Name: detection of enterohemorrhagic escherichia coli colonization in murine host by non-invasive in vivo bioluminescence system
Uploaded: 2018-04-09T12:30:00.000+00:00
Duration: 6 min 20 s
Description: Watch this Scientific Journal Video about Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System at JoVE.com

我们从医学研究部、美梅医疗中心 (台湾台南) 等地认识到, 帮助小鼠感染, 以及国立诚武大学实验动物中心的支持。这项工作得到了科技部长 (多数) 赠款 (多数 104-2321-006-019、105-2321-b-006-011、and106-2321-B-006-005) 的支持。

注意: 根据疾病控制和预防中心 (CDC) 生物安全指导 (https://www.cdc.gov/), 大肠杆菌 O157:H7 是生物安全等级 2 (BSL-2) 病原体。因此, 所有涉及肠出血性大肠杆菌的实验程序都必须在 BSL-2 设施中进行。在进行实验时, 要穿上实验室的大衣和手套。在认证的生物安全柜 (BSC) 工作。对实验台进行70% 乙醇消毒实验。所有接触 (或潜在接触) 肠出血性大肠杆菌的仪器或设备应使用70% 乙醇或漂白剂进行消毒。污染 (或可能污染的) 废物应小心密封和蒸压。如有必要, 戴上口罩、护眼、双手套或连身裤。6周大的 C57BL/6 雌性小鼠在国立成武大学实验动物中心 (NCKU) 购买和维护。动物实验由 NCKU 的机构动物护理和使用委员会批准 (批准号 104-039)。
1. Bioluciferase 标记的大肠杆菌生成
<ol><li>混合50的脱氧核糖核酸 (脱氧核糖核酸), 1 ul 每10微米向前和反向底漆, 2 ul 2.5 毫米 deoxynucleotides (dNTPs), 2 ul 10x 缓冲器, 0.2 ul DNA 聚合酶, 和无菌的双蒸馏水 (ddH2O) 到最后一卷 20 ul, 以放大抗卡那霉素卡带, nptII基因24。DNA 模板来自 pBSL18024, 它是从国家 BioResource 项目 (NBRP) 购买的。PCR 条件列在表 1中, 并且底漆序列在表 2中可用。</li>
	<li>将 PCR 产品结扎在套件协议之后的商业克隆载体上 (请参见材料表)。</li>
	<li>将nptII基因片段从克隆向量中的NsiI和SmaI和克隆复制到 pBBR1MCS4 中, 由NsiI和ScaI从 pAKlux29中消化, 以创建卡那霉素抗性质粒,pWF27813。</li>
	<li>将luxCDABE操纵从表达质粒9、pAKlux2、 SpeIhf和ScaI的荧光素酶和克隆到SpeI-hf和SmaI中消化 pWF278 生成卡那霉素抗性和荧光素酶表达质粒, pWF27913。 注: 对于质粒提取物和切除片段纯化, 分别采用商业质粒萃取和凝胶萃取试剂盒。为酵素消化, 混合 100 ng 脱氧核糖核酸, 1 ul 10x 缓冲器, 1 ul 每选择的制约酵素和无菌 ddH2O 到总容量 10 ul, 并且孵化在37°c 为 2.5-3 h。</li>
	<li>将 pWF279 质粒转化为大肠杆菌 O157:H7 EDL933 主管细胞, 用2500伏电穿孔4毫秒。</li>
	<li>将转化后的细菌细胞在1毫升 Luria Bertani (LB) 培养基中孵育1小时, 37 摄氏度。</li>
	<li>板上的细菌在 LB 琼脂板块补充50µg/毫升卡那霉素在37摄氏度, 16-18 小时。</li>
	<li>第二天, 通过体内成像机检查板的发光信号。选择一个单一的殖民地从板块和文化它在3毫升 LB 补充50µg/毫升卡那霉素在37°c 16-18 h。</li>
	<li>在无菌 ddH2O 中稀释100% 甘油, 制备细菌培养基, 使30% 甘油溶液。</li>
	<li>冷冻卡那霉素耐药性的大肠杆菌O157:H7 EDL933 细菌, 窝藏荧光素酶质粒作为一个细菌库存的螺帽 cryovial 作为1:1 的细菌培养率和30% 甘油溶液-80 摄氏度。甘油的最后浓度是15%。</li>
</ol>2. 生物发光大肠杆菌制备口服疫苗
注: 在图 1中介绍了大肠杆菌制备和小鼠口服饲实验程序的时间流程图, 以帮助实验性的准备工作。
<ol><li>条纹大肠杆菌O157:H7 EDL933 细菌将荧光素酶质粒放到 LB 琼脂板上, 其50µg/毫升卡那霉素从-80 摄氏度的库存。在37摄氏度培养 16-18 小时的细菌。</li>
	<li>选择一个单一的殖民地从隔夜板块和文化在3毫升 LB 培养基与50µg/毫升卡那霉素 16-18 小时在37摄氏度孵化器在 220 rpm。</li>
	<li>亚文化细菌 (1:100 稀释) 到卡那霉素 (50 µg/毫升) 含有 LB 汤 2.5-3 小时在37摄氏度孵化器 200 rpm。(例如, 添加2毫升的夜间培养细菌到200毫升 LB 培养基与卡那霉素 (50 µg/毫升) 补充)。</li>
	<li>孵育 2.5-3 小时的细菌, 并测量 600 nm (OD600) 的光学密度值, 直到该值介于0.9 到1之间。细菌细胞数的密度约为 108菌落形成单位 (CFU)/毫升。</li>
	<li>离心机重新培养的细菌在 8000 x g 30 分钟, 4 摄氏度。</li>
	<li>在不搅动细菌颗粒的情况下去除上清液, 用100毫升0.9% 无菌生理盐水用温和搅拌法清洗颗粒。</li>
	<li>重复步骤2.5 和2.6。</li>
	<li>清洗后, 离心机的细菌培养在 8000 x g 30 分钟, 4 摄氏度, 并轻轻地丢弃上清。</li>
	<li>将颗粒凝结成100倍, 0.9% 无菌生理盐水。例如, 如果200毫升细菌是离心, 添加2毫升正常生理盐水悬浮颗粒。</li>
	<li>确认细菌 CFU (它应该是大约 109 CFU/100 ul 在正常盐水冷凝后) 通过10倍的系列稀释25电镀浓缩细菌。</li>
</ol>3. 小鼠口服饲肠出血性大肠杆菌
<ol><li>用链霉素水 (5 克/升) 治疗6周大的雌性 C57BL/6 小鼠24小时。</li>
	<li>24小时后, 在饲前, 改用普通饮水24小时。用常规水治疗24小时后, 小鼠可以口服饲肠内大肠杆菌。</li>
	<li>通过拉回柱塞, 在注射器中填充 100 ul 肠出血性大肠杆菌。确保注射器内没有气泡。用手指对着注射器取出气泡。</li>
	<li>轻轻举起动物, 小心地放在笼子的顶端。</li>
	<li>小心地抓住老鼠的尾巴, 动物会抓住笼子的顶部, 试图离开。</li>
	<li>用拇指和食指抓住动物颈部和背部松弛的皮肤, 以防止老鼠头部移动, 轻轻地抑制老鼠。</li>
	<li>按住鼠标垂直位置插入饲针, 确保鼠标头固定和垂直。</li>
	<li>将饲针插入嘴后的小鼠嘴里, 向下移动到食道和胃部。</li>
	<li>当插入的针是在鼠标的一半或2/3 长度, 注射 100 ul 大肠杆菌, 其中包含约 109 CFU 细胞。</li>
</ol>4. 可视化
<ol><li>口服饲后, 检测1和2天的发光信号。</li>
	<li>在研究动物的生物发光信号之前, 麻醉将它们放入2.5% 异氟醚的腔内, 1.5 升/分钟氧。</li>
	<li>等待 2-5 分钟, 直到所有老鼠失去知觉, 停止移动。动物已经准备好体内检测生物发光。</li>
	<li>通过体内成像系统检测和成像动物的生物发光信号。请参阅5节 "数据采集" 软件操作。在成像过程中, 所有动物都在持续供应2.5% 异氟醚, 1.5 升/分氧。<ol>
<li>单击 "文件" > "实时", 然后手动将焦点放在鼠标上。</li>
		<li>选择曝光时间和激光强度。</li>
		<li>单击 "获取 > 捕获"。</li>
	</ol>
</li>
	<li>对于体外成像, 弄死小鼠颈椎脱位, 并清除感染小鼠的整个肠道. 用体内成像系统将肠道置于9厘米培养皿和图像中。该设置与 "体内" 映像相同, 但视图字段设置为-b。有关详细信息, 请参阅5节 "数据获取"。 注意: 颈椎脱位的方法: 用一只手抓住尾巴, 把笼子顶部的老鼠控制住, 这样动物就能抓住笼子。将标记笔或拇指和第一手指放在头骨底部的颈部后部。用手或物体快速向前推进, 同时抑制头部, 用手握住尾巴向后拉。 仔细检查以确认呼吸骤停, 没有心跳。</li>
</ol>5. 数据采集
注意: 用于数据采集的软件列在材料表中。
<ol><li>要获取图像, 请打开软件的购置控制面板 (图 2)。</li>
	<li>选择 "发光"、"照片" 和 "叠加"。</li>
	<li>将曝光时间设置为 "自动"。将 Binning 设置为 "介质"。</li>
	<li>设置ƒ/停止为1的发光和8的照片。ƒ/停止控制 CCD 探测器接收的光量。</li>
	<li>根据感兴趣的图像字段设置视图字段。选项 "D" 可以容纳五6周大的老鼠, 并在同一时间图像。"C" 可以在一个领域中想象三6周大的老鼠。</li>
	<li>一旦鼠标/样品准备好成像, 点击 "获取" 图像获取。</li>
	<li>打开已获取的图像数据。</li>
	<li>打开 "工具面板" 面板 (图 3)。</li>
	<li>选择 ROI 工具。我们建议圆圈 (最左边的一个) 来范围在图像上的发光区域 (图 4)。</li>
	<li>单击 "测量 ROIs" (铅笔图标) 以测量表面发光强度 (图 3)。"ROI 度量" 面板和量化值显示 (图 5)。</li>
	<li>使用 "ROI 度量" 面板左上角的 "配置测量" 来选择所需的值/信息 (图 6), 否则单击 "导出" 以导出此数据表并另存为. csv 文件 (图 5)。</li>
	<li>使用列 "总通量 (p/秒)" 的值作为. csv 文件中的发光强度量化。</li>
</ol>

<ol>
	<li>Pennington, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+O157.">Escherichia coli O157.</a> Lancet. 376 (9750), 1428-1435 (2010).</li><li>Mayer, C. L., Leibowitz, C. S., Kurosawa, S., Stearns-Kurosawa, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shiga+toxins+and+the+pathophysiology+of+hemolytic+uremic+syndrome+in+humans+and+animals.">Shiga toxins and the pathophysiology of hemolytic uremic syndrome in humans and animals.</a> Toxins (Basel). 4 (11), 1261-1287 (2012).</li><li>Tarr, P. I., Gordon, C. A., Chandler, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shiga-toxin-producing+Escherichia+coli+and+haemolytic+uraemic+syndrome.">Shiga-toxin-producing Escherichia coli and haemolytic uraemic syndrome.</a> Lancet. 365 (9464), 1073-1086 (2005).</li><li>Obrig, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Escherichia+coli+Shiga+Toxin+Mechanisms+of+Action+in+Renal+Disease.">Escherichia coli Shiga Toxin Mechanisms of Action in Renal Disease.</a> Toxins (Basel). 2 (12), 2769-2794 (2010).</li><li>Nguyen, Y., Sperandio, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enterohemorrhagic+E.+coli+(EHEC)+pathogenesis.">Enterohemorrhagic E. coli (EHEC) pathogenesis.</a> Front Cell Infect Microbiol. 2, 90 (2012).</li><li>Wiles, S., Robertson, B. D., Frankel, G., Kerton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+monitoring+of+in+vivo+colonization+and+clearance+dynamics+by+light-emitting+bacteria.">Bioluminescent monitoring of in vivo colonization and clearance dynamics by light-emitting bacteria.</a> Methods Mol Biol. 574, 137-153 (2009).</li><li>Hutchens, M., Luker, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+bioluminescence+imaging+to+the+study+of+infectious+diseases.">Applications of bioluminescence imaging to the study of infectious diseases.</a> Cell Microbiol. 9 (10), 2315-2322 (2007).</li><li>Rhee, K. 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=Determination+of+spatial+and+temporal+colonization+of+enteropathogenic+E.+coli+and+enterohemorrhagic+E.+coli+in+mice+using+bioluminescent+in+vivo+imaging.">Determination of spatial and temporal colonization of enteropathogenic E. coli and enterohemorrhagic E. coli in mice using bioluminescent in vivo imaging.</a> Gut Microbes. 2 (1), 34-41 (2011).</li><li>Karsi, A., Lawrence, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broad+host+range+fluorescence+and+bioluminescence+expression+vectors+for+Gram-negative+bacteria.">Broad host range fluorescence and bioluminescence expression vectors for Gram-negative bacteria.</a> Plasmid. 57 (3), 286-295 (2007).</li><li>Lane, M. C., Alteri, C. J. S., Smith, S. N., Mobley, L. H. <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+flagella+is+coincident+with+uropathogenic+Escherichia+coli+ascension+to+the+upper+urinary+tract.">Expression of flagella is coincident with uropathogenic Escherichia coli ascension to the upper urinary tract.</a> Proc Natl Acad Sci U S A. 104 (42), 16669-16674 (2007).</li><li>Roxas, J. 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=Enterohemorrhagic+E.+coli+alters+murine+intestinal+epithelial+tight+junction+protein+expression+and+barrier+function+in+a+Shiga+toxin+independent+manner.">Enterohemorrhagic E. coli alters murine intestinal epithelial tight junction protein expression and barrier function in a Shiga toxin independent manner.</a> Lab Invest. 90 (8), 1152-1168 (2010).</li><li>Siragusa, G. R., Nawotka, K., Spilman, S. D., Contag, P. R., Contag, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Real-Time Monitoring of Escherichia coli O157:H7 Adherence to Beef Carcass Surface Tissues with a Bioluminescent Reporter. , (1999).</li><li>Kuo, C. 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=Mutation+of+the+Enterohemorrhagic+Escherichia+coli+Core+LPS+Biosynthesis+Enzyme+RfaD+Confers+Hypersusceptibility+to+Host+Intestinal+Innate+Immunity+In+vivo.">Mutation of the Enterohemorrhagic Escherichia coli Core LPS Biosynthesis Enzyme RfaD Confers Hypersusceptibility to Host Intestinal Innate Immunity In vivo.</a> Front Cell Infect Microbiol. 6, 82 (2016).</li><li>Wiles, 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=Organ+specificity,+colonization+and+clearance+dynamics+in+vivo+following+oral+challenges+with+the+murine+pathogen+Citrobacter+rodentium.">Organ specificity, colonization and clearance dynamics in vivo following oral challenges with the murine pathogen Citrobacter rodentium.</a> Cell Microbiol. 6 (10), 963-972 (2004).</li><li>Wiles, S., Pickard, K. M., Peng, K., MacDonald, T. T., Frankel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+bioluminescence+imaging+of+the+murine+pathogen+Citrobacter+rodentium.">In vivo bioluminescence imaging of the murine pathogen Citrobacter rodentium.</a> Infect Immun. 74 (9), 5391-5396 (2006).</li><li>Contag, C. H., Contag, P. R., Mullins, J. I., Spillman, S. D., Stevenson, D. K., Benaron, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photonic+detection+of+bacterial+pathogens+in+living+hosts.">Photonic detection of bacterial pathogens in living hosts.</a> Mol Microbiol. 18 (4), 593-603 (1995).</li><li>Hardy, J., Francis, K. P., DeBoer, M., Chu, P., Gibbs, K., Contag, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+replication+of+Listeria+monocytogenes+in+the+murine+gall+bladder.">Extracellular replication of Listeria monocytogenes in the murine gall bladder.</a> Science. 303 (5659), 851-853 (2004).</li><li>Kaniga, K., Sory, M. P., Delor, I., Saegerman, C., Limet, J. N., Cornelis, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+Yersinia+enterocolitica+in+Murine+and+Bovine+Feces+on+the+Basis+of+the+Chromosomally+Integrated+luxAB+Marker+Gene.">Monitoring of Yersinia enterocolitica in Murine and Bovine Feces on the Basis of the Chromosomally Integrated luxAB Marker Gene.</a> Appl Environ Microbiol. 58 (3), 1024-1026 (1992).</li><li>Trcek, J., Fuchs, T. M., Trulzsch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Yersinia+enterocolitica+invasin+expression+in+vitro+and+in+vivo+using+a+novel+luxCDABE+reporter+system.">Analysis of Yersinia enterocolitica invasin expression in vitro and in vivo using a novel luxCDABE reporter system.</a> Microbiology. 156 (Pt 9), 2734-2745 (2010).</li><li>Morin, C. E., Kaper, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+stabilized+luciferase-expressing+plasmids+to+examine+in+vivo-induced+promoters+in+the+Vibrio+cholerae+vaccine+strain+CVD+103-HgR.">Use of stabilized luciferase-expressing plasmids to examine in vivo-induced promoters in the Vibrio cholerae vaccine strain CVD 103-HgR.</a> FEMS Immunol Med Microbiol. 57 (1), 69-79 (2009).</li><li>Law, R. J., Gur-Arie, L., Rosenshine, I., Finlay, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+in+vivo+model+systems+for+studying+enteropathogenic+Escherichia+coli+infections.">In vitro and in vivo model systems for studying enteropathogenic Escherichia coli infections.</a> Cold Spring Harb Perspect Med. 3 (3), a009977 (2013).</li><li>Ritchie, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+Models+of+Enterohemorrhagic+Escherichia+coli+Infection.">Animal Models of Enterohemorrhagic Escherichia coli Infection.</a> Microbiol Spectr. 2 (4), EHEC-0022-2013 (2014).</li><li>Chou, T. 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=Enterohaemorrhagic+Escherichia+coli+O157:H7+Shiga-like+toxin+1+is+required+for+full+pathogenicity+and+activation+of+the+p38+mitogen-activated+protein+kinase+pathway+in+Caenorhabditis+elegans.">Enterohaemorrhagic Escherichia coli O157:H7 Shiga-like toxin 1 is required for full pathogenicity and activation of the p38 mitogen-activated protein kinase pathway in Caenorhabditis elegans.</a> Cell Microbiol. 15 (1), 82-97 (2013).</li><li>Alexeyev, M. F., Shokolenko, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini-Tnl+0+transposon+derivatives+for+insertion+mutagenesis+and+gene+delivery+into+the+chromosome+of+Gram-negative+bacteria.">Mini-Tnl 0 transposon derivatives for insertion mutagenesis and gene delivery into the chromosome of Gram-negative bacteria.</a> Gene. 160 (1), 59-62 (1995).</li><li>Wiegand, I., Hilpert, K., Hancock, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agar+and+broth+dilution+methods+to+determine+the+minimal+inhibitory+concentration+(MIC)+of+antimicrobial+substances.">Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances.</a> Nat Protoc. 3 (2), 163-175 (2008).</li><li>Pansare, V., Hejazi, S., Faenza, W., Prud'homme, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+Long-Wavelength+Optical+and+NIR+Imaging+Materials:+Contrast+Agents,+Fluorophores+and+Multifunctional+Nano+Carriers.">Review of Long-Wavelength Optical and NIR Imaging Materials: Contrast Agents, Fluorophores and Multifunctional Nano Carriers.</a> Chem Mater. 24 (5), 812-827 (2012).</li><li>Heim, R., Cubitt, A. B., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+green+fluorescence.">Improved green fluorescence.</a> Nature. 373 (6516), 663-664 (1995).</li><li>Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clearer+vision+for+in+vivo+imaging.">A clearer vision for in vivo imaging.</a> Nat Biotechnol. 19 (4), 316-317 (2001).</li><li>Frangioni, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+near-infrared+fluorescence+imaging.">In vivo near-infrared fluorescence imaging.</a> Current Opinion in Chemical Biology. 7 (5), 626-634 (2003).</li><li>Collins, J. W., 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=Citrobacter+rodentium:+infection,+inflammation+and+the+microbiota.">Citrobacter rodentium: infection, inflammation and the microbiota.</a> Nat Rev Microbiol. 12 (9), 612-623 (2014).</li><li>Mallick, E. M., 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+novel+murine+infection+model+for+Shiga+toxin-producing+Escherichia+coli.">A novel murine infection model for Shiga toxin-producing Escherichia coli.</a> J Clin Invest. 122 (11), 4012-4024 (2012).</li><li>Petty, N. 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=The+Citrobacter+rodentium+genome+sequence+reveals+convergent+evolution+with+human+pathogenic+Escherichia+coli.">The Citrobacter rodentium genome sequence reveals convergent evolution with human pathogenic Escherichia coli.</a> J Bacteriol. 192 (2), 525-538 (2010).</li><li>Kovach, M. E., Elzer, P. H., Hill, D. S., Robertson , G. T., Farris, M. A., Roop, R. M., Peterson, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four+new+derivatives+of+the+broad-host-range+cloning+vector+pBBR1MCS,+carrying+different+antibiotic-resistance+cassettes.">Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes.</a> Gene. 166 (1), 175-176 (1995).</li><li>Galen, J. E., Nair, J., Wang , J. Y., Wasserman, S. S., Tanner, M. K., Sztein , M. B., Levine, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+Plasmid+Maintenance+in+the+Attenuated+Live+Vector+Vaccine+Strain+Salmonella+typhiCVD+908-htrA.">Optimization of Plasmid Maintenance in the Attenuated Live Vector Vaccine Strain Salmonella typhiCVD 908-htrA.</a> Infect Immun. 67 (12), 6424-6433 (1999).</li><li>Francis, K. P., 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=Visualizing+pneumococcal+infections+in+the+lungs+of+live+mice+using+bioluminescent+Streptococcus+pneumoniae+transformed+with+a+novel+gram-positive+lux+transposon.">Visualizing pneumococcal infections in the lungs of live mice using bioluminescent Streptococcus pneumoniae transformed with a novel gram-positive lux transposon.</a> Infect Immun. 69 (5), 3350-3358 (2001).</li><li>Goldwater, P. N., Bettelheim, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+enterohemorrhagic+Escherichia+coli+(EHEC)+infection+and+hemolytic+uremic+syndrome+(HUS).">Treatment of enterohemorrhagic Escherichia coli (EHEC) infection and hemolytic uremic syndrome (HUS).</a> BMC Med. 10, (2012).</li></ol>

作者没有什么可透露的。

据报道, 用荧光素酶质粒转化出的肠出血性大肠杆菌已被用于检查其在宿主或基因表达式中的定位在体内8,11,12。在这里展示的小鼠模型也被报道来检测在小鼠宿主8中的肠大肠杆菌的殖民时间和定位。然而, 我们提供了如何管理肠内大肠杆菌疫苗接种小鼠灌的详细协议, 以及如何精心准备口腔饲的生物发?...

大肠杆菌 O157:H7 是一种病原体, 引起腹泻1, HS2, 溶血症3, 甚至急性肾功能衰竭4通过污染的水或食物。肠出血性大肠杆菌是一个致病性 enterobacterium 和殖民的胃肠道的人1。当出血性大肠杆菌第一次坚持宿主肠道上皮, 他们通过 III. 型分泌系统 (T3SS) 将定植因子注入宿主细胞, 该类型作为分子注射器诱导附加和谦逊 (a/E) 病变随后执行附着力 (殖民化)5。enterocyte 避嫌 (李) 致病性岛5的轨迹编码了这些涉及 A/E 病变形成的基因。
生物发光是一种光产生化学反应, 荧光素酶催化其基底荧光素生成可见光 6.这种酶法通常要求存在氧或三磷酸腺苷 (ATP)6。生物发光成像 (BLI) 允许研究人员的可视化和量化的寄主病原体相互作用的活动物7。BLI 可以通过以下生物发光细菌来表征活体动物的细菌感染周期, 它们迁移到并侵入不同的组织7;这揭示了感染的动态发展。此外, 动物的细菌负荷与生物发光信号8有关;因此, 简单直接地估计实验动物的病理状况是一种方便的指标。
这里使用的质粒包含荧光素酶操纵, luxCDABE, 这是从细菌Photorhabdus luminescens编码其自己的荧光素酶基板7,9。通过将这种荧光素酶表达质粒转化为细菌, 可以通过观察活动物中的这些生物发光细菌来监测其定植和感染过程。总体而言, BLI 和生物发光标记的细菌允许研究人员监测细菌数量和位置, 细菌的生存能力与抗生素/治疗治疗, 和细菌基因表达感染/殖民化6,7. 报告了许多致病细菌, 表达了luxCDABE操纵检查其感染周期和/或感染中的基因表达。这些细菌, 包括致肾盂肾炎大肠杆菌 10, 出血性大肠杆菌8, 11, 12, 13,致病大肠杆菌 (EPEC)8 , 柠檬酸杆菌rodentium14,15, 沙门氏菌伤寒16, 李斯特氏李斯特氏增生17, 耶尔森氏菌小肠结肠炎 18, 19,和霍乱弧菌20已被记录在案。
已开发了几个实验模型, 以促进研究肠大肠杆菌的殖民化在体外和体内21,22,23。然而, 缺乏适当的动物模型来研究肠内大肠杆菌的定植在体内, 从而导致缺乏细节。为促进对肠内大肠杆菌定植机制的研究,在体内, 建立动物模型来观察和量化活体动物的肠外培养是一种无创的方法是很有价值的。
这篇手稿描述了一个老鼠大肠杆菌的殖民模型, 使用一个生物发光表达系统来监测大肠杆菌在活寄主时间的殖民化。小鼠灌接种生物发光标记的大肠杆菌, 并在小鼠身上检测到无侵入性的体内成像系统13中的发光信号。感染了生物发光标记的大肠杆菌病的小鼠在2天后的肠道感染后显示出明显的生物发光信号, 这表明这些细菌在感染后2天后在宿主肠道内被殖民。前体图像数据表明, 这种殖民化在小鼠的盲肠和结肠中特别存在。利用该模型, 通过体内成像系统, 在活体宿主中检测出生物发光大肠杆菌的定植, 研究肠道细菌定植的详细机制, 从而促进对大肠杆菌诱发的生理和病理改变。

<table><tbody><tr><td>Shaker incubator</td><td>YIH DER</td><td>LM-570R</td><td>bacteria incubation&amp;nbsp;</td></tr><tr><td>Orbital shaking incubator</td><td>FIRSTEK</td><td>S300</td><td>bacteria incubation&amp;nbsp;</td></tr><tr><td>pBSL180</td><td></td><td></td><td>source of nptII gene</td></tr><tr><td>pAKlux2</td><td></td><td></td><td>source of luxCDABE operon</td></tr><tr><td>T&amp;amp;A Cloning Kit</td><td>Yeastern Biotech</td><td>FYC001-20P</td><td>use for TA cloning&amp;nbsp;</td></tr><tr><td>&lt;em&gt;Nsi&lt;/em&gt; I</td><td>NEB</td><td>R0127S</td><td>use for plasmid cloning&amp;nbsp;</td></tr><tr><td>&lt;em&gt;Sca&lt;/em&gt; I</td><td>NEB</td><td>R0122S</td><td>use for plasmid cloning&amp;nbsp;</td></tr><tr><td>&lt;em&gt;Spe&lt;/em&gt; I-HF</td><td>NEB</td><td>R0133S</td><td>use for plasmid cloning&amp;nbsp;</td></tr><tr><td>&lt;em&gt;Sma&lt;/em&gt; I&amp;nbsp;</td><td>NEB</td><td>R0141S</td><td>use for plasmid cloning&amp;nbsp;</td></tr><tr><td>T4 ligase</td><td>NEB</td><td>M0202S</td><td>use for plasmid cloning&amp;nbsp;</td></tr><tr><td>Ex Taq</td><td>TaKaRa</td><td>RR001A</td><td>use for PCR amplification</td></tr><tr><td>10X Ex Taq Buffer</td><td>TaKaRa</td><td>RR001A</td><td>use for PCR amplification</td></tr><tr><td>dNTP Mixture&amp;nbsp;</td><td>TaKaRa</td><td>RR001A</td><td>use for PCR amplification</td></tr><tr><td>PCR machine</td><td>applied Biosystem&amp;nbsp;</td><td>2720 thermal cycler&amp;nbsp;</td><td>&amp;nbsp;for PCR amplification</td></tr><tr><td>Glycerol</td><td>SIGMA</td><td>G5516-1L</td><td>use for bacteria stocking solution</td></tr><tr><td>NaCl</td><td>Sigma</td><td>31434-5KG-R</td><td>chemical for making LB medium, 10 g/L</td></tr><tr><td>Tryptone</td><td>CONDA pronadisa</td><td>Cat 1612.00</td><td>chemical for making LB medium, 10 g/L</td></tr><tr><td>Yeast Extract powder</td><td>Affymetrix</td><td>23547-1 KG</td><td>chemical for making LB medium, 5 g/L</td></tr><tr><td>Agar</td><td>CONDA pronadisa</td><td>Cat 1802.00</td><td>chemical for making LB agar</td></tr><tr><td>kanamycin&amp;nbsp;</td><td>Sigma</td><td>K4000-5G</td><td>antibiotics, use for seleciton</td></tr><tr><td>streptomycin&amp;nbsp;</td><td>Sigma</td><td>S6501-100G</td><td>antibiotics, eliminate the microbiota in mice</td></tr><tr><td>EDL933 competent cell</td><td>Homemade</td><td></td><td>method is on supplemental document&amp;nbsp;</td></tr><tr><td>Electroporator</td><td>MicroPulser</td><td></td><td>for electroporation</td></tr><tr><td>Electroporation Cuvettes</td><td>Gene Pulser/MicroPulser</td><td>1652086</td><td>for electroporation</td></tr><tr><td>High-speed centrifuge</td><td>Beckman Coulter</td><td>Avanti, J-26S XP</td><td>use for centrifuging bacteria&amp;nbsp;</td></tr><tr><td>Fixed-Angle Rotor</td><td>Beckman Coulter</td><td>JA25.5</td><td>use for centrifuging bacteria&amp;nbsp;</td></tr><tr><td>Fixed-Angle Rotor</td><td>Beckman Coulter</td><td>JLA10.5</td><td>use for centrifuging bacteria&amp;nbsp;</td></tr><tr><td>centrifuge bottles</td><td>Beckman Coulter</td><td>REF357003</td><td>use for centrifuging bacteria&amp;nbsp;</td></tr><tr><td>centrifuge bottles</td><td>Thermo Fisher scientific</td><td>3141-0500</td><td>use for centrifuging bacteria&amp;nbsp;</td></tr><tr><td>eppendorf biophotometer plus&amp;nbsp;</td><td>eppendorf</td><td>AG 22331 hamburg</td><td>for measuring the OD600 value of bacteria</td></tr><tr><td>C57BL/6 mice&amp;nbsp;</td><td>Laboratory Animal Center of NCKU</td><td></td><td></td></tr><tr><td>lab coat, gloves</td><td></td><td></td><td>for personnel protection&amp;nbsp;</td></tr><tr><td>isoflurane&amp;nbsp;</td><td>Panion &amp;amp; BF Biotech Inc.</td><td>G-8669</td><td>for mice anesthesia, pharmaceutical grade</td></tr><tr><td>1ml syringe&amp;nbsp;</td><td></td><td></td><td>use for oral gavage of mice</td></tr><tr><td>Reusable 22 G ball-tipped feeding needle</td><td></td><td>&amp;phi;0.9 mm X L 50 mm</td><td>use for oral gavage of mice</td></tr><tr><td>surgical&amp;nbsp; scissors&amp;nbsp;</td><td></td><td></td><td>use for mice experiment</td></tr><tr><td>Xenogen IVIS 200 imaging system</td><td>Perkin Elmer</td><td>IVIS spectrum</td><td>use for bioluminescent image capture&amp;nbsp;</td></tr><tr><td>Living Image Software</td><td>Perkin Elmer</td><td>version 4.1</td><td>use for quantifying the image data</td></tr></tbody></table>

detection of enterohemorrhagic escherichia coli colonization in murine host by non-invasive in vivo bioluminescence system

肠出血性大肠杆菌 (肠出血性大肠杆菌) O157:H7, 是 causesdiarrhea、出血性结肠炎 (HS) 和溶血尿毒症综合征 (溶血尿毒症) 的食源病原体, 可殖民到人类肠道。为了研究肠内大肠杆菌定植的详细机制在体内,必须有动物模型来监测和量化大肠杆菌的定植。通过将生物发光表达质粒转化为肠出血性大肠杆菌来监测和定量活寄主中大肠杆菌的定植, 我们在这里展示了一种小鼠大肠杆菌的定植模型。用生物发光标记的大肠杆菌接种的动物, 通过非侵入性的体内成像系统检测, 显示了小鼠强烈的生物发光信号。感染后1和2天后, 生物发光信号仍可在受感染的动物身上检测到, 这表明在寄主中至少有2天发生了大肠杆菌的殖民。我们还表明, 这些发光的肠外大肠杆菌定位到小鼠肠道, 特别是在盲肠和结肠, 从前体图像。这种鼠肠大肠杆菌定植模型可以作为一种工具, 以提高目前的知识, 肠出血性大肠杆菌的殖民机制。

我们通过口服饲对6周大的雌性 C57BL/6 小鼠进行生物发光标记的肠内毒素 (~ 109细菌细胞) 的管理。在1小时内对小鼠进行口服大肠杆菌疫苗接种后, 通过体内成像系统检查动物的荧光信号, 如图 7所示。结果表明, 饲小鼠具有生物发光标记的大肠杆菌的强烈发光信号。我们检查了2天后感染的信号。如图 8A所示, 接种了?...

Watch this Scientific Journal Video about Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System at JoVE.com

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

提出了一种利用生物发光标记细菌对肠出血性大肠杆菌 (大肠杆菌) 进行殖民化的小鼠模型的详细协议。在活体动物中, 通过非侵入性的体内成像系统来检测这些生物发光细菌, 可以提高我们目前对大肠杆菌定植的认识。

非侵入性 体内生物发光系统检测小鼠宿主肠出血性大肠杆菌的定植

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

detection-enterohemorrhagic-escherichia-coli-colonization-murine-host

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

9.4K 次观看.  National Cheng Kung University. 提出了一种利用生物发光标记细菌对肠出血性大肠杆菌 (大肠杆菌) 进行殖民化的小鼠模型的详细协议。在活体动物中, 通过非侵入性的体内成像系统来检测这些生物发光细菌, 可以提高我们目前对大肠杆菌定植的认识。

视频: Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

用荧光素酶在小鼠细菌性感染的形象

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

科研

免疫与感染

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

在体外和体内生物发光记者人类胚胎干细胞的基因显像

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

生物学

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite this similarity, these bacteria show extreme variations in characteristics like growth behavior, intensity of light emission or regulation of bioluminescence. The method presented here is a newly developed assay that combines recording of cell growth and bioluminescent light emission intensity over time utilizing a plate reader. The resulting growth and light emission characteristics can be linked to important features of the respective bacterial strain, such as quorum sensing regulation. The cultivation of a range of bioluminescent bacteria requires a specific medium (e.g., artificial sea water medium) and defined temperatures. The easy to handle, non-bioluminescent standard-research bacterium Escherichia coli (E. coli), on the other hand, can be cultivated inexpensively in high quantities in laboratory scale. Exploiting E. coli by introducing a plasmid containing the whole lux operon can simplify experimental conditions and additionally opens up many possibilities for future applications. The expression of all lux genes utilizing an E. coli expression strain was achieved by construction of an expression plasmid via Gibson cloning and insertion of four fragments containing seven lux genes and three rib genes of the lux-rib operon into a pET28a vector. E. coli based lux gene expression can be induced and controlled via Isopropyl-&#946;-D-thiogalactopyranosid (IPTG) addition resulting in bioluminescent E. coli cells. The advantages of this system are to avoid quorum sensing regulation restrictions and complex medium compositions along with non-standard growth conditions, such as defined temperatures. This system enables analysis of lux genes and their interplay, by the exclusion of the respective gene from the lux operon, or even addition of novel genes, exchanging the luxAB genes from one bacterial strain by another, or analyzing protein complexes, such as luxCDE.

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

Bioluminescent bacteria regulate light production through a variety of mechanisms, such as quorum sensing. This novel method allows the in situ investigation of bioluminescence and the correlation of light emission to cell density. An artificial bioluminescent Escherichia coli system allows the characterization of lux operons, Lux proteins, and their interplay.

就地生物发光细菌细胞密度与光发射的测量与相关性研究

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite ...

生物化学

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

生物发光细菌成像<em在体内</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

在活体成像系统（IVIS）的神经侵入性脑炎病毒的检测

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

4D多模态成像<em&gt;柠檬酸rodentium</em在小鼠感染

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several animal models have been generated, including the widely used orthotopic mouse models, where cancer cells are injected into the mammary fat pad. However, these models cannot help monitor tumor growth kinetics and metastatic colonization. Cutting-edge tools to monitor cancer cells in real time in mice will significantly advance the understanding of tumor biology. 
Here, breast cancer cell lines stably expressing luciferase and green fluorescent protein (GFP) were established. Specifically, this technique contains two sequential steps initiated by measuring the luciferase activity in vitro and followed by the implantation of the cancer cells into mammary fat pads of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. After the injection, both the tumor growth and metastatic colonization are monitored in real time by the noninvasive bioluminescence imaging system. Then, the quantification of GFP-expressing metastases in the lungs will be examined by fluorescence microscopy to validate the observed bioluminescence results. This sophisticated system combining luciferase and fluorescence-based detection tools evaluates cancer metastasis in vivo, which has great potential for use in breast cancer therapeutics and disease management.

Here, we describe a noninvasive monitoring method involving luciferase and green fluorescent protein expression in various breast cancer cell lines. This protocol provides a technique to monitor tumor formation and metastatic colonization in real time in mice.

使用生物发光监测小鼠乳腺癌生长和转移集落形成

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several ...

癌症研究

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

结合<em&gt;在体内</em&gt;光学及μCT成像监视感染，炎症和骨解剖在骨科植入物感染的小鼠

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

医学

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.

增强型绿色荧光蛋白表达大肠杆菌评价小鼠腹腔巨噬细胞吞噬细胞增多

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a ...

Longitudinal Follow-Up of Urinary Tract Infections and Their Treatment in Mice using Bioluminescence Imaging

Urinary tract infections (UTI) rank among the most common bacterial infections in humans and are routinely treated with empirical antibiotics. However, due to increasing microbial resistance, the efficacy of the most used antibiotics has declined. To find alternative treatment options, there is a great need for a better understanding of the UTI pathogenesis and the mechanisms that determine UTI susceptibility. In order to investigate this in an animal model, a reproducible, non-invasive assay to study the course of UTI is indispensable.
For years, the gold standard for the enumeration of bacterial load has been the determination of Colony Forming Units (CFU) for a particular sample volume. This technique requires post-mortem organ homogenates and serial dilutions, limiting data output and reproducibility. As an alternative, bioluminescence imaging (BLI) is gaining popularity to determine the bacterial load. Labeling pathogens with a lux operon allow for the sensitive detection and quantification in a non-invasive manner, thereby enabling longitudinal follow-up. So far, the adoption of BLI in UTI research remains limited.
This manuscript describes the practical implementation of BLI in a mouse urinary tract infection model. Here, a step-by-step guide for culturing bacteria, intravesical instillation and imaging is provided. The in vivo correlation with CFU is examined and a proof-of-concept is provided by comparing the bacterial load of untreated infected animals with antibiotic-treated animals. Furthermore, the advantages, limitations, and considerations specific to the implementation of BLI in an in vivo UTI model are discussed. The implementation of BLI in the UTI research field will greatly facilitate research on the pathogenesis of UTI and the discovery of new ways to prevent and treat UTI.

This manuscript describes the intravesical administration of uropathogenic bacteria with a lux operon to induce a urinary tract infection in mice and subsequent longitudinal in vivo analysis of the bacterial load using bioluminescence imaging.

使用生物发光成像对小鼠尿路感染及其治疗的纵向随访

Urinary tract infections (UTI) rank among the most common bacterial infections in humans and are routinely treated with empirical antibiotics. However, ...

非侵入性体内生物发光系统检测小鼠宿主肠出血性大肠杆菌的定植

摘要

探索更多视频

此视频中的章节

用荧光素酶在小鼠细菌性感染的形象

在体外和体内生物发光记者人类胚胎干细胞的基因显像

就地生物发光细菌细胞密度与光发射的测量与相关性研究

生物发光细菌成像

在活体成像系统（IVIS）的神经侵入性脑炎病毒的检测

4D多模态成像

使用生物发光监测小鼠乳腺癌生长和转移集落形成

结合

增强型绿色荧光蛋白表达大肠杆菌评价小鼠腹腔巨噬细胞吞噬细胞增多

使用生物发光成像对小鼠尿路感染及其治疗的纵向随访

非侵入性 体内生物发光系统检测小鼠宿主肠出血性大肠杆菌的定植

摘要

探索更多视频

此视频中的章节

用荧光素酶在小鼠细菌性感染的形象

在体外和体内生物发光记者人类胚胎干细胞的基因显像

就地生物发光细菌细胞密度与光发射的测量与相关性研究

生物发光细菌成像

在活体成像系统（IVIS）的神经侵入性脑炎病毒的检测

4D多模态成像

使用生物发光监测小鼠乳腺癌生长和转移集落形成

结合

增强型绿色荧光蛋白表达大肠杆菌评价小鼠腹腔巨噬细胞吞噬细胞增多

使用生物发光成像对小鼠尿路感染及其治疗的纵向随访

非侵入性体内生物发光系统检测小鼠宿主肠出血性大肠杆菌的定植