产生稳定的转基因C.使用微量注射线虫

Published 8/15/2008
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Summary

本视频演示,到线虫的性腺创建转基因动物的显微注射技术。

Cite this Article

Copy Citation

Berkowitz, L. A., Knight, A. L., Caldwell, G. A., Caldwell, K. A. Generation of Stable Transgenic C. elegans Using Microinjection. J. Vis. Exp. (18), e833, doi:10.3791/833 (2008).

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Abstract

转基因线虫可以很容易地创建,通过显微注射DNA质粒的解决方案进入性腺1。质粒DNA的重新排列,形成稳定遗传,但不以同样的效率 2实际的染色体染色体外concatamers。一个共同感兴趣的基因注入一个明显的表型标记,如ROL - 6或GFP,在解剖显微镜下,允许转基因动物的选择。可能是外源基因表达的细胞定位研究的原始推动者。此外,转基因可以由不同的组织特异性的启动子,以评估在特定的细胞或组织的基因产物的作用。这项技术有效地推动所有组织线虫的生殖细胞或早期胚胎基因表达 3 。转基因动物的创建是广泛使用的实验范式。本视频演示了显微注射过程产生转基因的蠕虫。此外,选择和维护稳定的转基因线虫线描述。

Protocol

表达质粒的构建

需要两个质粒:感兴趣的基因组织特异表达的第二个可选择的改造标记之一。

实验质粒

  1. 选择一个感兴趣的组织/细胞类型,例如,神经或肌肉特异性启动子表达的启动子。如果是同一组织内的多个基因的表达,在网关系统(Invitrogen)的质粒发起人卡带都可以使用。这种方法允许任何利益recombinational克隆4子下游的基因插入,一般情况下,上游序列2-5 KB足够的正确和准确的表达。

  2. 感兴趣的基因的cDNA克隆的方法有两种:
    1. 传统的克隆用限制性内切酶。
    2. 为Gateway克隆,它是使用包含网关ATT乙 recombinational序列的引物PCR扩增。扩增的cDNA第一重组到捐助载体pDONR201创建入门载体,然后转化到E.大肠杆菌菌株DH5a。入门载体是继成功的重组体的选择和小型预习DNA提取,重组到含有启动子目标的载体,以创建表达质粒。 recombinational克隆的详情,可从Invitrogen公司的网关手册或在考德威尔等5

选择标记质粒

转基因标记质粒的例子包括:ROL - 6或使用的体壁肌肉 UNC - 54启动子驱动荧光蛋白的表达。这些标记质粒通常可以从研究界内,根据请求。

微量注射线虫

制备

  1. 被注入的两个质粒DNA进行隔离。定量,并在50 ng /μL的每一个1:1的比例混合在一起。

  2. 琼脂垫做准备:
    1. 直到溶解在水中的2%琼脂糖溶液;加热或微波。
    2. 在工作​​台上设置了22 × 50 mm的盖玻片。
    3. 使用巴斯德吸管,放置在一个玻璃盖一个熔化的琼脂糖下降,立即放置在一个90度的第二个玻璃盖,在下拉到第一。重复其他几个盖玻片。夷为平地的琼脂糖光盘的直径应为15-20毫米之间。
    4. 后琼脂糖凝固(这个只需要时刻),取出盖玻片,并允许垫完全风干(几个小时到过夜)。
    5. 在室温下存放在玻璃盖盒垫。

  3. DNA溶液的针装载机:

    在中间明火加热,并迅速拉至约两倍的长度从100μL的毛细管针装载机。 毛细管冷却后的两半分开被抢购。库存10-20注射针头装载机。商店直立的离 ​​心管架。谨慎使用不刺穿自己点。

  4. 进行显微注射​​针:

    针是由一个特殊的毛细管,其中包含一个内部的玻璃纤维长丝作为DNA溶液的灯芯。这些都是拉一针拉拔机上。我们用一个Narishige PP - 830拉拔,热定型的24.8。几个针拉的时间和存储在一个小塑料盒。多针,可随时“装”上的橡皮泥地带长期储存。

  5. 针尖“断路器”:

    的显微注射针的尖端拉那么细,它实际上是在它的封闭式,并应使用玻璃盖的小碎片,破开。这些都是准备纸巾包裹在一个18 × 18毫米的玻璃盖,轻轻施加压力,直到它断裂成几件。一个有用的大小的碎片大约有3 x 4毫米。

  6. 野生型蠕虫(N2),成长到成年早期阶段使用的标准程序6注射。准备约100正确上演蠕虫/构造注入。

程序

  1. 打开主阀是连接到一个微量注射针臂和支架的氦罐。关闭调节阀,让气体进入线,使35磅的压力。注意使用脚踏板,可以释放的气体。

  2. 一针器插入到一个标准口pipetter管(玻璃毛细管的容器中),并制定了少量的质粒混合物(1μL)。

  3. 针器小心放入后端的注射针头,轻轻插入方式通过的长度针,直到阻力感觉。轻轻吹驱逐的DNA溶液,然后取出装载机。

  4. 显微注射的手臂插入针头,小心地放置在小的内部橡胶垫片。

  5. 放在琼脂板上的一个边缘的玻璃盖针破陶片。盖的碎片,以及琼脂板的整个表面,与卤烃油。将倒置显微镜舞台上的琼脂垫。

  6. 查看使用4X的目标和明视场照明领域的中心针的位置,但还没有降低进油。此外,中心盖在玻璃碎片的内缘的位置,然后专注于IT(仍在使用4X目标)。下入油针,将玻璃盖的碎片一样焦平面。提高放大倍率切换到40X目标。重新碎片的边缘,如果有必要重新定位针尖。

  7. 要开拓尖端的注射针头,轻轻地轻推针对盖玻璃碎片的边缘,而在同一时间,采用短脉冲的气体通过脚踏板。中针会充分破开液体的小水滴时逃脱针年底的。多余的液体流动,由于过大开幕,是不可取的,因为它会杀死动物。

  8. 从舞台删除琼脂板,提高了针,但不改变X或Y轴位置。滑动阶段,从下面的针,取出琼脂垫,并将其放置到在解剖显微镜。垫转移到一种蠕虫病毒,油面以下。如果蠕虫蠕动,轻轻抚摩它,直到它接触琼脂垫。潮湿的蠕虫,要坚持干琼脂糖。

  9. 注塑
    1. 快速放置搬上舞台的琼脂垫背面,中心视野的蠕虫病毒。
    2. 下针到蠕虫的同一平面上的重点。
    3. 霍夫曼照明(对比度过滤器的类型)过滤器切换。这使得蠕虫的形态细节成为可见。如果Nomarski / DIC光学上倒置的范围,将工作以及。使用40X的目标,着眼于“颗粒感”合胞中心的蠕虫病毒性腺生殖核下方的“蜂窝”模式,选择定位于一侧面临的针虫的性腺是。
    4. 微调针的位置,直到针尖还重点(性腺的“颗粒感”同一平面)。
    5. 轻轻插入针和性腺中心申请的气体压力驱逐到性腺手臂液滴的DNA或两个简短的脉冲。你应该观察整个远端性腺的液体传播的波。不要以为更多的液体是更好,因为过多的液体流入近端性腺手臂弯曲,可以关闭卵母细胞生产。如果可能的话,根据蠕虫的位置上,以同样的方式注入其他性腺手臂。
    6. 重要的是迅速开展工作,同时注入了一种蠕虫病毒,它可以很容易变干。注入第二性腺臂的决定后的速度有多快,你可以​​重新定位针和蠕虫依赖。整个过程应该最好少于1-2分钟。

  10. 从舞台上拆下的玻璃盖,放在解剖范围。应用上直接注入蠕虫(22MM KH 2 PO 4,42mm的NA 2 HPO 4,85MM氯化钠,1MM 硫酸镁 )M9的缓冲液加入1μl(这可能需要淹没的卤烃油的表面下面的枪头)。缓冲区应补充水分的蠕虫病毒,然后将浮琼脂糖垫表面。蠕虫传输到正规板使用了蠕虫挑。琼脂糖垫可重复使用数次,直到它已成为缓冲区和蠕虫不再坚持过于饱和。

转基因选择

  1. 注入蠕虫中,P 0代,被转移到个人新鲜,细菌接种板(60毫米),并允许重现。通常情况下,注入动物孵育2-3天,直到后代出现在20 ° C。

  2. 的F 1后代观察转基因标记用荧光体视显微镜(如荧光)的证据。每个荧光,载波F 1,动物分别克隆到一个新的板块(因为每个F 1的后代,被认为是一个独特的行)。允许的F 1雌雄同体重现。 ,同样,这通常是在20 ° C为2-3天,直到后代出现。

  3. 观察以及转基因动物的F 2代。 F 2,继承和表达的转基因阵列的动物被认为是一个“稳定” ​​行。

    注:在F 1年龄有可能是很多的转基因动物,但他们并不稳定。 F 1的转基因动物的大多数人不会传播到F 2稳定线。

    注意:要控制在基因的拷贝数变异,产生三个独立的每个构造注入稳定线的最低

  4. 每个独立的稳定线应保持作为一个单独的蠕虫。每一行可以传播几个转基因动物在每一代的新盘,这必须使用荧光体视显微镜,如果选择标记荧光。

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Discussion

执行此过程时,重要的是要记住:
- 直接注入到性腺中心
- 没有注入过多的液体
- 迅速开展工作,以防止dessication。

如果进针,如它被打破过大的问题,这是最好的改造,而不是试图以比不太理想的针注入新鲜针,。

代转基因蠕虫有许多应用,如:
- 突变基因的鉴定,在方法调用转化救援
- 截短蛋白的表达蛋白结构域的映射,通过
- 一个细胞和疾病的进程的基因的作用表征。

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Acknowledgements

我们要感谢考德威尔实验室所有成员的合作精神。运动障碍在实验室的研究已经得到了巴赫曼 - 斯特劳斯肌张力障碍及帕金森基金会,美国帕金森基金会,美国帕金森病协会,阿拉巴马州帕金森氏病协会,迈克尔J.福克斯帕金森病研究基金会,和本科生科研。

Materials

Name Type Company Catalog Number Comments
Agarose Ultrapure Invitrogen 15510-027
Halocarbon Oil, Voltalef Hulle 10S elfatochem, France
Coverglass 18x18 mm Fisher Scientific 12-548-A
Coverglass 20x30 mm Fisher Scientific 12-548-5A
Coverglass 22x50 mm Fisher Scientific 12-545-E
100 ul Capillary VWR international 53432-921
Glass Capillary for Needles "Kwik-Fil" World Precision Instruments, Inc. 1B100F-4
Needle Puller Tool Narishige International Model PP-830
microINJECTOR System Tritech Research, Inc. MINJ-1000 Scope, Stage, Manipulator
Dissecting, with Fluorescence Microscope Nikon Instruments SMZ800
Dissecting Microscope Nikon Instruments SMZ645

DOWNLOAD MATERIALS LIST

References

  1. Stinchcomb, D. T., Shaw, J. E., Carr, S. H., Hirsh, D. Extrachromosomal DNA transformation of Caenorhabditis elegans. Mol. Cell. Biol. 5, 3484-3496 (1985).
  2. Mello, C. C., Kramer, J. M., Stinchcomb, D., Ambros, V. Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. EMBO J. 10, 3959-3970 (1991).
  3. Kelly, W. G., Xu, S., Montgomery, M. K., Fire, A. Distinct requirements for somatic and germline expression of a generally expressed Caenorhabditis elegans gene. Genetics. 146, 227-238 (1997).
  4. Cao, S., Gelwix, C. C., Caldwell, K. A., Caldwell, G. A. Torsin-mediated neuroprotection from cellular stresses to dopaminergic neurons of C. elegans. J Neurosci. 25, 3801-3812 (2005).
  5. Caldwell, G. Integrated Genomics: A Discovery-Based Laboratory Course. John Wiley and Sons. West. Sussex, England. (2006).
  6. Brenner, S. The genetics of Caenorhabditis elegans. Genetics. 77, 71-94 (1974).

Comments

11 Comments

  1. Dear Dr. Laura A,   I am very much interested to work with you as a Researcher. Regarding myself, I would like to inform you that I did my Ph.D. degree from the Faculty of Science, Hiroshima University, Japan. I completed my M.Phil. M.Sc. and B.Sc. (Honours) degree from the University of Rajshahi, Bangladesh. Now I am working on toxic response and behavior of C. elegans as a postdoctoral researcher at Pusan National University, S. Korea.    I would be glad if you would kindly accept me as a Researcher in your Laboratory and provide me a fellowship or any financial support.   I look forward to your kind reply at your earliest convenience.   With best regards   Sincerely yours, Dr. M. Golam Mortuza Present Address Postdoctoral Researcher Lab of Ecology and Behavior System Division of Biological Sciences Pusan National University Busan 609-735, S. Korea E-mail: mortuzaru@yahoo.com   Permanent Address Professor Department of Zoology Rajshahi University Rajshahi 6²05, BANGLADESH E-mail: mortuza@ru.ac.bd

    Reply
    Posted by: Anonymous
    April 15, 2009 - 2:41 AM
  2. Hi, i am learning microinjection in C. elegans and am having problems recovering the worms.  They seem to recover fine initially and are thrashing around in the buffer on the plate, but after a day when I look back they have all died on the plate where they were placed.  I don't know what is wrong, do you have any suggestions? Thanks. Jane

    Reply
    Posted by: Anonymous
    May 22, 2009 - 1:57 PM
  3. Dear Jane,
    Death post injection is likely from two causes. The first is that the worms spent too much time stuck down on the agar pad before being hydrated off. Often they will be alive (barely) but then die. Reducing the time they spend stuck down will stop this source of death. As you become more adept and quicker with the process this will improve. The second cause of post injection death is from the worm getting stabbed too much- either from being injected in the wrong place (intestine), being injected too deeply (the &#x²01C;shish-kabob&#x²01D; mistake) or using too big of a needle. Often one can tell if this is the problem when the next day the injected worm has exploded out its guts. Make sure you are using a very tiny, fine tip in the needle and only barely enter under the cuticle into the gonad. Again these will improve with practice.

    One last thought- when you transfer the worm from the injection pad to the plate sometimes oil comes along with it. Gently move the worm out from the oil droplet onto the food. This will help it recover.
    Good luck!

    Laura

    Reply
    Posted by: Anonymous
    May 26, 2009 - 1:18 PM
  4. Hi Laura,
    Thanks so much for replying to my message. I think my problem is that I am always injecting in the wrong place. Occasionally they explode after injection, in which case I know I have injected too much or stabbed too much. My needle is very fine and is penetrating quite easily but only every once in a while dŒs it look like I have hit the gonad. I am trying to line it up so that I can see the nuclei on either side, but I still seem to miss most of the time. It mostly seems to be in the body of the worm. Do you have any tips for how I can improve my accuracy of hitting the gonad?

    Reply
    Posted by: Anonymous
    May 28, 2009 - 3:20 PM
  5. Dear Jane,
    I was trying to figure out how to help you visualize exactly where to inject. Here are some additional pointers and details from the video.

    I discuss how the dead center best spot for injecting is in the grainy interior of the gonad. Depending on your optics this may be very faint and hard to see. This graininess is like sand compared to the &#x²01C;pebble&#x²01D;-like appearance of the intestine. The gonad interior has also been described as a fine &#x²01C;velvetly&#x²01D; texture.

    If you go back and watch the video at time 8:08 that worm is a little too dry- see how the embryos (which don&#x²019;t dry out) are standing out more.
    In frame 8:²7 a nice view of the gonad with the arrow is shown. You can see an impression of the germ nuclei, but the &#x²01C;graininess&#x²01D; isn&#x²019;t too visible.
    Starting at 8:4² there is also a nice view of the animal and its major features. The lower left portion curving up towards the left is intestine. On the upper side moving towards the right are oocytes from the distal arm of the gonad. In the middle, between the oocytes and the intestine, lies a view of the proximal gonad exactly where it should be injected. At 8:53 the needle seems to be positioned correctly but what you see upon injection is the oocytes expanding outward, indicating that the needle tip wasn&#x²019;t positioned properly.
    At 9:06 shows a perfect gonad hit.

    Also, I try (if possible) to position the worm such that I am injecting closer to parallel to the worm rather than perpendicular to it. The force needed to puncture the cuticle when coming in at a 90o angle to the long axis of the worm is higher and results in a greater chance of going through into the intestine or even &#x²01C;shish-kabob&#x²01D;. I usuall try to position the worm and the needle at 15-30o angles to each other. For example see 8:53 & 9:06 (preferred) vs 8:57

    Hope these will help you.
    Laura

    Reply
    Posted by: Laura B.
    June 15, 2009 - 11:47 AM
  6. Hello!

    I am currently designing an automated system for c.elegans injections that involves circulating worms through microchannel in a PDMS chip and positioning them for injection from a microneedle. One of the important aspects of our design is to fabricate microneedles which can more easily puncture the worm's cuticle due to smaller needle size.

    However, we have been unable to find any reference, print or electronic, that has measured the force needed to puncture the worm. This force is important in choosing how thin our needles can be, and of course the force dŒs vary with needle tip size. We may need to experimentally determine this "puncture strength" ourselves as the first to ever do so, but I am wondering if you might know of anyone who has measured similar parameters that we can at least get a ballpark place to start.

    Thanks so much!

    -- Andrew

    Reply
    Posted by: Anonymous
    November 22, 2009 - 5:52 PM
  7. Andrew,

    We are not aware of the type of precise information on puncture strength or penetration forces required for C. elegans, as we are molecular biologists, but your question reminded me of a method I remembered (and once tried without success) from the late 1990s. The technology you describe is likely much improved or can be. Check out this paper and maybe contact the authors regarding your question.

    Genetic transformation of nematodes using arrays of micromechanical piercing structures.
    Hashmi S, Ling P, Hashmi G, Reed M, Gaugler R, Trimmer W.
    Biotechniques. 1995 Nov;19(5):766-70.

    Best of luck with your experiments. If you are ever interested in us to beta-test something as collaborators, please let us know.

    Guy

    Reply
    Posted by: Anonymous
    November 22, 2009 - 8:38 PM
  8. Hello,

    I am currently trying to inject double stranded DNA as an alternative approach to do tissue specific knockdown. I am following a protocol in which DNA sense and antisense fused to tissue specific promoter are injected together with a marker. According to the protocol, I should inject the PCR product directly and at the concentration of 100 ng/ul. However, when I injected this mixture into the gonad, most of the worms don't survive more than one day, which is not the case with the normal injection for generating the transgenic line.

    DŒs someone have any experience with this and can suggest me something that I should change or incorporate in my protocol to make the injection work?

    Thanks before,
    Yemima

    Reply
    Posted by: Anonymous
    June 6, 2011 - 12:21 PM
  9. We would like to start doing microinjections in our lab and I am would like to know where you acquired the halocarbon oil. In the Wormbook microinjection protocol, Series 700 Halocarbon oil from Halocarbon Products in NJ was used. Do you know how this compares to the Voltalef halocarbon oil that you used?

    Thank you very much for your help and for the wonderful instructional video!

    -Erin

    Reply
    Posted by: Erin M.
    March 13, 2013 - 1:40 PM
  10. Dear Erin,
    We no longer can get the Voltalef oil and have too switched to using Halocarbon oil 700 (but ours is from Sigma). It is a bit thinner than the Voltalef, but it works as well, if not even a little better. Glad you like the video. Good luck with the injections. --Laura

    Reply
    Posted by: Laura B.
    March 13, 2013 - 2:59 PM
  11. Thank you so much!

    -Erin

    Reply
    Posted by: Erin M.
    March 17, 2013 - 11:51 AM

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