JoVE   
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Biology

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Neuroscience

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Immunology and Infection

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Clinical and Translational Medicine

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Bioengineering

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Applied Physics

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Chemistry

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Behavior

  
You do not have subscription access to articles in this section. Learn more about access.

  JoVE Environment

|   

JoVE Science Education

General Laboratory Techniques

You do not have subscription access to videos in this collection. Learn more about access.

Basic Methods in Cellular and Molecular Biology

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms I

You do not have subscription access to videos in this collection. Learn more about access.

Model Organisms II

You do not have subscription access to videos in this collection. Learn more about access.

Essentials of
Neuroscience

You do not have subscription access to videos in this collection. Learn more about access.

Automatic Translation

This translation into Chinese was automatically generated through Google Translate.
English Version | Other Languages

 JoVE Biology

蠕虫病毒实验室介绍:从培养蠕虫突变

*1, *1, 1

1Department of Biology, University of Texas at Arlington

* These authors contributed equally
Article
    Downloads Comments Metrics

    You must be subscribed to JoVE to access this content.

    This article is a part of   JoVE Biology. If you think this article would be useful for your research, please recommend JoVE to your institution's librarian.

    Recommend JoVE to Your Librarian

    Current Access Through Your IP Address

    You do not have access to any JoVE content through your current IP address.

    IP: 54.82.144.55, User IP: 54.82.144.55, User IP Hex: 911380535

    Current Access Through Your Registered Email Address

    You aren't signed into JoVE. If your institution subscribes to JoVE, please or create an account with your institutional email address to access this content.

     

    Summary

    与表型的缺陷突变体的筛选是一个简单的方法识别基因功能,在一个给定的生物过程。在这篇文章中我们将介绍如何对文化自由的生活的蠕虫(如,

    Date Published: 1/11/2011, Issue 47; doi: 10.3791/2293

    Cite this Article

    Chaudhuri, J., Parihar, M., Pires-daSilva, A. An Introduction to Worm Lab: from Culturing Worms to Mutagenesis. J. Vis. Exp. (47), e2293, doi:10.3791/2293 (2011).

    Abstract

    这个协议描述的程序,以保持线虫在实验室,以及如何mutagenize他们使用两种可供选择的的方法:甲基磺酸乙酯(EMS)和4,5“,8 trimethylpsoralen结合紫外线(TMP /紫外线)。线虫是因为其简单的身体计划和交配系统,这是自我施肥雌雄同体和男性可以产生数以百计的每动物后代组成强大的生物遗传学的研究系统。线虫是保持中含有的细菌草坪的琼脂板,可以很容易地从一个板块转移到另一台使用一挑。 EMS是常用的一种烷化剂诱发点突变和小缺失,而TMP /紫外线主要是诱导删除。根据正在使用的线虫的物种,环境管理体系和TMP的含量进行优化。为了隔离隐性突变线虫Pristionchus pacificus,F2代的动物视觉筛选表型。为了说明这些方法,我们诱变蠕虫和不协调(UNC),矮胖(DPY)和变压器(TRA)突变体。

    Protocol

    第1部分:准备线虫的生长介质(NGM)的Petri板

    实验线虫, 线虫 P. pacificus通常在6厘米的Petri板含有线虫的生长介质(NGM)1实验室培养。蠕虫是保持在20 ° C,但可以根据线虫的物种和实验设计,在一定温度范围内(4 ° C - 25 ° C之间)孵育。为了准备与NGM板,标准的无菌技术已被用来防止真菌和细菌的污染。以下是协议准备NGM板:

    1. 称取蛋白胨2克,琼脂15克,2.4克氯化钠2.72克KH 2 PO 4 2升锥形瓶中,使体积至1 L与水,高压灭菌器,并允许它冷却到60 ° C水浴中。
    2. 添加0.8毫升的1 M 氯化钙 ,胆固醇(5毫克/毫升乙醇中),1米硫酸镁 。为了防止真菌和细菌的污染,1 ml链霉素(100毫克/毫升)和1毫升制霉菌素(10毫克/毫升),可以被添加到每公升介质。
    3. 使用Unispense机和保持无菌条件下,倒入所需的板媒体。板应装到约2 / 3 。一个标准的直径为60毫米的钢板,需要约10毫升的媒体。不要板块移动,直到琼脂完全gelified,否则会有琼脂表面凹凸不平,使其难以集中的体视显微镜上的蠕虫。
    4. 一旦干燥,板材可立即接种或储存在4℃,直至与细菌播种彗星。

    第2部分:准备OP50细菌和播种NGM的Petri板

    P. pacificus是在实验室培养大肠杆菌 OP50应变喂养。 OP50上NGM板的有限增长,从而形成一个薄的草坪,方便的可视的蠕虫1。要成长的OP50文化,使用LB -肉汤:

    1. 添加5克,2.5克酵母提取物和2.5 g氯化钠bactotryptone螺丝帽瓶500毫升,用蒸馏水和高压灭菌容至500 ml。
    2. 同一个OP50单菌落接种肉汤的无菌条件下培养板,让它成长一夜之间在37 ° C。
    3. LB - OP50是随时可以使用,可以储存在4 ° C几个月,如果保持无菌。每当种子板块需要无菌一个小烧杯,倒入少量的肉汤,这将有助于防止污染的股票文化。
    4. 种子板,分配到60毫米直径的NGM的Petri板和漩涡传播的OP50 100-120μL。
    5. 孵育这些种子草坪生长板在室温下过夜。这些种子的板,现在可以存储长达3个星期在4 ° C。它们存储在一个倒置的位置,有助于防止快速干燥。

    第3部分:一挑

    在本节中,我们介绍了如何挑一个蠕虫。挑用于蠕虫从一个位置转移到另一个。挑30计90%的铂金10%铱丝,虽然一些研究人员可能更喜欢稍有不同的金属成分:

    1. 剪下约3-4厘米的导线,巴斯德吸管(其尖端被打破到一个较小的长度)的提示,然​​后0.5厘米内入玻璃密封在一个本生灯。从玻璃突起线的长度大约是3-3.5厘米,但可以根据个人喜好而有所不同。
    2. 拼合线是凸出使用锤子或钳子的结束。然后夷为平地的部分向上弯曲,形成了瓢。
    3. 用沙纸,以防止破坏性的蠕虫或琼脂,理顺拿起锋利的边缘。

    第4部分:采摘蠕虫

    1. 挑蠕虫,火焰消毒的铂丝和拖细菌草坪扁平尖挑大衣与稠粘细菌的一角,小心不要穿刺或损坏的琼脂表面。
    2. 使用立体显微镜,很轻轻一刷直到蠕虫被拾起棍子上挑的细菌的蠕虫病毒/蠕虫对粘提示。
    3. 一旦蠕虫的挑尖,转移它立即到新盘,通过触摸或滑动的挑尖对新盘的细菌草坪和蠕虫抓取。必须小心不损坏板面的琼脂,其他的蠕虫爬进孔,使其难以检索。如果蠕虫停留挑太久,它可能变干。

    第5部分:EMS诱变

    乙基甲烷磺酸盐(EMS)诱导突变在基因组中的2的广谱。突变体可以识别的缺陷,如产蛋,肌肉缺损S,性别决定的dauer形成,行为,和性腺形成。为EMS诱变的协议是类似 C描述线虫 1。

    1. 准备500毫升1N氢氧化钠(或更多)。
    2. 需4-5,直径6厘米的Petri板的蠕虫病毒,其中包含几百蠕虫幼虫少年第三阶段(J3),洗掉的蠕虫使用2-3毫升无菌每盘M9。
    3. 在无菌的,一次性的15毫升离心管收集的所有的蠕虫。离心5-7分钟,或在1500 XG直到蠕虫形成一个颗粒。
    4. 吸出液体,并用2毫升M9的重新暂停蠕虫。 M9的一个试管中2毫升添加20μL的环境管理体系和动摇解散。然后,添加这种蠕虫悬挂和漩涡轻轻2毫升EMS解决方案。 EMS的终浓度为47毫米。注意:EMS是一个非常强烈的诱变剂,并应在通风橱中处理。在这个诱变剂接触的所有材料(手套,滴管,提示),应被视为与1N氢氧化钠不活跃的EMS。双层手套穿在整个EMS诱变过程。根据您的机构的指导方针,处置危险废物的。
    5. 将离心管中,并用封口膜覆盖的上限;水平放置在通风柜,让孵育3.5小时的蠕虫。管也可以放在低速摇臂。
    6. 打开管,并用封口膜覆盖的上限;蠕虫下沉的垂直位置,直到孵化的地方。
    7. 吸出上清液,并丢弃到一个烧杯/烧瓶中含有1N氢氧化钠。这将可灭活该环境管理体系。
    8. 加入5毫升M9的蠕虫,倒置管的25-30倍,洗5-7分钟,或蠕虫,离心机在1500 XG直到蠕虫形成一个颗粒。吸出上清液,并丢弃到1N氢氧化钠烧杯。
    9. 重复至少3次洗涤步骤。
    10. 最后一次洗涤后,重新暂停少量的M9(〜500μL)的蠕虫病毒颗粒,并滴入含有OP50细菌草坪的NGM板出来。在这一点上,你不再需要工作在化学罩。
    11. 让液体干燥几分钟,然后挑选健康的J4的幼虫转移到新鲜的种子板块。根据您所在机构的危险废物准则丢弃原板。
    12. 让诱变蠕虫自我受精。使用立体显微镜下,屏幕上的F2代表型为隐性突变体的利益。

    第6部分:补骨脂素诱变

    在本节中,我们描述了长波长紫外线(UV)一起使用TMP的突变过程。 TMP /紫外线诱变的方法被广泛用于诱导缺失突变在基因 3 。

    1. 4-5,直径6厘米的Petri板收集到15 mL离心管前面介绍的步骤中的蠕虫病毒。
    2. 添加10毫升M9的反转20次,在1500 XG洗5分钟的蠕虫和离心机。弃上清,重复洗涤,共3次。吸出液体和重新暂停在10倍左右,M9的体积含有30微克/毫升的TMP的蠕虫。吸取20μL到2毫升M9的TMP(3毫克/毫升)。环境管理体系一样,TMP是一种强力致癌物质,必须采取适当的安全措施来处理。为了防止在紫外线照射下正确的眼睛穿的潜在风险,应使用。必须在适当的生物危害袋被丢弃的一次性。
    3. 在黑暗中孵育15分钟的低转速的摇杆(40芬欧)在室温下管。设置覆盖的管垂直为10分钟,让所有的蠕虫下沉。
    4. 从巴斯德吸管管的底部取出蠕虫和它们转移到一个非种子选手NGM板。丢弃以下机构的危险化学品的指引上清。
    5. 保持在暗盘,直到多余的川芎嗪板吸收。
    6. 使用紫外线强度计,设置之间的长波紫外线源的距离和钢板等,对钢板的强度是500微 / 平方厘米4。删除铝盖,盖,盖,板,含有50秒川芎嗪治疗蠕虫的长波紫外线辐射暴露。
    7. 盖,在室温下5小时,在黑暗中的蠕虫。
    8. 挑选和健康的J4的幼虫转移到新鲜的种子板块。丢弃以下机构的危险化学品的指引的原板。
    9. 使用立体显微镜,屏幕F2代利益的突变表型。

    第7部分:代表结果

    一些在体育pacificus(可以通过EMS或TMP /紫外线)诱变产生的突变表型,如图1所示。每个突变体具有特征性的的表型,野生型动物,很容易分辨。从蠕虫被处理的诱变剂,30 J3/J4从亲代(P0)的幼虫被放置在单独的板块,并让其产卵,这是F1代。从板块表现出健康的F1子代,共1000 F1S被转移到个别板块和F2代表型的筛选。矮胖5,6,不协调6和变压器7表型的动物被发现。

    图1
    1。Pristionchus pacificus野生型和突变型的动物。 A. WT雌雄同体B. WT男三DPY突变D。 UNC突变E. TRA突变。前两名面板显示野生型雌雄同体(左)和男(右)的动物,在余下的面板不同的突变体进行比较。

    Subscription Required. Please recommend JoVE to your librarian.

    Discussion

    突变是一个广泛采用的技术,在各种实验模式生物基因研究。通常用于突变实验,以确定一个基因的2的功能。这里所描述的突变程序可以使用,不仅为体育pacificus,但也为C。线虫和其他相关线虫物种。在这里,我们介绍两种方法,环境管理体系和TMP /紫外线诱变。

    EMS是一种化学物质,诱导点突变或整个基因组的3,8小缺失。补骨脂突变(TMP / UV)主要诱发原因可能断裂,在紫外线照射下引起的DNA缺失突变。删除片段通常在长度为3 kb的范围为0.10至15。

    我们筛选的表型是比较常见的找到。在屏幕使用EMS F2动物每400板块,一个板块一个DPY,UNC或茶。 DPY可确认其异常运动和茶,有一个男的表型和XX染色体型7 短尺寸7日,联合国军司令部。 TMP /紫外线诱变的产生与环境管理体系 3相比,突变体的频率较低,但它会产生更大的缺失。

    线虫,第四阶段后期幼虫或年轻成人是最有效的的发展阶段,因为他们有生殖线核的最大数量进行诱变。因此,它是有用的,开始第四阶段的幼虫许多家长人口与同步的实验。一个简单的方法是同步文化与鸡蛋,而不是成虫新的文化。在下面的一代,大多数蠕虫将大约在同一时代。隐性突变,可以检测到通过检查F1个人,这将产生约百分之二十五个纯合子的后代。

    可进一步利用线虫的基因屏幕上的权力,通过修改设计的屏幕9。雌雄同体的生活方式和快速生成时间相结合,使线虫的遗传学研究的理想之选。

    Subscription Required. Please recommend JoVE to your librarian.

    Disclosures

    没有利益冲突的声明。

    Acknowledgements

    这项工作已经由美国国家科学基金会资助的IOB#0615996。

    References

    1. Brenner, S. Genetics. 77, (1), 71-71 (1974).
    2. Anderson, P. Methods Cell Biol. 48, 31-31 (1995).
    3. Yandell, M. D., Edgar, L. G., Wood, W. B. Proceedings of the National Academy of Sciences of the United States of America. 91, (4), 1381-1381 (1994).
    4. Pires-daSilva, A. WormBook. 1-1 (2005).
    5. Kenning, C., Kipping, I., Sommer, R. J. Genesis. 40, (3), 176-176 (2004).
    6. Sommer, R. J., Carta, L. K., Kim, S. Y. Fundamental and Applied Nematology. 19, (6), 511-511 (1996).
    7. Pires-daSilva, A., Sommer, R. J. Genes & development. 18, (10), 1198-1198 (2004).
    8. Wei, A., Yuan, A., Fawcett, G. Nucleic acids research. 30, (20), 110-110 (2002).
    9. Jorgensen, E. M., Mango, S. E. Nat Rev Genet. 3, (5), 356-356 (2002).

    Comments

    5 Comments

    I liked the explanation and the beauty of the description. It would be quite helpful for the beginners like us to learn and execute worm handling techniques.
    Reply

    Posted by: AnonymousJune 29, 2011, 6:03 AM

    If you would like to see JoVE at your institution, please let your librarian know about us.
    Reply

    Posted by: AnonymousJune 29, 2011, 9:49 AM

    Thank you for your feedback, Sonu. We're glad that the article was of practical use to you.
    Reply

    Posted by: AnonymousJune 29, 2011, 9:45 AM

    Great video. We are new to nematode work and so love the opportunity to learn from experienced nematode researchers in videos such as yours on JOVE. I would have therefore liked to see more detail about some of the identifications of the worms, for example at 9min 38sec you describe the worm in Panel A as being a wild type hermaphrodite while the worm in panel B you describe as a wild type male. They both look identical in the video however. What is the basis for these identifications? The three mutations as well in Panels C-D you also state the name of but it is not clear how you came to identify that this is what they were. Lastly at 3min 49sec you say to perform mutagenesis using EMS you used worms at the third juvinile stage. How do you tell what stage a worm is at and obtain entire dishes of worms at this stage, wouldn't they be of varying ages?
    Reply

    Posted by: Haydn A.December 17, 2013, 1:47 AM

    At this magnification it is indeed difficult to distinguish the two sexes. If you pause the video at 9 min 38 sec you will notice that the male is thinner than the hermaphrodite and that it has a white stripe running along the entire body in the ventral side. At higher magnification, which is not shown in the video, you will notice that the end of the tail is longer and thinner in the hermaphrodite compared to males. You will also see a pair of sclerotized structures in the male tail that are named spicules, which are used to open the vulva of hermaphrodites (see electron micrograph in http://dev.biologists.org/content/130/6.cover-expansion and DIC microscopy in http://www.ncbi.nlm.nih.gov/pubmed/15155582).
    The mutants in Panels C-D are based on their morphology and behavior. The dumpy mutants are shorter, the unc mutant shown in the video is very thin and moves only the head, and the transformer mutant is a XX animal with male phenotype. A more thorough description of the transformer phenotype in P. pacificus is in http://www.ncbi.nlm.nih.gov/pubmed/15155582.
    When doing the mutagenesis, usually we collect worms from a plate that has animals of all stages. Only at the end of the treatment (e.g., Part 5, step 11), we choose the stage of the worms that we want. The stage of the worm is determined by its relative size.
    Reply

    Posted by: Andre P.December 19, 2013, 5:19 AM

    Post a Question / Comment / Request

    You must be signed in to post a comment. Please or create an account.

    Metrics

    Waiting
    simple hit counter