荧光标记病毒的结构，如绿色荧光蛋白（GFP），根据内源性病毒启动子控制有利于病毒感染的可视化和推广使用荧光板读者。 <ol><li>种子LCRT细胞进入培养板，留下一个井用于计数。种子细胞，使得它们将是80％-90％汇合的一天之后。使用10 5 CEL浓度LS /毫升（每孔100μl），以在96孔平底板一天后产生所需的汇合。 </li><li>第二天，预暖的1×PBS，1×胰蛋白酶，在37℃水浴中开始实验10分钟，以事先完全和无血清培养基。 </li><li>从计数井和冲洗细胞用5ml的1×PBS的摇动它在井的表面吸出培养基。 </li><li>冲洗后，吸出PBS孵育细胞用2ml 1X胰蛋白酶直到细胞分离从烧瓶（〜2分钟）。 </li><li>将细胞重悬于完全培养基的适当体积，以使用血细胞计数器的可数的范围内，得到的细胞密度。为了确保准确的细胞计数，彻底混合细胞悬浮液通过上下吹打接种血细胞计数仪之前。 </li><li>确定在感染所需复数（MOI）所需的感染病毒原液的体积。 所需帕确形成单位（PFU）=镀细胞的数量* MOI（PFU /细胞） 病毒股票的数量=所需需要PFU /病毒滴度的股票（PFU /毫升） </li><li>制备病毒接种物在管中的无血清培养基。加入接种到细胞前通过涡旋或移液调匀。 </li><li>感染细胞1小时，于37℃，在此之后施加DMEM + 1％FBS的维持覆盖。 </li><li>扫描板1和后两天感染（PI）以可视化的GFP荧光。 </li></ol></li><li>病毒的细胞毒性 注:低光条件下进行的刃天青细胞毒性测定为化合物是光敏的。只有良好的培养基应包括纠正背景荧光。 <ol><li>制备刃天青的5％（体积/体积）中的1×PBS溶液中。混合该溶液通过移液。 </li><li>从细胞中吸出培养基，并应用5％刃天青溶液。包括一个孔含有介质只以校正背景荧光。 </li><li>孵育细胞30分钟，在37 °C，后它使用荧光酶标仪（激发波长530，发射595纳米）读取荧光。 </li><li>分析相对于未感染的控制校正背景荧光数据。 </li></ol></li></ol> 3.住房和处理<ol><li>住房和饮食<ol><li>房子棉鼠分别减少内斗中含灭鼠床上用品（1/8"玉米芯床上用品），PVC管材不超过8英寸，nestlets为充实（ 图2）的部分聚碳酸酯老鼠笼。 </li><li>使用可固定钢篮坐在凌驾笼子，并包含啮齿类动物食物和一瓶水。 注意:此笼设置将允许动物的安全和容易捕捉，与载置对至关重要轿厢的存在的端富集管。 </li></ol></li><li>处理<ol><li>处理棉鼠在早晨之前，轮由动物设施的技术人员，以避免激动人心它们之前的过程。 </li><li>在所有的手续，穿上厚厚的皮手套的保护。 </li><li>作为动物主要停留在富集管，使用它们在常规清洗大鼠转移到一个新的笼子。可替代地，略微打开笼子，以允许处理程序以在到达他们的手，该动物随后可以通过洁肤皮肤只是肩膀上方和向下推抑制。实践中注意不要过度使用武力作为动物可能咬自己的舌头。 </li><li>要有耐心，并且使用一个稳定的手，动物有很强的飞行和战斗回应，并会尽量避免捕捉运行，并跳出笼子。重要的是，不要在尾部处理动物会发生脱套。 </li><li>陷阱在他们的富集管过直接处理的动物。这大大减少伤害和逃避。 </li></ol></li></ol> 4.捕捉和麻醉<ol><li>捕获<ol><li>穿着厚厚的皮手套保护在所有的程序离子。 </li><li>使用带有孔的空气和一个盖，麻醉诱导室足够大，以适应在容器和装在气体输出软管（ 图3）一鼻锥体的大型的透明塑料容器。 </li><li>在对工作，以使该过程更高效，对动物的曝光时间减少到异氟醚，吸入麻醉剂。确保一位研究人员负责开启和更换钢盖在笼子和感应室的盖（处理器＃1）及其联营公司是负责管和运输的感应室动物的捕捉（处理器＃ 2）。 </li><li>将笼子放在一个平面上，并取下外盖。抬起钢送纸托盘稍微缓慢操纵富集管所以它与保持架的两侧，并针对背面平行。如果有必要，使用对象来操纵富集管，而无需打开笼子，避免搅动动物（手柄R·1）。 </li><li>如果动物变得激动，离开管子，以便有足够的时间进行动物放松，在管（处理器＃1）再次定居。 </li><li>慢慢地，故意抬起，从浓缩管钢盖最远的边缘，保持在笼子里接触的另一端。作的空间足够大的塑料容器（处理程序＃2）。 </li><li>在一个平稳，快速运动，充实管凌驾推动塑料容器。保持容器保持器的侧面的接触，捕集该动物在管。作为快速执行步骤4.1.6和4.1.7越好（处理程序＃2）。 </li><li>除去钢馈纸盘并给塑料容器盖，处理程序＃2（处理程序＃1）。滑动的塑料盖的笼和容器的侧面之间，是注意到在这个过程中，被困动物的附属物。不要密封容器，因为这将使下一步更困难（处理器＃2）。 </li></ol></li><li> AnesthesIA <ol><li>确保该容器保持闭合和运输动物到感应腔。快速地放置在腔室中的动物并取出容器盖在一个流体运动（处理程序＃2）。打开并立即更换感应室盖（处理器＃1）。 </li><li>打开异氟烷向感应室（5升/分钟）的流量和监视动物为嗜睡的迹象，在该点迅速从管和容器，无论是从感应腔室移除滑动动物，以促进气体循环。 </li><li>当鼠被完全麻醉，将其移动到工作表面，并放置在鼻子和嘴到鼻锥（ 图3）。当它是反应迟钝有力脚趾捏鼠完全麻醉。 </li><li>放置在动物的眼睛兽医凡士林眼药膏，以防止干燥和擦伤。这是因为棉鼠有大眼睛，可以容易感染，如果伤害发生的重要一步。 </li> &lt;LI&gt;小心地监控和保持恒定的呼吸率，并确保该动物的鼻子保持在鼻锥整个过程中嵌合。适当调整异氟烷的流速。所需的异氟醚的量麻醉各动物会有所不同。 </li><li>程序后，返回动物笼子里，并确保它重新获得充分的流动性和胸骨斜卧。 </li></ol></li></ol> 5.准备LCRT细胞皮下肿瘤形成的注意:一个T-150烧瓶LCRT（90％汇合），得到大约2×10 7个细胞。碱对所需的细胞的总数所需的T-150烧瓶的数目。种子额外烧瓶，以确保得到所需的细胞的总数量，以及容纳电池的制备和那些需要额外的注射过程中丢失。置于冰上细胞尽可能延长细胞活力。 <ol><li>收获细胞，吸出培养基，从该烧瓶中的ð冲洗细胞用5毫升1X PBS的。 </li><li>抽吸PBS孵育细胞用2ml的1×胰蛋白酶直到细胞从烧瓶中（〜2分钟）分离。 </li><li>重悬的细胞在8ml培养基（总共为10ml的细胞悬浮液），并轻轻地吸液管上下打散细胞团块。继续从另外的烧瓶收获细胞。 </li><li>池中的所有细胞悬浮液到一个锥形管，大约4的T-150S每50毫升锥形管中。 </li><li>离心管，在160×g下 10分钟，在4℃。 </li><li>抽吸培养基并悬浮在PBS中的适当体积（10毫升的PBS元T-150）的细胞沉淀到使用血细胞计数器的可数的范围内，得到的细胞密度。为了确保准确的细胞计数，彻底混合细胞悬浮液通过上下吹打加载血细胞计数仪之前。 </li><li>计算细胞总数: 细胞的总数目收获=细胞计数（细胞/ ml）×悬浮体积（ml） </li><li>确定细胞悬浮液所需的所有注射​​体积。使每个实验2-3额外的剂量。总共皮下注射5×10 5 LCRT细胞将形成在3-4天内可触及的肿瘤。 所需的细胞总数= 5×10 5个单元×剂量的总数 体积的细胞悬液需要（毫升）=（要求*总和注射量的总细胞）/（细胞收获的总数） </li><li>吸管细胞悬浮液的所需体积成锥形管含有PBS中并充分混合。等分试样单独注射（100微升）进Eppendorf管中。在注射过程保持在冰管。 </li></ol> 6.注射注:带有两个研究者执行程序，一个用于执行，而其他显示器动物的呼吸速率和一般状况，同时在麻醉下注射。使用胰岛素注射器（29克X 1/2"，0.3毫升）的所有注射​​和一个新的针每只动物。 <ol><li>皮下注射<ol><li>捕捉和麻醉的动物（第4节）。 </li><li>剃须使用快船注射部位。棉鼠毛皮是厚并要求一个尖锐微调以获得一光滑的表面，用于注射。清洁注射部位使用棉棒70％的乙醇，并允许它在继续之前完全蒸发。 </li><li>负载注射器（29克X 1/2"，0.3毫升）与细胞制定缓慢而稳步增长。如果泡沫是很明显的甩尾一些力注射器。一旦气泡在顶部推柱塞直到液体是在针的顶端。 </li><li>解除皮肤注射部位（称为皮肤隆起），并插入针头斜面朝上。确保针头在皮肤下自由移动，以避免肌内注射。 </li><li>排出注射器的内容物均匀和缓慢。下拔出针头斜面的一面。 </li></ol></li><li>瘤内注射<ol><li>捕获ð麻醉的动物（第4节）。 </li><li>清洁注射部位使用棉棒70％的乙醇，并允许它在继续之前完全蒸发。 </li><li>负载注射器（29克X 1/2&#39;，0.3毫升）与病毒接种物由制定缓慢而稳定地握住针在直立位置。如果泡沫是很明显的甩尾一些力注射器。一旦气泡在顶端推然后柱塞直到液体是在针的顶端。 </li><li>插入针头斜面朝上进入肿瘤并排出注射器的内容物均匀和缓慢地移动的同时在针中的风扇状图案，部分撤回针头的每个移动之前，防止肿瘤的裂伤。下拔出针头斜面的一面。 注:皮下LCRT肿瘤生长快，在5-7天达到约100 立方毫米。此外，坏死和出血性中心常常形成肿瘤的表面上在几天内，并要求CAreful监测（ 图4）。 </li></ol></li><li>腹腔注射<ol><li>捕捉和麻醉的动物（第4节）。 </li><li>使用棉签清洁注射部位用70％乙醇，并允许它在继续之前完全蒸发。 </li><li>负载注射器（29克X 1/2"，0.3毫升）与药物通过制定缓慢而稳步增长。如果泡沫是很明显的甩尾一些力注射器。一旦气泡在顶端推然后柱塞直到液体是在针的顶端。 </li><li>针插入腹部的右下象限。向后拉柱塞，以确保血液或粪便不吸移，这表示所述针的不正确放置。如果发生这种情况，撤回的针头和准备一个新的注射器。当针被正确放置，排出注射器的内容物均匀和缓慢。 </li></ol></li></ol> 7.肿瘤切除和剖检<ol><li>收集和清理人升工具用70％乙醇之前，动物安乐死。 </li><li>通过所需的方法安乐死的动物，CO 2吸入（2升/分钟5-10分钟）的建议。检查动物的身体状况有任何异常。 </li><li>将背斜卧的动物上的夹层板，清洁用70％乙醇的动物。 </li><li>使用镊子解除肌肤的小腹。用剪刀通过皮肤和肌肉切割和使内侧切口运行动物的长度（肛门至下巴）。 </li><li>通过使两个切口，1横向向上胸腔的侧面和一个横跨胸骨暴露心脏和肺切的肋笼。检查肺的裂片为任何转移27,29。 </li><li>审查所有器官的异常，并记录在颜色，大小和一致性的任何变化。如果有必要，切割器官用手术刀来检查内部组织。具体来说，检查肝，肾，脾，胃肠道。 </li><li>检查淋巴结转移的膨大和27,29。 </li><li>收集肿瘤，使侧翼切口上面和下面的肿瘤，使得皮肤能够拉离体用镊子。虽然紧紧握住用镊子皮肤，用手术刀在肿瘤和真皮（ 图5）之间切割仔细切除肿瘤。 </li><li>立即将肿瘤中的10％中性缓冲福尔马林的标记的容器。 </li><li>取决于肿瘤的大小，允许1-2天（≤2毫米，小）或5-6天（&gt; 2毫米，大）用于制备切片用于组织学分析（ 图6）之前的固定。 </li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+analysis+of+an+oncolytic+herpesvirus+encoding+granulocyte-macrophage+colony+stimulating+factor+for+the+treatment+of+malignant+melanoma.">Critical analysis of an oncolytic herpesvirus encoding granulocyte-macrophage colony stimulating factor for the treatment of malignant melanoma.</a> Journal of Oncolytic Virotherapy. 3, 11-20 (2014).</li><li>Jones, C., Chowdhury, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+biology+of+bovine+herpesvirus+type+1+(BHV-1),+its+role+as+a+cofactor+in+the+bovine+respiratory+disease+complex+and+development+of+improved+vaccines.">A review of the biology of bovine herpesvirus type 1 (BHV-1), its role as a cofactor in the bovine respiratory disease complex and development of improved vaccines.</a> Anim Health Res Rev. 8 (2), 187-205 (2007).</li><li>Jones, C., Chowdhury, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bovine+herpesvirus+type+1+(BHV-1)+is+an+important+cofactor+in+the+bovine+respiratory+disease+complex.">Bovine herpesvirus type 1 (BHV-1) is an important cofactor in the bovine respiratory disease complex.</a> Vet Clin North Am Food Anim Pract. 26 (2), 303-321 (2010).</li><li>Hushur, O., Takashima, Y., Matsumoto, Y., Otsuka, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restriction+of+bovine+herpesvirus+1+(BHV-1)+growth+in+non-permissive+cells+beyond+the+expression+of+immediate+early+genes.">Restriction of bovine herpesvirus 1 (BHV-1) growth in non-permissive cells beyond the expression of immediate early genes.</a> J Vet Med Sci. 66 (4), 453-455 (2004).</li><li>Cuddington, B. P., Dyer, A. L., Workenhe, S. T., Mossman, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncolytic+bovine+herpesvirus+type+1+infects+and+kills+breast+tumor+cells+and+breast+cancer-initiating+cells+irrespective+of+tumor+subtype.">Oncolytic bovine herpesvirus type 1 infects and kills breast tumor cells and breast cancer-initiating cells irrespective of tumor subtype.</a> Cancer Gene Ther. 20 (5), 282-289 (2013).</li><li>Cuddington, B. P., Mossman, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permissiveness+of+Human+Cancer+Cells+to+Oncolytic+Bovine+Herpesvirus+1+Is+Mediated+in+Part+by+KRAS+Activity.">Permissiveness of Human Cancer Cells to Oncolytic Bovine Herpesvirus 1 Is Mediated in Part by KRAS Activity.</a> J Virol. 88 (12), 6885-6895 (2014).</li><li>Small, E. 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=A+phase+I+trial+of+intravenous+CG7870,+a+replication-selective,+prostate-specific+antigen-targeted+oncolytic+adenovirus,+for+the+treatment+of+hormone-refractory,+metastatic+prostate+cancer.">A phase I trial of intravenous CG7870, a replication-selective, prostate-specific antigen-targeted oncolytic adenovirus, for the treatment of hormone-refractory, metastatic prostate cancer.</a> Mol Ther. 14 (1), 107-117 (2006).</li><li>Freytag, S. O., 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=Phase+I+study+of+replication-competent+adenovirus-mediated+double+suicide+gene+therapy+for+the+treatment+of+locally+recurrent+prostate+cancer.">Phase I study of replication-competent adenovirus-mediated double suicide gene therapy for the treatment of locally recurrent prostate cancer.</a> Cancer Res. 62 (17), 4968-4976 (2002).</li><li>Benjamin, R., Helman, L., Meyers, P., Reaman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+I/II+dose+escalation+and+activity+study+of+intravenous+injections+of+OCaP1+for+subjects+with+refractory+osteosarcoma+metastatic+to+lung.">A phase I/II dose escalation and activity study of intravenous injections of OCaP1 for subjects with refractory osteosarcoma metastatic to lung.</a> Hum Gene Ther. 12 (12), 1591-1593 (2001).</li><li>Prince, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cotton+Rat+in+Biomedical+Research.">The Cotton Rat in Biomedical Research.</a> Animal Welfare Information Center Newsletter. 5 (2), 3-5 (1994).</li><li>Tsai, J. C., Garlinghouse, G., McDonnell, P. J., Trousdale, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+animal+model+of+adenovirus-induced+ocular+disease.+The+cotton+rat.">An experimental animal model of adenovirus-induced ocular disease. The cotton rat.</a> Arch Ophthalmol. 110 (8), 1167-1170 (1992).</li><li>Ginsberg, H. 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=A+mouse+model+for+investigating+the+molecular+pathogenesis+of+adenovirus+pneumonia.">A mouse model for investigating the molecular pathogenesis of adenovirus pneumonia.</a> Proc Natl Acad Sci U S A. 88 (5), 1651-1655 (1991).</li><li>Russell, S. J., Peng, K. W., Bell, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncolytic+virotherapy.">Oncolytic virotherapy.</a> Nat Biotechnol. 30 (7), 658-670 (2012).</li><li>Mossman, K. L., Saffran, H. A., Smiley, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+ICP0+mutants+are+hypersensitive+to+interferon.">Herpes simplex virus ICP0 mutants are hypersensitive to interferon.</a> J Virol. 74 (4), 2052-2056 (2000).</li><li>Mossman, K. L., Smiley, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+ICP0+and+ICP34.5+counteract+distinct+interferon-induced+barriers+to+virus+replication.">Herpes simplex virus ICP0 and ICP34.5 counteract distinct interferon-induced barriers to virus replication.</a> J Virol. 76 (4), 1995-1998 (2002).</li><li>Hummel, J. L., Safroneeva, E., Mossman, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+ICP0-Null+HSV-1+and+interferon+signaling+defects+in+the+effective+treatment+of+breast+adenocarcinoma.">The role of ICP0-Null HSV-1 and interferon signaling defects in the effective treatment of breast adenocarcinoma.</a> Mol Ther. 12 (6), 1101-1110 (2005).</li><li>Papp, Z., Middleton, D. M., Mittal, S. K., Babiuk, L. A., Baca-Estrada, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+immunization+with+recombinant+adenoviruses:+induction+of+immunity+and+protection+of+cotton+rats+against+respiratory+bovine+herpesvirus+type+1+infection.">Mucosal immunization with recombinant adenoviruses: induction of immunity and protection of cotton rats against respiratory bovine herpesvirus type 1 infection.</a> J Gen Virol. 78 (11), 2933-2943 (1997).</li><li>Papp, Z., Babiuk, L. A., Baca-Estrada, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+pre-existing+adenovirus-specific+immunity+on+immune+responses+induced+by+recombinant+adenovirus+expressing+glycoprotein+D+of+bovine+herpesvirus+type+1.">The effect of pre-existing adenovirus-specific immunity on immune responses induced by recombinant adenovirus expressing glycoprotein D of bovine herpesvirus type 1.</a> Vaccine. 17 (7-8), 933-943 (1999).</li><li>Mittal, S. K., 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=Induction+of+systemic+and+mucosal+immune+responses+in+cotton+rats+immunized+with+human+adenovirus+type+5+recombinants+expressing+the+full+and+truncated+forms+of+bovine+herpesvirus+type+1+glycoprotein+gD.">Induction of systemic and mucosal immune responses in cotton rats immunized with human adenovirus type 5 recombinants expressing the full and truncated forms of bovine herpesvirus type 1 glycoprotein gD.</a> Virology. 222 (2), 299-309 (1996).</li><li>Steel, J. C., 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=Syngeneic+Cotton+Rat+Cancer+Model+for+Replicating+Adenoviral+Vectors.">Syngeneic Cotton Rat Cancer Model for Replicating Adenoviral Vectors.</a> Molecular Therapy. 13 (1), 123 (2006).</li><li>Toth, K., 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=Cotton+rat+tumor+model+for+the+evaluation+of+oncolytic+adenoviruses.">Cotton rat tumor model for the evaluation of oncolytic adenoviruses.</a> Hum Gene Ther. 16 (1), 139-146 (2005).</li><li>Toth, K., Spencer, J. F., Wold, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocompetent,+semi-permissive+cotton+rat+tumor+model+for+the+evaluation+of+oncolytic+adenoviruses.">Immunocompetent, semi-permissive cotton rat tumor model for the evaluation of oncolytic adenoviruses.</a> Methods Mol Med. 130, 157-168 (2007).</li><li>Steel, J. C., 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=Immunocompetent+syngeneic+cotton+rat+tumor+models+for+the+assessment+of+replication-competent+oncolytic+adenovirus.">Immunocompetent syngeneic cotton rat tumor models for the assessment of replication-competent oncolytic adenovirus.</a> Virology. 369 (1), 131-142 (2007).</li><li>Workenhe, S. T., 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=Immunogenic+HSV-mediated+oncolysis+shapes+the+antitumor+immune+response+and+contributes+to+therapeutic+efficacy.">Immunogenic HSV-mediated oncolysis shapes the antitumor immune response and contributes to therapeutic efficacy.</a> Mol Ther. 22 (1), 123-131 (2014).</li><li>Sobol, P. T., 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=Adaptive+antiviral+immunity+is+a+determinant+of+the+therapeutic+success+of+oncolytic+virotherapy.">Adaptive antiviral immunity is a determinant of the therapeutic success of oncolytic virotherapy.</a> Mol Ther. 19 (2), 335-344 (2011).</li><li>Prince, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cotton+Rat+in+Biomedical+Research.">The Cotton Rat in Biomedical Research.</a> Animal Welfare Information Center Newsletter. 5 (2), (1994).</li></ol>

The authors acknowledge there are no financial conflicts of interest related to this research.

Cotton rats are highly excitable and have a strong flight response. Therefore, special care should be taken to minimize any undue stress on the animal. The cage setup described will allow for safe and easy capture of the animals, with the placement of the enrichment tube being of the utmost importance. When setting up cages, ensure that the enrichment tubes meet the size and shape requirements, and are placed in proper orientation in the cage. It is also important to ensure that any technicians who might be aiding in ani...

溶瘤病毒（OV）通过利用正常和肿瘤细胞1,2-之间的生化差异选择性地复制在肿瘤细胞中。有两种类型的OVS:那些不需要的突变来实现选择性溶瘤，称为天然存在的野生型病毒和那些必须被改造以实现选择性溶瘤。在给定的肿瘤类型突变的集合确定为一个OV 2的选择生长优势正常细胞的性质。使用OVS的安全和利益已被证明在临床试验中3-7。尽管在溶瘤病毒疗法的领域中的先进存在临床前和临床结果之间的间隙，这表明需要更好的模型来评价OVS的抗肿瘤功效。 牛疱疹病毒1型（BHV-1）是疱疹病毒家族的一个成员，并且Alphaherpesviridae亚科。 BHV-1 initi茨牛呼吸系统疾病综合症在牛，体现在各种各样的症状类似感冒8,9。 BHV-1结合使用的HSV-1的附件和条目受体如硫酸乙酰肝素和的Nectin-1 10。然而，它在Nectin-2的10的地方结合CD155。 BHV-1具有一个非常窄的宿主范围，使得其不能有效地进入并启动复制在正常和转化的鼠细胞3,4,10。这使得使用常规的鼠模型的有问题的。 BHV-1的溶瘤能力已被证明在体外 11,12。 BHV-1已经显示出从各种组织学起源，包括乳腺癌细胞和乳腺癌起始细胞11,12的启动在复制和杀死人肿瘤细胞。然而，BHV-1的抗肿瘤能力，必须在体内的免疫活性宿主的范围内进行评价。 人类腺病毒（广告），对于这有57种血清型鉴定，最常见的导致呼吸系统疾病的人。溶瘤腺病毒载体已被评估为他们的抗肿瘤功效与若干前进进入临床试验阶段13-15。尽管有前途的临床前数据，临床效果已经达到人们的期望。人肿瘤异种移植模型通常用于研究Ad载体的抗肿瘤功效，尽管它们表现出减弱的免疫应答的病毒16,17。此外，同基因小鼠模型是非允许对广告感染，使得使用这些模型不切实际17,18宿主的免疫应答的评估。 宿主免疫系统已被列为最具影响力的机制，OVS引起肿瘤细胞死亡19。耐受和非耐受的肿瘤相关抗原之间的抗肿瘤反应（TAA）的型号而异，并且可以极大地影响OV疗法的成功。在HSV-1 OV KM100（ICP0 N212VP16 在 1814年20）20,21引起肿瘤消退中的80％的荷瘤小鼠中的鼠多瘤中间T抗原乳腺癌模型22。然而，在HER-2 / neu的型号，KM100的抗肿瘤功效在同系小鼠的20％完全消退和肿瘤停滞之间在转基因变化，HER-2耐受小鼠。总之，这些数据突出的利用动物模型最能概括人类的免疫景观，充分了解哪些功能确定治疗的成功充分评估OVS的重要性。 棉鼠（Sigmodon hispidus），原产于北美和南美，是最常用的呼吸道合胞病毒感染模型（如5审查）。棉鼠中也使用抗BHV-1接种研究，他们概括与牛呼吸道疾病6,23相关联的病理。此外，BHV-1感染棉鼠是免疫原性，诱导持续粘膜和系统免疫应答6,23-25。细胞系已被来自自发纤维肉瘤和乳腺（LCRT）和骨（CCRT和VCRT），分别为26的骨肉瘤。棉鼠已被用来评估溶瘤腺病毒载体在体内的效力，因为它们很容易受到感染的广告，并表现出相似的病理学人类27-29。对于OVS的临床前评价使用免疫受损模型不仅少指示对治疗的临床反应，但它们不能顾及在溶瘤病毒疗法30,31免疫系统的作用。因此，同系和乳腺癌和骨肉瘤的肿瘤耐受棉鼠模型是相关的模型，其中，以评估OVS，如BHV-1和Ad的临床前功效不能使用常规的小鼠模型的研究。

<table><tbody><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium&amp;nbsp;</td><td>Gibco</td><td>11965-092</td><td>May use any brand&amp;nbsp;</td></tr><tr><td>1X Phosphate Buffered Saline&amp;nbsp;</td><td></td><td></td><td>Can prepare in lab, filter to sterilize</td></tr><tr><td>200 mM L-glutamine</td><td>Gibco</td><td>25030164</td><td>May use any brand</td></tr><tr><td>100x Antibiotic-Antimycotic&amp;nbsp;</td><td>Gibco</td><td>15240-062</td><td>May use any brand</td></tr><tr><td>Fetal bovine serum</td><td>Quality Biological Inc.</td><td>110-001-101HI</td><td>May use any brand</td></tr><tr><td>T-150cm&lt;sup&gt;2&lt;/sup&gt; tissue culture flask</td><td>Fisher Scientific</td><td>14-826-80</td><td>May use any brand</td></tr><tr><td>1X TypLE Express</td><td>Life Technologies</td><td>12604-013</td><td></td></tr><tr><td>12-well cell culture plate, flat bottom</td><td>Fisher Scientific</td><td>08-772-29</td><td>May use any brand, must be tissue culture treated</td></tr><tr><td>alamarBlue</td><td>Life Technologies</td><td>DAL1025</td><td>May use an alternative reagent for determination of cell viability</td></tr><tr><td>8640 Teklad 22/5 Rodent diet</td><td>Harlan&amp;nbsp;</td><td>8640</td><td></td></tr><tr><td>1/8&amp;rdquo; corncob rodent bedding</td><td>Harlan</td><td>7092</td><td></td></tr><tr><td>Nestlets</td><td>Ancare</td><td>-</td><td>Made of pulped virgin cotton fiber, dust-free and autoclavable</td></tr><tr><td>50 mL Conical tubes</td><td>Fisher Scientific</td><td>14-432-22</td><td>May use any brand, must be sterile</td></tr><tr><td>Isoflurane USP, 99.9 %, inhalation anesthetic</td><td>Pharmaceutical Partners of Canada Inc.</td><td>M60302</td><td></td></tr><tr><td>70% Ethanol</td><td></td><td></td><td>Can prepare in lab</td></tr><tr><td>10 % Neutral Buffered Formalin</td><td>Sigma-Aldrich</td><td>HT501128</td><td>May use any brand</td></tr><tr><td>NAPCO NapFlow 1200 Class II A/B3 Biosafety Microbiological Safety Cabinet (cell culture hood)</td><td>NAPCO</td><td>Model used not currently available</td><td>May use any brand</td></tr><tr><td>Thermo Fisher Scientific Precision Heated Water Bath</td><td>Fisher Scientific</td><td>Model used not currently available&amp;nbsp;</td><td>May use any brand</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Reichert Bright-line Hemacytometer</td><td>Sigma-Aldrich</td><td>Z359629</td><td>May use any brand</td></tr><tr><td>Typhoon Trio BioAnalyzer&amp;nbsp;</td><td>GE Healthcare Life Sciences</td><td>Model used not currently available&amp;nbsp;</td><td>May use any fluorescence plate reader</td></tr><tr><td>Tecan Safire&lt;sup&gt;2&lt;/sup&gt; Multi-detection Microplate Reader</td><td>Tecan</td><td>Model used not currently available&amp;nbsp;</td><td>May use any fluorescence plate reader</td></tr><tr><td>Allegra 6R benchtop centrifuge</td><td>Beckman Coulter</td><td>366816</td><td>May use any brand</td></tr><tr><td>Table Top Anaesthesia machine</td><td>VetEquip</td><td>Model used not currently available&amp;nbsp;</td><td>May use any brand, must be portable</td></tr><tr><td>Wahl Peanut Mini Clippers</td><td>Wahl</td><td></td><td>May use any brand of small clippers</td></tr><tr><td>Insulin syringes 29 G x 1/2&#039;, 0.3 mL</td><td>BD</td><td>329464</td><td>May use any brand. Insulin syringes are recommended as they make injections easier through the rat&amp;rsquo;s tough skin.&amp;nbsp;</td></tr><tr><td>Cotton swabs</td><td>MedPro</td><td>018-425</td><td>May use any brand</td></tr><tr><td>Sharp-Pointed Dissecting Scissors</td><td>Fisher Scientific</td><td>8940</td><td>May use any brand</td></tr><tr><td>Dissecting Tissue Forceps</td><td>Fisher Scientific</td><td>13-812-41</td><td>May use any brand</td></tr></tbody></table>

handling of the cotton rat in studies for the pre-clinical evaluation of oncolytic viruses

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact. For this strategy to be successful, recent studies have shown that involvement of the host immune system is essential. Therefore, oncolytic virotherapy should be evaluated within the context of an immunocompetent model. Furthermore, the study of antitumor therapies in tolerized animal models may better recapitulate results seen in clinical trials. Cotton rats, commonly used to study respiratory viruses, are an attractive model to study oncolytic virotherapy as syngeneic models of mammary carcinoma and osteosarcoma are well established. However, there is a lack of published information on the proper handling procedure for these highly excitable rodents. The handling and capture approach outlined minimizes animal stress to facilitate experimentation. This technique hinges upon the ability of the researcher to keep calm during handling and perform procedures in a timely fashion. Finally, we describe how to prepare cotton rat mammary tumor cells for consistent subcutaneous tumor formation, and how to perform intratumoral and intraperitoneal injections. These methods can be applied to a wide range of studies furthering the development of the cotton rat as a relevant pre-clinical model to study antitumor therapy.

由于棉鼠的极其激发的性质，是熟悉并利用优化，以减少动物的应力缓和在其作为临床前动物模型中使用的程序。采用适当的处理技术也将最大限度地减少风险，研究员。 当使用棉鼠当务之急是保持冷静。老鼠是非常激动，将试图逃跑的笼子。使用富集管和nestlets将最大限度地减少逃逸尝试。 图2示出了最佳的笼设置在棉鼠，包括富集管安置捕获提供帮助。此外?...

Watch this Scientific Journal Video about Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses at JoVE.com

Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses

Cotton rats are extremely excitable and have a strong flight-or-fight response. A handling method optimized to reduce the stress of the animals is described which will make cotton rats more accessible as a preclinical model.

处理棉鼠的研究为溶瘤病毒的临床前评估

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact.  For this strategy to be ...

handling-cotton-rat-studies-for-pre-clinical-evaluation-oncolytic

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

9.3K 次观看.  McMaster University. Cotton rats are extremely excitable and have a strong flight-or-fight response. A handling method optimized to reduce the stress of the animals is described which will make cotton rats more accessible as a preclinical model. 

视频: Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses

Genome-wide RNAi Screening to Identify Host Factors That Modulate Oncolytic Virus Therapy

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus replication. Here we present the application of this technology to uncovering host targets that specifically modulate the replication of Maraba virus, an oncolytic rhabdovirus, and vaccinia virus with the goal of enhancing therapy. While the protocol has been tested for use with oncolytic Maraba virus and oncolytic vaccinia virus, this approach is applicable to other oncolytic viruses and can also be utilized for identifying host targets that modulate virus replication in mammalian cells in general. This protocol describes the development and validation of an assay for high-throughput RNAi screening in mammalian cells, the key considerations and preparation steps important for conducting a primary high-throughput RNAi screen, and a step-by-step guide for conducting a primary high-throughput RNAi screen; in addition, it broadly outlines the methods for conducting secondary screen validation and tertiary validation studies. The benefit of high-throughput RNAi screening is that it allows one to catalogue, in an extensive and unbiased fashion, host factors that modulate any aspect of virus replication for which one can develop an in vitro assay such as infectivity, burst size, and cytotoxicity. It has the power to uncover biotherapeutic targets unforeseen based on current knowledge.

Here we describe a protocol for employing high-throughput RNAi screening to uncover host targets that can be manipulated to enhance oncolytic virus therapy, specifically rhabodvirus and vaccinia virus therapy, but it can be readily adapted to other oncolytic virus platforms or for discovering host genes that modulate virus replication generally.

全基因组的 rna 干扰筛查以确定调节溶瘤病毒治疗的宿主因素

High-throughput genome-wide RNAi (RNA interference) screening technology has been widely used for discovering host factors that impact&#160;virus ...

科研

癌症研究

A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while animals are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different forms of learning. Specifically, viral-mediated delivery is used to express a genetically modified inhibitory G-protein coupled receptor (the Designer Receptor) into a discrete brain region in the rodent. Three weeks later, when designer receptor expression levels are high, a pharmacological agent (the Designer Drug) is administered systemically 30 min prior to a specific behavioral session. The drug has affinity for the designer receptor and thus results in inhibition of neurons that express the designer receptor, but is otherwise biologically inert. The brain region remains silenced for 2-5 hr (depending on the dose and route of administration). Upon completion of the behavioral paradigm, brain tissue is assessed for correct placement and receptor expression. This approach is particularly useful for determining the contribution of individual brain regions to specific components of behavior and can be used across any number of behavioral paradigms.

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while rats are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different components of learning.

为远程沉默的神经活动在鼠害的方法在学习独立的阶段

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while animals are engaged in learning and ...

行为学

Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis. In recent years, oncolytic virotherapies have proven themselves to be potentially powerful tools to fight malignancy. Due to the unique dual blood supply in the liver, it is possible to apply therapies locally to orthotopic liver tumors, which are predominantly fed by arterial blood flow. We have previously demonstrated that hepatic arterial delivery of oncolytic viruses results in safe and efficient transduction efficiency of multifocal HCC lesions, resulting in significant prolongation of survival in immune competent rats. This procedure closely mimics the application of transarterial embolization in patients, which is the standard palliative care provided to many HCC patients. The ability to administer tumor therapies through the hepatic artery in rats allows for a highly sophisticated preclinical model for evaluating novel viral vectors under development. Here we describe the detailed protocol for microdissection of the hepatic artery for infusion of oncolytic virus vectors to treat orthotopic HCC.

Oncolytic virotherapies are under development as novel therapeutics for the treatment of hepatocellular carcinoma (HCC). Here we describe a method for locoregional therapy of HCC via hepatic arterial administration of oncolytic virus.

溶瘤病毒的Transarterial管理局大鼠原位肝癌的局部治疗

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis.  In recent years, oncolytic virotherapies have proven ...

医学

Generation of Transgenic Rats using a Lentiviral Vector Approach

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is an invaluable tool for gene function analysis in living organisms. The standard transgenesis method is based on microinjecting foreign DNA fragments into a pronucleus of a fertilized oocyte. This technique is widely used in mice but remains relatively inefficient and technically demanding in other animal species. The transgene can also be introduced into one-cell-stage embryos via lentiviral infection, providing an effective alternative to standard pronuclear injections, especially in species or strains with a more challenging embryo structure. In this approach, a suspension that contains lentiviral vectors is injected into the perivitelline space of a fertilized rat embryo, which is technically less demanding and has a higher success rate. Lentiviral vectors were shown to efficiently incorporate the transgene into the genome to determine the generation of stable transgenic lines. Despite some limitations (e.g., Biosafety Level 2 requirements, DNA fragment size limits), lentiviral transgenesis is a rapid and efficient transgenesis method. Additionally, using female rats that are mated with a fertile male strain with a different dominant fur color is presented as an alternative to generate pseudopregnant foster mothers.

This article aims to provide the methodology for lentiviral transgenesis in rat embryos using multiple injections of a virus suspension into the zygote perivitelline space. Female rats that are mated with a fertile male strain with a different dominant fur color is used to generate pseudopregnant foster mothers.

使用伦迪病毒载体方法生成转基因大鼠

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is ...

生物工程

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.

Methods for a wide-scale central nervous system gene delivery in the rat are covered. In this example, the purpose is to mimic a disease that affects the entire spinal cord. The widespread transduction can be used to deliver a therapeutic protein to the CNS from a one-time, peripheral administration.

腺相关病毒对大鼠的静脉管理方法和技巧及中枢神经系统转导的评价

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), ...

神经科学

A Rat Model of EcoHIV Brain Infection

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. Establishment of the EcoHIV infected rat model for studies of drug abuse and neurocognitive disorders, would be beneficial in the study of neuroHIV and HIV-1 associated neurocognitive disorders (HAND). In the present study, we demonstrate the successful creation of a rat model of active HIV infection using chimeric HIV (EcoHIV). First, the lentiviral construct of EcoHIV was packaged in cultured 293 FT cells for 48 hours. Then, the conditional medium was concentrated and titered. Next, we performed bilateral stereotaxic injections of the EcoHIV-EGFP into F344/N rat brain tissue. One week after infection, EGFP fluorescence signals were detected in the infected brain tissue, indicating that EcoHIV successfully induces an active HIV infection in rats. In addition, immunostaining for the microglial cell marker, Iba1, was performed. The results indicated that microglia were the predominant cell type harboring EcoHIV. Furthermore, EcoHIV rats exhibited alterations in temporal processing, a potential underlying neurobehavioral mechanism of HAND as well as synaptic dysfunction eight weeks after infection. Collectively, the present study extends the EcoHIV model of HIV-1 infection to the rat offering a valuable biological system to study HIV-1 viral reservoirs in the brain as well as HAND and associated comorbidities such as drug abuse.

Here, we present a protocol to establish a new rat model of active HIV infection using chimeric HIV (EcoHIV), which is critical for enhancing our understanding of HIV-1 viral reservoirs in the brain and offering a system to study HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse).

生态HIV脑感染的老鼠模型

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

监测内质网钙稳态使用<em&gt; Gaussia</em&gt;荧光素酶SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

生物学

Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk

Heart disease is the leading cause of morbidity and mortality worldwide. Due to the ease of handling&#160;and abundance of transgenic strains, rodents have become essential models for cardiovascular research. However, spontaneous lethal cardiac arrhythmias that often cause mortality in heart disease patients are rare in rodent models of heart disease. This is primarily due to the species differences in cardiac electrical properties between human&#160;and rodents and poses a challenge to the study of cardiac arrhythmias using rodents. This protocol describes an approach to enable efficient transgene expression in mouse and rat ventricular myocardium using echocardiography-guided intramuscular injections of recombinant virus (adenovirus and adeno-associated virus). This work also outlines a method to enable reliable assessment of cardiac susceptibility to arrhythmias using isolated, Langendorff-perfused mouse and rat hearts with both adrenergic and programmed electrical stimulations. These techniques are critical for studying heart rhythm disorders associated with adverse cardiac remodeling after injuries, such as myocardial infarction.

The present protocol describes methods for transgene expression in rat and mouse hearts by direct intramyocardial injection of the virus under echocardiography guidance. Methods for the assessment of the susceptibility of hearts to ventricular arrhythmias by the programmed electrical stimulation of isolated, Langendorff-perfused hearts are also explained here.

啮齿动物心脏中的病毒转基因表达和心律失常风险的评估

Heart disease is the leading cause of morbidity and mortality worldwide. Due to the ease of handling&#160;and abundance of transgenic strains, rodents ...

Establishing an Active HIV Infection in a Rat Model

Source: Li, H., McLaurin, et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/62137">A Rat Model of EcoHIV Brain Infection.</a> J. Vis. Exp. (2021).
This video demonstrates the procedure of injecting chimeric HIV viruses into the cortex of a rat&#39;s brain, where the virus RNA integrates into the host genome, replicates, and spreads to establish an active HIV infection in the experimental model.

在大鼠模型中建立活动性 HIV 感染

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuropathology

Infectious Diseases

Targeting Liver Tumors with Oncolytic Viruses via the Hepatic Artery

Source: Altomonte, J. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/53757">Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats.</a>&#160;J. Vis. Exp. (2016)
The video demonstrates locoregional delivery of oncolytic viruses through hepatic arterial injection as a therapeutic approach for hepatocellular carcinoma. These viruses selectively replicate in tumor cells, inducing cell lysis and releasing tumor cell-associated antigens. This process triggers immunogenic cell death, inducing an anti-tumor response.

处理棉鼠的研究为溶瘤病毒的临床前评估

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