The present study was supported by the NIH grants HL120947 (P.C.), HL103868 (P.C.), and the American Heart Association Grant-in-Aid (P.C.)

注:总论:执行涉及操纵在无菌条件下的蛋的所有程序，和无菌技术应相应地使用。预清洁所有的设备用70％乙醇后再使用。在一般情况下，流感病毒接种和收获应在BSL-2实验室中进行。但是，如果该协议被用于传播更多的致病性流感病毒（ 例如 ，流感大流行和大流行前的菌株，即危及家禽和牲畜，人感染高致病性禽流感毒株，1918年的H1N1病毒株），更高的生物安全水平，在某些情况下，生物安全防范措施和授权是必需的。当地生物安全委员会应批准涉及流感病毒的所有研究工作。 1.准备鸡蛋的接种<ol><li>从合适的供应商处获得的血清的无病原体（SPF）的新鲜受精禽卵。通常情况下，鸡卵被使用。鸡蛋应该是我们ED抵达后立即。 </li><li>抵达后的地方鸡蛋一个鸡蛋加湿孵化器与自动鸡蛋特纳经常转动鸡蛋。如果自动鸡蛋特纳不可用，手动旋转鸡蛋一天（一定要戴上手套并保留一切无菌地）至少2-3次。 </li><li>孵化卵在37℃和55-60％湿度为10-11天。 </li></ol> 2.鸡蛋烛<ol><li>用一个鸡蛋烛台检查对着明亮光线的鸡蛋在一个黑暗的房间通过后约7或8天的潜伏期烛检查鸡蛋不育。 <ol><li>擦蛋烛台，用70％的乙醇。经常更换手套和用70％乙醇消毒，以减少污染的病原体的风险。 </li><li>从孵化器中取出卵子，并将它们放在空的鸡蛋盒。 </li><li>关灯的房间。 </li><li>保持每个鸡蛋一体的大型月底在对烛台时间。 </li><li>观察蛋determinË如果是肥沃还是贫瘠。 注:细血管导致豆形胚应清晰可见。未受精的卵子会出现一个小血点有明显的蛋黄。 </li></ol></li><li>丢弃卵是受精，并返回可行蛋的孵化器。不要让鸡蛋孵化器外超过30分钟。如果一个鸡蛋的状态不能得到证实，将其标记，后来观察它。 </li></ol> 3.编写病毒的接种<ol><li>稀释的流感病毒原液至约10 3 -在无菌PBS中含有10mM HEPES（pH 7.4）中10 4 PFU / ml的。关键的一步:在使用前稀释病毒的股票，并保持病毒稀释冰上所有的时间。 </li></ol>通过尿囊路线4.流感病毒接种<ol><li>在病毒接种（10天或11）的一天，再次蜡烛鸡蛋和消除任何死胚。区分活胚胎通过找蛋内运动。 </li><li>从培养箱中取出三个鸡蛋，并安排在鸡蛋中与空气囊一个单独的支架。 </li><li>纪念鸡蛋用永久性记号笔的领空。 </li><li>洗蛋壳，用70％的乙醇的空气空间的上方。 </li><li>把鸡蛋与空气的空间在一个鸡蛋纸箱和成生物安全罩（BSL-2）。 </li><li>使用无菌18G针头打一个小孔，在外壳上每个鸡蛋的空气囊。要注意不要插入针过深，以免刺伤胚胎或蛋黄。 </li><li>拟定稀释流感病毒进入无菌1ml注射器，并附加一个22G针头。 </li><li>认真推进针注射器以45°角插入尿囊腔。 </li><li>注入0.2毫升稀释的流感病毒。 </li><li>重复注射与其他两个鸡蛋，然后丢弃的注射器和针头。 </li><li>密封带从胶枪（或熔融石蜡）一滴粘结剂的孔。 BË小心，胶水不漏蛋内。 </li><li>将鸡蛋放回蛋孵化器与空气空间指向向上。 </li><li>重复步骤4.2 - 4.12，直到所有的鸡蛋都已经接种。 </li></ol> 5.孵化时间<ol><li>孵育感染鸡蛋无需转动，在37℃和〜60％的湿度。 </li><li>选择最佳孵育时间取决于流感病毒的类型。孵化甲型流感48小时;孵育流感B和C为72小时。 </li></ol> 6.流感病毒收获<ol><li>经过潜伏期，寒意鸡蛋在4℃最少4小时（或O / N）杀死胚胎和收缩血管，减少污染的血液被感染的尿囊液的风险。这样做是为了防止红血细胞从吸收的病毒，从而降低了产率。可替代地，寒意鸡蛋30分钟在-20℃;然而，这会增加血液污染的风险。 </li><li>之后的鸡蛋被充分冷却，卵转移到生物安全罩。将蛋与气囊朝上牢固夹持。清洁蛋表面用70％的乙醇。 </li><li>用无菌剪刀打开上方的空气囊的蛋壳。周围的空气囊去掉外壳，注意不要破坏绒毛尿囊膜。 </li><li>打开绒毛尿囊膜，用无菌钝钳。轻轻移动胚胎和卵黄囊用小刮刀或汤匙放在一边。小心不要破裂的蛋黄。 </li><li>用1毫升的Eppendorf移液器，仔细收集尿囊液。如果需要的话，倾斜鸡蛋有点一边收集尽可能多的流体越好。 </li><li>组合来自所有鸡蛋尿囊液到50ml的塑料离心管中。保持病毒收获期间冰在任何时候都管。一个鸡蛋产量5 - 10毫升一个淡黄色的液体。 </li><li>旋转含有病毒的尿囊液在1000×g离心，在4 10分钟6℃到沉淀杂物。转移透明流体到一个新的50ml管中保持在冰上。配置鸡蛋放入一个BSL-2的废弃物容器。 </li></ol> 7.储存<ol><li>立即等分澄清尿囊液（ 例如，50，100或500微升等分试样）和单元冷冻在液氮中。传输冻病毒储存至-80℃冰箱长期保存。 </li></ol>

<ol>
	<li>Centers for Disease Control and Prevention (CDC). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimates+of+deaths+associated+with+seasonal+influenza+---+United+States,+1976-2007.">Estimates of deaths associated with seasonal influenza --- United States, 1976-2007.</a> MMWR. Morbidity and mortality weekly report. 59 (33), 1057-1062 (2010).</li><li>Neumann, G., Noda, T., Kawaoka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+and+pandemic+potential+of+swine-origin+H1N1+influenza+virus.">Emergence and pandemic potential of swine-origin H1N1 influenza virus.</a> Nature. 459 (7249), 931-939 (2009).</li><li>Tumpey, T. M., Garcia-Sastre, A., 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=Pathogenicity+of+Influenza+Viruses+with+Genes+from+the+1918+Pandemic+Virus:+Functional+Roles+of+Alveolar+Macrophages+and+Neutrophils+in+Limiting+Virus+Replication+and+Mortality+in+Mice.">Pathogenicity of Influenza Viruses with Genes from the 1918 Pandemic Virus: Functional Roles of Alveolar Macrophages and Neutrophils in Limiting Virus Replication and Mortality in Mice.</a> J Virol. 79 (23), 14933-14944 (2005).</li><li>Doherty, P. C., Turner, S. J., Webby, R. G., Thomas, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+and+the+challenge+for+immunology.">Influenza and the challenge for immunology.</a> Nat Immu. 7 (5), 449-455 (2006).</li><li>Webster, R. G., Govorkova, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuing+challenges+in+influenza.">Continuing challenges in influenza.</a> Ann N Y Acad Sci. 1323 (1), 115-139 (2014).</li><li>Szretter, K. J., Balish, A. L., Katz, 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=Unit+15G.1+Influenza:+propagation,+quantification,+and+storage.+.">Unit 15G.1 Influenza: propagation, quantification, and storage. .</a> Current protocols in microbiology. , (2006).</li><li>Short, K. R., Kroeze, E. J. B. V., Fouchier, R. A. M., Kuiken, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+influenza-induced+acute+respiratory+distress+syndrome.">Pathogenesis of influenza-induced acute respiratory distress syndrome.</a> Lancet Infect Dis. 14 (4), 57-69 (2014).</li><li>Ito, T., Suzuki, Y., 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=Differences+in+sialic+acid-galactose+linkages+in+the+chicken+egg+amnion+and+allantois+influence+human+influenza+virus+receptor+specificity+and+variant+selection.">Differences in sialic acid-galactose linkages in the chicken egg amnion and allantois influence human influenza virus receptor specificity and variant selection.</a> J Virol. 71 (4), 3357-3362 (1997).</li><li>Robertson, J. S., Nicolson, C., Major, D., Robertson, E. W., Wood, 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=The+role+of+amniotic+passage+in+the+egg-adaptation+of+human+influenza+virus+is+revealed+by+haemagglutinin+sequence+analyses.">The role of amniotic passage in the egg-adaptation of human influenza virus is revealed by haemagglutinin sequence analyses.</a> J Gen Virol. 74 (Pt 10), 2047-2051 (1993).</li><li>Gaush, C. R., Smith, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+and+plaque+assay+of+influenza+virus+in+an+established+line+of+canine+kidney+cells).">Replication and plaque assay of influenza virus in an established line of canine kidney cells).</a> Appl Microbiol. 16 (4), 588-594 (1968).</li></ol>

The authors have nothing to disclose.

流感导致疾病的一个大的全球负担，工作持续到了解肺损伤7的发病机制。以促进研究在这种致命的疾病，各种方法已经被开发来传播的流感病毒6。这里，我们描述了一种技术，以产生流感病毒在鸡卵。这种方法的优点是，它是高度可重复的，并导致大量的高滴度流感病毒的股票，这往往是必需的体外研究。 大多数实验室将能够快速，轻松地开发该协?...

甲型流感仍然是一个重大威胁人类健康。这是一个潜在的破坏性呼吸系统疾病有大型的全球性负担造成了全球每年1 500,000人死亡。流感病毒是在家庭正粘病毒和携带8负义单链RNA在它们的基因组1,2。病毒基因组的高可变性（ 即抗原"漂移"）防止长期免疫力。此外，流感是抗病毒的药物3日益抗性。 2009年甲型H1N1流感大流行强调所有流感病（流感病毒，抗病毒性，推迟疫苗的生产）相关的具有挑战性的问题。该疫苗配方是每年由世界卫生组织为最有可能致病的流感病毒株（分别为H1N1，H3N2和乙型流感）4确定。因为这种方法是基于预测流感病毒株S，致病菌株偶尔错误地标识和疫苗的有效性急剧下降。此外，新型流感病毒株的出现可以绕开造成流行病2这些预防计划。 一个通用流感疫苗的创作一直难以捉摸5。因此，研究必须继续为了解肺损伤的发病机制。到设施的实验室研究，几种方法已经被开发用于病毒分离和传播6。人流感病毒可以在多种哺乳动物细胞基质进行放大。然而，在大批量生成高滴度病毒最好在胚卵实现。以下协议描述的技术为A型流感病毒的传播和存储，从现有的病毒股票。

<table><tbody><tr><td>Phosphate buffered saline (PBS)</td><td>Cellgro&amp;nbsp;</td><td>21-040-CV</td><td></td></tr><tr><td>serum pathogen-free (SPF) fertilized chicken eggs&amp;nbsp;</td><td>VALO BioMedia</td><td></td><td></td></tr><tr><td>humidified egg incubator&amp;nbsp;</td><td>FARM iNNOVATORS</td><td>Model 2100</td><td></td></tr><tr><td>automatic egg turner</td><td>FARM iNNOVATORS</td><td>Model 3200</td><td></td></tr><tr><td>egg candler</td><td>FARM iNNOVATORS</td><td>Model 3300&amp;nbsp;</td><td></td></tr><tr><td>1 ml syringe</td><td>BD Bioscience</td><td>309659</td><td></td></tr><tr><td>18G needle</td><td>BD Bioscience</td><td>305196</td><td></td></tr><tr><td>20G / 22G needle</td><td>BD Bioscience</td><td>305176 / 305156</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Ethanol&gt;99.5%</td><td>Sigma-Aldrich</td><td>459844</td><td>diluted to 70% using water</td></tr><tr><td>glue gun &quot;Ad tech Hi Temp Project Pro&quot;</td><td>Ad tech, Adhesive Technologies, Inc.</td><td>Model 1105</td><td></td></tr><tr><td>glue &quot;Ad tech MINI SIZE, Multi Temp&quot;</td><td>Ad tech, Adhesive Technologies, Inc.</td><td>220-34ZIP30</td><td></td></tr><tr><td>MDCK cells</td><td>ATCC</td><td>CRL-2935</td><td></td></tr><tr><td>Washed Pooled Turkey Red Blood Cells, 10%</td><td>Lampire Biological Laboratories</td><td>724908</td><td></td></tr></tbody></table>

influenza virus propagation in embryonated chicken eggs

流感与约36000人死亡，每年有超过20万的住院在美国的关联。新的流感病毒株引起的突变，并重新分类连续出现复杂的病毒控制，并有必要的新颖的药物和疫苗的永久发展。流感的实验室为基础的研究中，需要对病毒的传播有可靠和具有成本效益的方法。在这里，一个全面的协议是为A型流感病毒的传播在肥沃的鸡蛋，它始终产生高滴度的病毒股票。简言之，血清的无病原体（SPF）的受精鸡蛋孵育在37℃和55-60％湿度下10 - 11天。在此期间，胚胎发育，可以很容易地用一个鸡蛋烛台监测。病毒接种是通过注射病毒原液的成使用的针的尿囊腔。后在37℃下孵育2天，将蛋冷冻至少4小时，在4℃。上方的空气囊和尿囊膜蛋壳然后小心地打开，并含有病毒的尿囊液收获。流体从碎屑清除通过离心，等分并转移至-80℃长期储存。大量（5-10毫升的病毒含有每蛋流体）和高病毒效价通常与该协议实现作出了鸡蛋的病毒制备我们的有利方法的使用，特别是用于在体外研究需要大量病毒在其中需要同样的病毒股票的高剂量。

多种方法已经发展到滴定流感病毒。当选择适当的方法之一的考虑是，某些确定总颗粒无论活力（ 例如 ，血凝素测定法），而其他人都基于感染性的，这将评估存活的病毒颗粒（ 例如 ，空斑测定）6。此处，尿囊液的病毒滴度从接种小鼠-适应的流感病毒（A / PR / 8/34）鸡卵收集空斑测定（ 图1）和血凝试验（ 图2）进行测定。 流...

Watch this Scientific Journal Video about Influenza Virus Propagation in Embryonated Chicken Eggs at JoVE.com

Influenza Virus Propagation in Embryonated Chicken Eggs

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

在鸡胚的流感病毒传播

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

influenza-virus-propagation-in-embryonated-chicken-eggs

Research

JoVE Journal

Immunology and Infection

42.9K 次观看.  Cedars-Sinai Medical Center. Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

视频: Influenza Virus Propagation in Embryonated Chicken Eggs

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

质粒DNA重组流感病毒的一代

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

科研

免疫与感染

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Rescue of Recombinant Newcastle Disease Virus from cDNA

Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA2-5. Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs.

Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.

从救援的cDNA重组新城疫病毒

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

Production of High-Titer Recombinant Newcastle Disease Virus from Allantoic Fluid

Newcastle disease virus (NDV), also known as avian orthoavulavirus serotype-1, is a negative sense, single-stranded RNA virus that has been developed both as an oncolytic virus and a viral-vectored vaccine. NDV is an attractive therapeutic and prophylactic agent due to its well-established reverse genetics system, potent immunostimulatory properties, and excellent safety profile. When administered as an oncolytic virus or a viral-vectored vaccine, NDV elicits a robust antitumor or antigen-specific immune response, activating both the innate and adaptive arms of the immune system. 
Given these desirable characteristics, NDV has been evaluated in numerous clinical trials and is one of the most well-studied oncolytic viruses. Currently, there are two registered clinical trials involving NDV: one evaluating a recombinant NDV-vectored vaccine for SARS-CoV-2 (NCT04871737), and a second evaluating a recombinant NDV encoding Interleukin-12 in combination with Durvalumab, an antiPD-L1 antibody (NCT04613492). To facilitate further advancement of this highly promising viral vector, simplified methods for generating high-titer, in vivo-grade, recombinant NDV (rNDV) are needed. 
This paper describes a detailed procedure for amplifying rNDV in specified pathogen-free (SPF) embryonated chicken eggs and purifying rNDV from allantoic fluid, with improvements to reduce loss during purification. Also included are descriptions of the recommended quality control assays, which should be performed to confirm lack of contaminants and virus integrity. Overall, this detailed procedure enables the synthesis, purification, and storage of high-titer, in vivo-grade, recombinant, lentogenic, and mesogenic NDV for use in preclinical studies.

Here we provide a detailed procedure for production, purification, and quantification of high-titer recombinant Newcastle disease virus. This protocol consistently yields &#62; 6 &#215; 109 plaque-forming units/mL, providing virus quantities appropriate for in vivo animal studies. Additional quality control assays to ensure safety in vivo are described.

从尿囊液中生产高滴度重组新城疫病毒

Newcastle disease virus (NDV), also known as avian orthoavulavirus serotype-1, is a negative sense, single-stranded RNA virus that has been developed both ...

癌症研究

Using Chicken Embryo as a Powerful Tool in Assessment of Developmental Cardiotoxicities

Chicken embryos are a classical model in developmental studies. During the development of chicken embryos, the time window of heart development is well-defined, and it is relatively easy to achieve precise and timely exposure via multiple methods. Moreover, the process of heart development in chicken embryos is similar to mammals, also resulting in a four-chambered heart, making it a valuable alternative model in the assessment of developmental cardiotoxicities. In our lab, the chicken embryo model is routinely used in the assessment of developmental cardiotoxicities following exposure to various environmental pollutants, including per- and polyfluoroalkyl substances (PFAS), particulate matter (PMs), diesel exhaust (DE) and nano materials. The exposure time can be freely selected based on the need, from the beginning of development (embryonic day 0, ED0) all the way to the day prior to hatch. The major exposure methods include air-cell injection, direct microinjection, and air-cell inhalation (originally developed in our lab), and the currently available endpoints include cardiac function (electrocardiography), morphology (histological assessments) and molecular biological assessments (immunohistochemistry, qRT-PCR, western blotting, etc.). Of course, the chicken embryo model has its own limitations, such as limited availability of antibodies. Nevertheless, with more laboratories starting to utilize this model, it can be used to make significant contributions to the study of developmental cardiotoxicities.

Chicken embryos, as a classical developmental model, are used in our lab to assess developmental cardiotoxicities following exposure to various environmental contaminants. Exposure methods and morphological/functional assessment methods established are described in this manuscript.

使用鸡胚胎作为评估发育性心脏病的有力工具

Chicken embryos are a classical model in developmental studies. During the development of chicken embryos, the time window of heart development is ...

发育生物学

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

单细胞转染鸡胚

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

生物学

In Ovo Intravascular Injection in Chicken Embryos

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering exogenous materials into chicken embryos has a great advantage for studying gene function, transgenic breeding, and chimera preparation during embryonic development. Here we show the method of in ovo intravascular injection whereby exogenous materials such as plasmid vectors or modified primordial germ cells (PGCs) can be transferred into donor chicken embryos at early developmental stages. The results show that the intravascular injection through the dorsal aorta and head allows injected materials to diffuse into the whole embryo through the blood circulatory system. In the presented protocol, the efficacy of exogenous plasmid and lentiviral vector introduction, and the colonization of injected exogenous PGCs in the recipient gonad, were determined by observing fluorescence in the embryos. This article describes detailed procedures of this method, thereby providing an excellent approach to studying gene function, embryo and developmental biology, and gonad-chimeric chicken production. In conclusion, this article will allow researchers to perform in ovo intravascular injection of exogenous materials into chicken embryos with great success and reproducibility.

In Ovo Intravascular Injection in Chicken Embryos

The overall goal of this paper is to describe how to perform in ovo intracellular injection of exogenous materials into chicken embryos. This approach is very useful to study the developmental biology of chicken embryos.

在 奥沃 鸡胚血管内注射

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

使用人类流感病毒感染的斑马鱼模型到屏幕抗病毒药物和表征宿主免疫细胞反应

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious public health issues and suggest the possibility of an epidemic. However, our molecular understanding of the virus is rudimentary, and it is necessary to study the biological properties of its envelope proteins as therapeutic targets and to develop strategies to control infection. We developed a solid viral pseudotyped particle (pp) platform to study avian influenza virus, including the functional analysis of its hemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins, the reassortment characteristics of the HAs and NAs, receptors, tropisms, neutralizing antibodies, diagnosis, infectivity, for the purposes of drug development and vaccine design. Here, we describe an experimental procedure to establish pps with the envelope glycoproteins (HA, NA) from two influenza A strains (HAPI H5N1 and 2013 avian H7N9). Their generation is based on the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins into a pp. In addition, we also detail how these pps are quantified with RT-qPCR, and the infectivity detection of native and mismatched virus pps depending on the origin of the HAs and NAs. This system is highly flexible and adaptable and can be used to establish viral pps with envelope glycoproteins that can be incorporated in any other type of enveloped virus. Thus, this viral particle platform can be used to study wild viruses in many research investigations.

This protocol describes an experimental process to produce high-titer infectious viral pseudotyped particles (pp) with envelope glycoproteins from two influenza A strains and how to determine their infectivity. This protocol is highly adaptable to develop pps of any other type of enveloped viruses with different envelope glycoproteins.

从高致病性H5N1病毒和禽流感病毒中生产含有包络糖蛋白的高蒂特感染性流感伪型颗粒

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious ...

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

Transfection is a technical process through which genetic material, such as DNA and double-stranded RNA, are delivered into cells to modify the gene of interest. Currently, transgenic technology is becoming an indispensable tool for the study of Eimeria, the causative agents of coccidiosis in poultry and livestock. This protocol provides a detailed description of stable transfection in eimerian parasites: purification and nucleofection of sporozoites or second-generation merozoites, and in vivo propagation of transfected parasites. Using this protocol, we achieved transfection in several species of Eimeria. Taken together, nucleofection is a useful tool to facilitate genetic manipulation in eimerian parasites.

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

Here, we provided a method to achieve stable transfection of chicken Eimeria parasites by nucleofecting sporozoites or second-generation merozoites. Genetically modified eimerian parasites expressing heterologous antigenic genes could be used as vaccine delivery vehicles.

鸡寄生虫的成核和活体繁殖

Transfection is a technical process through which genetic material, such as DNA and double-stranded RNA, are delivered into cells to modify the gene of ...

在鸡胚的流感病毒传播

摘要

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此视频中的章节

质粒DNA重组流感病毒的一代

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

从救援的cDNA重组新城疫病毒

从尿囊液中生产高滴度重组新城疫病毒

使用鸡胚胎作为评估发育性心脏病的有力工具

单细胞转染鸡胚

在奥沃鸡胚血管内注射

使用人类流感病毒感染的斑马鱼模型到屏幕抗病毒药物和表征宿主免疫细胞反应

从高致病性H5N1病毒和禽流感病毒中生产含有包络糖蛋白的高蒂特感染性流感伪型颗粒

鸡寄生虫的成核和活体繁殖

在鸡胚的流感病毒传播

摘要

探索更多视频

此视频中的章节

质粒DNA重组流感病毒的一代

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

从救援的cDNA重组新城疫病毒

从尿囊液中生产高滴度重组新城疫病毒

使用鸡胚胎作为评估发育性心脏病的有力工具

单细胞转染鸡胚

在 奥沃 鸡胚血管内注射

使用人类流感病毒感染的斑马鱼模型到屏幕抗病毒药物和表征宿主免疫细胞反应

从高致病性H5N1病毒和禽流感病毒中生产含有包络糖蛋白的高蒂特感染性流感伪型颗粒

鸡寄生虫的成核和活体繁殖

在奥沃鸡胚血管内注射