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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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 animal maintenance are informed of the specific requirements for housing. Taking these precautions will reduce agitation of the animal prior to performing procedures. It is also advisable that procedures be performed in the morning, before any handling by technicians or animal facility staff. Commencing procedures with calm animals will aid in their capture and handling.
The use of nestlets is highly recommended. In the wild, these rodents make their nests out of cotton so they are more comfortable when given the option to build them in their cages. A nest can also aid in animal capture, such that the presence of the animal in the enrichment tube may no longer be required. In this case, the plastic container may be placed directly on top of the animal from above. This action is of particular use as an experiment progresses as the rats may adapt to avoid the traditional capture method.
When handling cotton rats it is of the utmost importance that the researcher remain calm and make deliberate movements. These animals can sense unease and will respond to the perceived emotional state of the handler. Wearing thick leather gloves, following the procedures as described and approaching them calmly and with confidence will protect from bite injuries.
There are conflicting reports in the literature regarding the response of cotton rats to anesthetics 26,32. Sedation of cotton rats for extended periods of time using isoflurane is not recommended 32. We have found that, with careful monitoring, animals can be sedated using isoflurane for approximately 15-20 minutes without adverse effects. In our experience the rats respond rapidly to isoflurane and are fully sedated within several minutes. Likewise, waking occurs in less than a minute in most cases. However, anesthetizing an animal under intense stress alters their response to isoflurane, such that during waking they experience a lack of hind limb motor control and will stumble around the cage. This effect does not appear to have any lasting impact as the animal can execute proper motor control within several minutes of recovery. 
When preparing the LCRT cells for tumor injections, it is imperative that the cells be kept on ice whenever possible to prolong viability. Mix the cells with a large bore pipet to avoid shearing the cells. Furthermore, to ensure that all tumors grow at a similar rate we recommend staggering the preparation of multiple cell suspensions when more than five animals are being injected. This will avoid slower growth of tumors in animals injected last due to decreased viability of the LCRT cells. 
Excising the tumor tissue from the skin using a scalpel better maintains the tumor architecture and vasculature for histology in comparison to using tweezers to pinch the tumor from the skin. Although perfusion with phenobarbital will best preserve tissues for histopathology, obtaining a license for barbiturates was a limiting factor and was not performed. Thus, necropsies were performed immediately following euthanasia to preserve tissues. In our experience, this allowed for an accurate assessment of the pathology associated with our treatment. 
Overall, the major limiting factor is the requirement for two researchers for handling procedures. Although the procedures can be performed by one handler, the process is greatly facilitated, and the stress of the animals significantly reduced, using two handlers. These procedures require practice and careful planning. Allow sufficient time to carry out methods calmly and deliberately. There exists significant stigma about the aggressive nature of these animals and the difficulty in their handling. This article serves to make these animals more accessible to researchers by detailing a simple, established handling method that effectively minimizes stress and injury to both the researcher and the animals. Using the methods we have described, cotton rats represent an excellent animal model for the study of many immune therapies, including oncolytic virotherapy.

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，工作在一个小房间如果可能的话在夺回来帮助。如果发生逃逸，等待动物冷静下来，保持静止，然后覆盖它与明确的捕获容器或盖上戴手套的手，小心不要过度使用武力。 在对比小鼠，棉鼠具有细长吻瓦特HICH需要不同的鼻子嵌合递送麻醉气体。 图3示出了鼻锥工程化以适当地适合棉鼠和最大化递送异氟烷。使用橡胶膜作为嵌合可能导致创伤的大鼠的鼻子。 如果可能的话获得丢弃的动物练习之前，尝试他们在大鼠注射技术（这些并不需要其他研究人员，棉鼠或其他）。这将允许研究者获得熟悉针头和如何安全地处理它们。胰岛素注射器建议在棉鼠注射作为自己的皮肤厚韧相比于鼠标。然而，一个较大的针（21g的值x 1&#39;），可用于在注射肿瘤细胞，以避免对细胞活力的损失是由于在注射过程中细胞剪切。安全注意事项应遵循，如不重新盖上针头和妥善处置进入锐器容器。 该injec存活的肿瘤细胞的灰是正确的肿瘤的形成很重要的。 图1A示出了可以用于注射制备成棉鼠LCRT细胞的健康单层。在比较图1B示出LCRT细胞具有低的可行性，而不应被用于注射。它使用了一种用于注射细胞计数时，以验证肿瘤细胞活力，如台盼蓝染色法是重要的。 从LCRT细胞所形成的肿瘤快速生长和坏死中心常形成（ 图4A）。正因为如此，肿瘤的形成应仔细监测以避免溃疡（ 图4B）。如果发生溃疡的动物应该被牺牲掉，以避免感染，甚至死亡的败血症。 通常最好通过组织学分析研究的抗肿瘤治疗的效果。这需要肿瘤验尸切除。维持肿瘤组织的完整性将水库ULT的样品是在体内的肿瘤的更准确的表示中， 图5示出了切除术通过该肿瘤被小心地从使用手术刀和镊子周围组织分开。通过使用镊子可能破裂的肿瘤或破坏肿瘤组织的完整性，影响正确的组织学分析从周围组织拉强行去除肿瘤。肿瘤的致密且高度血管结构，如在图6中看到的那样，通过该切除术保持。这是在对各种影响肿瘤脉管系统的治疗分析重要的，因为与很多OVS的情况。 <img alt="图1" src="/files/ftp_upload/52232/52232fig1highres.jpg" /> 图1:健康LCRT细胞明视场显微镜图像（A）表型（〜90％存活）LCRT细胞准备准备注入棉鼠。LCRT细胞（B）不良型不适合注入。圆形的细胞是死的或濒死（由箭头表示）。图像被抓获放大10倍;比例尺= 1毫米。 <img alt="图2" src="/files/ftp_upload/52232/52232fig2highres.jpg" /> 图2:笼安装，便于棉鼠捕获的例子对笼子和包容nestlets有助于动物捕获年底富集管的优化配置。 <img alt="图3" src="/files/ftp_upload/52232/52232fig3highres.jpg" /> 图3:麻醉鼻锥递送异氟烷来麻醉棉鼠嵌合制造鼻锥体适合的棉鼠细长吻，以确保准确递送异氟烷气体的无创伤的鼻子。 <i毫克ALT ="图4"SRC ="/文件/ ftp_upload / 52232 / 52232fig4highres.jpg"/&gt; 图4:在一个皮下LCRT肿瘤坏死组织（A）的肿瘤组织的坏死的早期阶段。动物应仔细监测，以避免发展为（B）全开溃疡。应该被牺牲的动物，如果肿瘤溃烂感染和败血症会导致。 <img alt="图5" src="/files/ftp_upload/52232/52232fig5highres.jpg" /> 图5:皮下LCRT肿瘤为组织学上切除一棉鼠的侧翼的皮下肿瘤从皮肤用手术刀以维持肿瘤组织的完整性小心除去，从而提供了用于组织学分析的一个更好的代表性肿瘤架构。 <img alt="图6" src="/files/ftp_upload/52232/52232fig6highres.jpg"/&gt; 图6:从皮下LCRT肿瘤病理组织切片用苏木和伊红（H＆E）石蜡切片检查LCRT肿瘤组织的形态。图像捕捉，放大20倍;比例尺= 1毫米。

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

Nanyang Technological University

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

Humanized NOG Mice for Intravaginal HIV Exposure and Treatment of HIV Infection

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes the establishment of a human immune system in adult NOG mice. Here, we explain all the practical steps from isolation of umbilical cord blood derived human CD34+ cells and their subsequent intravenous transplantation into the mice, to the manipulation of the model through HIV infection, combination antiretroviral therapy (cART), and blood sampling. Approximately 75,000 hCD34+ cells are injected intravenously into the mice and the level of human chimerism, also known as humanization, in the peripheral blood is estimated longitudinally for months by flow cytometry. A total of 75,000 hCD34+ cells yields 20%&#8211;50% human CD45+ cells in the peripheral blood. The mice are susceptible to intravaginal infection with HIV and blood can be sampled once weekly for analysis, and twice monthly for extended periods. This protocol describes an assay for quantification of plasma viral load using droplet digital PCR (ddPCR). We show how the mice can be effectively treated with a standard-of-care cART regimen in the diet. The delivery of cART in the form of regular mouse chow is a significant refinement of the experimental model. This model can be used for preclinical analysis of both systemic and topical pre-exposure prophylaxis compounds as well as for testing of novel treatments and HIV cure strategies.

We have developed a protocol for the generation and evaluation of a humanized and human immunodeficiency virus-infected NOG mouse model based on stem cell transplant, intravaginal human immunodeficiency virus exposure, and droplet digital PCR RNA quantification.

用于阴道内艾滋病毒暴露和艾滋病毒感染的人性化 NOG 小鼠

Humanized mice provide a sophisticated platform to study human immunodeficiency virus (HIV) virology and to test antiviral drugs. This protocol describes ...

免疫与感染

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. ...

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 ...

生物学

Establishing an Active HIV Infection in a Rat Model

Source: Li, H., McLaurin, et al. <a href="https://app.jove.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://app.jove.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.