我们要感谢考德威尔实验室所有成员的合作精神。 March of Dimes的职业生涯从美国国家科学基金会奖，以海关总署从一个罗勒奥康纳学者奖，以及来自霍华德休斯医学研究所的阿拉巴马大学本科生科研的科学项目资助，资助的神经元的兴奋和癫痫研究中的考德威尔实验室。

涕灭威曝光范式<ol><li>在第一天，确保至少30个年轻的成年阶段各基因型和每个复制的蠕虫将于第二天，涕灭威的检测。这是最好的，如果实验者选择了50多个L4阶段到新鲜NGM板蠕虫（最好没有制霉菌素），其中含有大肠杆菌作为食物来源的大肠杆菌 （最好OP50），并成长为12-24小时，他们在一个一致的和宽松的温度（20 ° C至22 ° C是最好的，虽然25℃是好的）。 </li><li>第二天，用70％乙醇（酒精）和30％DDH 2 O的100毫米的涕灭威原液传播到定义的卷NGM减去制霉菌素片适量，涕灭威，达到了预期的涕灭威的浓度。我们坚持用电镀0.5毫米涕灭威100毫米涕灭威的37.5微升到7.5毫升NGM板。允许的涕灭威牌干在室温下大约30-60分钟。这是没有必要打击的盖子。另外，涕灭威，可以添加到NGM，并储存在4 ° C一个星期。 &nbsp;</li><li> E.干燥，板卷一致后大肠杆菌 （最好OP50）到每个涕灭威盘的中心和干燥，在室温30-60分钟。我们一贯板25μL的OP50，创建一个足够规模的食品草坪保持不集中在一个小点的蠕虫人满为患。 &nbsp;</li><li>当食物草坪干燥，人们可以进行涕灭威的检测。由于涕灭威的检测的主观性质，它是强烈建议，实验进行“盲目”。主实验者的同事可以重新标签与被检测的蠕虫的原板。同样，同事可能会转移蠕虫立即从原来的板块，以加密形式涕灭威板之前启动一个计时器。如果实验者预计特定菌株的蠕虫病毒特征的表型，如uncoordination，化验，然后还必须是一个类似的表型，以减少偏差的控制。此外，它是最好的，如果实验者实验平行利于规范实验的一个野生型菌株，以及一个耐药菌株和过敏应变，。实验者应努力去分析每一个复制的蠕虫一致。我们一贯分析30单基因型为每个复制的蠕虫。我们也执行至少有三个，每个实验重复。一个有经验的实验者应能分析时间至少有6株。 &nbsp;</li><li>计数以一致的方式催促每一个铂丝蠕虫瘫痪的蠕虫病毒的数量。我们一贯的头部和产品的两倍，两次共三个小时，每30分钟的尾巴的蠕虫病毒。咽抽水停止，也可能被用来定义瘫痪，但只有当实验者采用瘫痪，所有检测的一致的定义。此外，这是值得注意的，一些蠕虫，尤其是那些耐涕灭威，可能会尝试抓取板。在这种情况下，实验者可以传播一致的棕榈酸，蠕虫运动的一种物理性屏障，周围的涕灭威板。我们传播25μL棕榈酸10毫克/毫升乙醇。 </li></ol>云台曝光范式<ol><li>在第一天，确保至少30个年轻的成年阶段各基因型和每个复制的蠕虫将于第二天云台检测。这是最好的，如果实验者选择了50多个L4阶段到新鲜NGM板蠕虫（最好没有制霉菌素），其中含有大肠杆菌作为食物来源的大肠杆菌 （最好OP50），并成长为12-24小时，他们在一个一致的和宽松的温度（20 ° C至22 ° C是最好的，虽然25℃是好的）。 </li><li>第二天，0.5克云台/ mL的DDH云台2 O的原液。传播到NGM减去制霉菌素板定义的卷云台适量，达到理想的云台浓度。因为我们喜欢为PTZ剂量反应分析，我们到7.5毫升NGM板的板不同云台的金额。例如，一个可能板云台原液的37.5微升到7.5毫升NGM板，使5毫克云台/ mL的溶液。允许云台板，约60-120分钟在室温下干燥。这是没有必要打击的盖子。另外，云台可直接添加到NGM的，但应立即固化后用于检测。云台的稳定性大大高于涕少。因此，年纪较大的云台板的分析是不可靠的。云台干燥器，干燥剂在使用前应存放在-20 ° C。我们宁愿放弃后开放，以确保稳定的PTZ约两个月。 </li><li> E.干燥，板卷一致后大肠杆菌 （最好OP50）到中心的每个云台板和干燥，在室温30-60分钟。我们一贯板25μL的OP50，创建一个足够规模的食品草坪保持不集中在一个小点的蠕虫人满为患。 &nbsp;</li><li>当食物的草坪是干的，可进行云台检测。虽然云台检测不到涕灭威分析主观，仍强烈建议，实验进行“盲目”。主实验者的同事可以重新标签与被检测的蠕虫的原板。同样，同事可能会转移蠕虫立即从原来的板块，以加密形式云台板之前启动一个计时器。如果实验者预计特定菌株的蠕虫病毒特征的表型，如uncoordination，化验，然后还必须是一个类似的表型，以减少偏差的控制。此外，它是最好的，如果实验者检测野生型菌株，以及前或全身抽搐株并行，帮助规范实验，。实验者应努力去分析每一个复制的蠕虫一致。我们一贯分析30单基因型为每个复制的蠕虫。我们也执行至少有三个，每个实验重复。一个有经验的实验者应能分析时间至少3株。然而，云台诱发惊厥高峰期通常在15-30分钟之间，然后的频率和强度减弱蠕虫瘫痪，或在某些情况下，适应环境的药物。 </li><li>计数“癫痫样”抽搐蠕虫每隔30分钟一小时的总人数。这是最好的得分与前抽搐，我们称之为“头乙”的蠕虫病毒的数量，分别从蠕虫全身抽搐，全身瘫痪，我们称其为“补药”，或组合前抽搐BWM瘫痪，我们称之为“强直阵挛性”。大多数蠕虫的一个单一的基因型，表现出只有一种惊厥。然而，强直阵挛性蠕虫往往成为完全补品，所以应该继续过去30分钟的检测，即使所有的蠕虫已经展出抽搐。此外，这是值得注意的，一些蠕虫病毒可能会试图抓取板。在这种情况下，实验者可能蔓延的云台板周围的棕榈酸。我们传播25μL棕榈酸10毫克/毫升乙醇。 </li></ol>涕灭威和云台的选择线虫突触传递的突变体的行为反应<table style="height: 157px;" border="1"><tbody><tr><th>突变体的名称</th><th>突触的作用</th><th>行为无药物</th><th>行为反应的涕灭威（野生型N2相比） </th><th>到云台的行为反应</th></tr><tr><td> TOM - 1（ok188） </td><td>抑制性突触传递</td><td>不协调</td><td>加强瘫痪率</td><td>没有区别，从野生型</td></tr><tr><td> UNC - 43（n498n1186） </td><td>复杂</td><td>不协调</td><td>加强瘫痪率</td><td>全身惊厥</td></tr><tr><td> UNC - 25（e156） </td><td>促进GABA的传输</td><td>不协调</td><td>加强瘫痪率</td><td>前抽搐，全身瘫痪</td></tr><tr><td> SNB - 1（md247） </td><td>促进突触传递</td><td>不协调</td><td>减按瘫痪</td><td>前抽搐</td></tr><tr><td> UNC - 4（E120） </td><td>促进乙酰胆碱的传输</td><td>不协调</td><td>减按瘫痪</td><td>没有区别，从野生型</td></tr></tbody></table>神经兴奋剂，涕灭威，乙酰胆碱酯酶抑制剂，和GABA受体拮抗剂，戊四氮（PTZ），可用于在互补的方式来表征线虫突触传递的突变体。过剩的兴奋性乙酰胆碱（ACH）在乙酰胆碱传输的负调控因子的有害突变累积的蠕虫体壁神经肌肉接头（NMJs）（如TOM - 1和UNC - 43）积极的监管机构或抑制GABA的传输（例如，UNC - 25）。相反，蠕虫NMJs兴奋胆碱水平都在积极的监管机构一般突触传递（例如，SNB - 1）或ACH -特定的传输基因（如UNC - 4）减少有害突变。当相比，野生型N2蠕虫，NMJs升高兴奋胆碱传输的突变蠕虫展览涕灭威，引起的瘫痪率增强，而降低兴奋性乙酰胆碱传输突变蠕虫表明，涕灭威引起的瘫痪率降低。虽然云台破坏线虫体壁肌肉的神经元同步，不像涕灭威，云台也拮抗GABA的抑制线虫头部肌肉。因此，涕灭威的敏感性不能准确地预测云台灵敏度。阴性或阳性regul的具体缺陷的突变蠕虫乙酰胆碱传输ATION云台存在野生型N2蠕虫是无法区分的，而突变蠕虫与一般的突触传递特定缺陷或抑制GABA的传输表现出强大的云台诱导前抽搐的正调控的缺陷。此外，UNC - 43丧失功能的突变体显示在云台存在全身抽搐，并可能有更多的突触传递异常，从而有助于其独特的药物反应。

<ol>
	<li>Mahoney, T. R., Luo, S., Nonet, 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=Analysis+of+synaptic+transmission+in+Caenorhabditis+elegans+using+an+aldicarb-sensitivity+assay.">Analysis of synaptic transmission in Caenorhabditis elegans using an aldicarb-sensitivity assay.</a> Nat. Protoc. 1, 1772-1777 (2006).</li><li>Williams, S. N., Locke, C. J., Braden, A. L., Caldwell, K. A., Caldwell, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epileptic-like+convulsions+associated+with+LIS-1+in+the+cytoskeletal+control+of+neurotransmitter+signaling+in+Caenorhabditis+elegans.">Epileptic-like convulsions associated with LIS-1 in the cytoskeletal control of neurotransmitter signaling in Caenorhabditis elegans.</a> Hum. Mol. Genet.. 13, 2043-2059 (2004).</li></ol>

当前的协议与 C涕灭威曝光线虫不允许实验者区分与特定的赤字乙酰胆碱在突触传递中的广义赤字的传输和突变体的突变体，突变体的两个类表现出耐涕灭威。同样，涕灭威不能用来确定突变GABA的传输或广义故障的具体赤字负调节乙酰胆碱的传输，因为这两个类的突变体表现出过敏到涕灭威。我们云台暴露实验，涕灭威暴露实验结果相结合，结果使研究人员能够更好地表征突触传递的突变体。 <p...

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<td>Aldicarb</td>
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<td>Pentylenetetrazole</td>
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paradigms for pharmacological characterization of c. elegans synaptic transmission mutants

线虫，线虫，已成为研究神经递质的权宜之计模型。线虫在动物模型中是独一无二的，解剖学和神经系统的连接已经从电子显微镜确定和完善了药理实验。在这段视频中，我们描述了如何两个互补的神经兴奋剂，涕灭威，乙酰胆碱酯酶抑制剂，和伽马氨基丁酸（GABA）受体拮抗剂，戊四氮（PTZ），可受聘具体特征线虫神经肌肉接头的信号（NMJs），方便我们对立的神经回路的了解。 302线虫的神经元，十九电机D型GABA能神经元支配体壁肌肉（BWMs），而GABA能神经元，称为RMEs，头部肌肉的支配。相反，三十九个运动神经元表达的兴奋性神经递质，乙酰胆碱（ACH），拮抗GABA的传输在BWMs协调运动。 GABA能和胆碱能运动神经元在体壁NMJs的对立性质最初由激光烧蚀和后来挟着涕灭威曝光。涕灭威在野生型蠕虫当然时间或剂量反应瘫痪急性暴露的结果。然而，兴奋胆碱传输损失抗性涕灭威，蠕虫NMJs少乙酰胆碱的积累，导致少BWMs刺激。与乙酰胆碱的特定或一般的突触功能的突变体，可观察到涕灭威的电阻。与对立的传输γ-氨基丁酸和乙酰胆碱，GABA的传输损失，或失败负调节乙酰胆碱的释放一致，赋予过敏涕灭威。虽然涕灭威曝光导致的许多蠕虫病毒的神经递质基因同源的隔离，涕灭威曝光单独不能有效地判断当时的角色线虫在特定的运动神经元的基因和途径。为此，我们引入了一个互补的实验方法，它使用云台。 神经传递突变体显示清晰的表型，有别于涕灭威，引起瘫痪，到PTZ。野生型蠕虫病毒，以及与乙酰胆碱的释放或接收的具体无能的突变体，并没有显示明显的敏感性云台。然而，氨基丁酸的突变体，以及一般的突触功能的突变体，显示在时间的课程或剂量反应的方式，前抽搐。到BWMs突变体不能负调控一般神经递质的释放，因此，分泌过量的乙酰胆碱，成为云台瘫痪。离散突变类的PTZ诱导的表型表明，涕灭威和线虫的PTZ曝光范式互补的做法，可能会加速我们的神经传递的理解。此外，视频演示了如何进行药理实验，应建立对线虫的研究相一致方法。

Watch this Scientific Journal Video about Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants at JoVE.com

Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants

本视频演示了如何利用互补的方法，研究线虫，线虫突触功能的神经兴奋剂，涕灭威和戊四氮（PTZ），。这种互补的方法也可能被用来阐明调节神经元的同步进化上保守的机制和癫痫和癫痫发作的影响。

为线虫突触传递的突变体的药理特性的范式

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the ...

paradigms-for-pharmacological-characterization-c-elegans-synaptic

Research

JoVE Journal

Biology

14.0K 次观看.  University of Alabama. 本视频演示了如何利用互补的方法，研究线虫，线虫突触功能的神经兴奋剂，涕灭威和戊四氮（PTZ），。这种互补的方法也可能被用来阐明调节神经元的同步进化上保守的机制和癫痫和癫痫发作的影响。

视频: Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

产生稳定的转基因C.使用微量注射线虫

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

科研

生物学

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

Neurocircuit检测癫痫发作癫痫突变果蝇</em

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

生成的基因 C。线虫通过基因枪转化

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

基本的<em&gt;线虫</em&gt;方法：同步和观察

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

线虫趋含量的

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

一种用于培养胚胎<em&gt; C。线虫</em&gt;细胞

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that exhibit partial and variable transmission rates of extrachromosomal arrays to the next generation. In addition, transgenic animals are mosaic and express the transgene in a variable number of cells. Extrachromosomal arrays can be integrated into the C. elegans genome using UV irradiation to establish nonmosaic transgenic strains with 100% transmission rate of the transgene. To that extent, F1 progenies of UV irradiated transgenic animals are screened for animals carrying a heterozygous integration of the transgene, which leads to a 75% Mendelian transmission rate to the F2 progeny. One of the challenges of this method is to distinguish between the percentage of transgene transmission in a population before (X% transgenic animals) and after integration (&#8805;75% transgenic F2 animals). Thus, this method requires choosing a nonintegrated transgenic line with a percentage of transgenic animals that is significantly lower than the Mendelian segregation of 75%. Consequently, nonintegrated transgenic lines with an extrachromosomal array transmission rate to the next generation &#8804;60% are usually preferred for integration, and transgene integration in highly transmitting strains is difficult. Here we show that the efficiency of extrachromosomal arrays integration into the genome is increased when using highly transmitting transgenic lines (&#8805;80%). The described protocol allows for easy selection of several independent lines with homozygous transgene integration into the genome after UV irradiation of transgenic worms exhibiting a high rate of extrachromosomal array transmission. Furthermore, this method is quite fast and low material consuming. The possibility of rapidly generating different lines that express a particular integrated transgene is of great interest for studies focusing on gene expression pattern and regulation, protein localization, and overexpression, as well as for the development of subcellular markers.

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome

This protocol describes a rapid and low material consuming procedure for the integration of transgenic extrachromosomal arrays into the Caenorhabditis elegans genome using ultra violet (UV) irradiation. Furthermore, this protocol is particularly well suited for transgenic lines that transmit extrachromosomal arrays at a high rate.

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

对缺血/再灌注在缺氧，饥饿型号<em&gt; C。线虫</em

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound responses and repair in this model have illuminated our understanding of the cytoskeletal and genomic responses to tissue damage. The two most commonly used methods to wound the C. elegans adult skin are pricks with microinjection needles, and local laser irradiation. Needle wounding locally disrupts the cuticle, epidermis, and associated extracellular matrix, and may also damage internal tissues. Laser irradiation results in more localized damage. Wounding triggers a succession of readily assayed responses including elevated epidermal Ca2+ (seconds-minutes), formation and closure of an actin-containing ring at the wound site (1-2 hr), elevated transcription of antimicrobial peptide genes (2-24 hr), and scar formation. Essentially all wild type adult animals survive wounding, whereas mutants defective in wound repair or other responses show decreased survival. Detailed protocols for needle and laser wounding, and assays for quantitation and visualization of wound responses and repair processes (Ca dynamics, actin dynamics, antimicrobial peptide induction, and survival) are presented.

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity.

对于皮肤伤人方法和测定的伤口响应<em&gt;温度。线虫</em

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

方法来评估肌肉的亚细胞中<em&gt;温度。线虫</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...