斑马鱼的胚胎显微注射来分析基因的功能

Biology
 

Summary

该视频演示如何啉或mRNA可以注射到斑马鱼的胚胎,在单细胞阶段减少或增加特定的基因产物在随后的发展水平。

Cite this Article

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Rosen, J. N., Sweeney, M. F., Mably, J. D. Microinjection of Zebrafish Embryos to Analyze Gene Function. J. Vis. Exp. (25), e1115, doi:10.3791/1115 (2009).

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Abstract

研究斑马鱼的优点之一是操纵在胚胎中的蛋白质水平的易用性和速度。 Morpholinos,这是与靶RNA的合成寡核苷酸反义互补,可以添加到胚胎,以减少一个特定的基因产物的表达。相反,处理的mRNA可以添加到胚胎,增加一个基因产物的水平。车辆无论是基因或吗啉胚胎显微注射。显微注射是高效,快速,让数百个胚胎每小时注入。该视频显示在显微注射的所有步骤。简单地说,鸡蛋收集后立即被解雇,一字排开反对在培养皿中的显微镜幻灯片。下一步,注射材料装入了一种细尖的针是连接到一个微量空气源,并微量控制的调整,以产生一个理想的注射量。最后,针陷入胚胎的卵黄和吗啉或mRNA被驱逐。

Protocol

第1部分:蛋的生产和收集

  1. 注射前的晚上,设立鱼繁殖与到位的分隔坦克。为了增加总蛋生产,鱼可以成立一个男性两名女性的比例如果需要。
  2. 第二天早晨,打开后,房间的灯,拉几个坦克的分隔,并允许不受干扰的交配时间约20分钟。
  3. 使用一个过滤器,收集从养殖笼具,蛋和蛋水冲洗。鸡蛋与蛋水倒入培养皿中,并清除未受精卵,并用移液管的碎片。鱼可以集结更大的坦克,以产生额外轮注射鸡蛋。鸡蛋收集的时间调整,以便最大的鸡蛋生产的数字,而让他们通过单细胞阶段。
  4. 放置在一个100mm的培养皿倒盖的显微镜幻灯片。使用移液管排队反对方形成一个单一的列的幻灯片的鸡蛋。按Kimwipe对一侧对面的鸡蛋,从幻灯片中删除多余的蛋水。 (图1A)

第2部分:针拉,装载和准备

  1. 随着微管拉马,拉进两针,并存储在150毫米培养皿一个1.0毫米OD玻璃毛细管,通过铺设橡皮泥斜坡。针可以提前拉。
  2. 回程针与3μL注射使用microloader吸管材料。直到有很少或根本没有泡沫,其余摇向针尖丸。
  3. 打开气源和微量。针插入到微量,并确保一个密封的房屋内。检查的显微操纵器是在一个适当的位置,让一个广泛的运动和调整。针尖带入飞机的显微镜,高走下舞台,和上最薄的提示地区为重点。用一双锐利的镊子捏针在一个点上,叶针窄到足以刺穿蛋壳和蛋黄,但仍然能够提供一个一致的珠子大小的。可用于计算每次注射量的矿物油微米的下降。当注射入油,珠直径0.1毫米的有500注射材料的PL(图1B);通常用于500 PL或1nL注射量。踩下脚踏板和监察珠的大小,而削减针和调整所需的注射压力。理想的注射量,将填补蛋量的约10%。针尖的质量是至关重要的,以缓解注射液的质量和结果的一致性。

第3部分:注射液

  1. 确保已不是过去的四细胞阶段的胚胎。理想的情况下,应在单细胞阶段的胚胎。
  2. 对鸡蛋列下针,在你的对面手的菜。
  3. 皮尔斯绒毛膜表面的任何破碎或撕裂卵黄囊看,进入一个平稳中风蛋黄。注入注料入蛋黄(图1C)。避免注入气泡或伸展蛋黄作为可致死胚胎。工作的路线,调整压力需要保持一致的珠子大小,使用枪头,消除看受精,或在注射过程中破坏的鸡蛋。
  4. 鸡蛋列完成后,用一个鸡蛋水的温柔流移动到一个干净的培养皿中注入鸡蛋。必要时重复。保持几个uninjected胚胎作为对照。在第一天结束时,去除死胚胎,并记录注射胚胎的数量。定期更换菜蛋水,以减少感染的机会。

第4部分:代表结果

根据胚胎被注入的是什么,可能生存在一个比他们uninjected的兄弟姐妹率较低。幸运的是,它很容易注入大量的,所以这是很少见的问题。这是正常的morphants表现出稍微推迟发展。

为了说明显微注射的结果,我们使用此协议来操作的蛋白质的玻璃心(HEG)水平。 HEG是一种跨膜蛋白,心脏的正常发展所需的。在其缺席的情况下,胚胎发育与巨头的流入大片和商会1心。我们对heg_e3i3_egfr1和heg_e4i4_egfr2,HEG,注入浓度在500微米的两个以前未描述的morpholinos。在受精后2天,出现uninjected兄弟控制正常,如预期(图2A和B)。 heg_e3i3_egfr1注入胚胎脑水肿(图2c)。虽然大多数这些morphants心中已经改变了形态,他们的商会是正常大小的(图2d)。胚胎注入heg_e4i4_egfr2展览变量的心脏缺陷。一些看似正常的,而其他的大量流入大片和适度扩大的心房(图2E和F)。 HEG突变,正如先前报道1,有一个巨大的扩大的流入道和心房(图2G和H) 。

我们也注入400纳克/μLHEG全长的cDNA转录的mRNA与野生型胚胎。而uninjected控制正常发展(图3A和B),胚胎注入HEG的mRNA表现出频谱范围从野生型的外观,严重的独眼畸形的表型,缩短主体轴,头部和身体坏死,和中线缺陷(图3C和D)。

图1。注入胚胎在培养皿显微镜幻灯片(一)针对一字排开。注射量是由放在微米矿物油注射。注射量500 PL,这是常用的,有一个直径为0.1毫米(二)。注射后,立即啉或mRNA作为一个蛋黄(三)圈点当场可见。

图2。uninjected胚胎在受精后2天,没有严重的缺陷(A)或心脏表型(二)。与吗啉heg_e3i3_egfr1注射的胚胎脑水肿(箭头),但通常大小的心腔(四)。与吗啉heg_e4i4_egfr2注入了一些胚胎扩大流入大片和心房(E,F)。 HEG突变体的严重扩大的流入大片和心房(G,H)。 (一),(三),(E)和(G)4X DIC的图像;(二),(四),(F)和(H)20X DIC的图像。 A =心房,=流入道。

图3,在受精后24小时,uninjected的胚胎有一个长期的,直身无坏死或中线(A)和(二)之间的神经管缺陷的两个明确的眼睛。 HEG的mRNA注入一些胚胎,相反,表现出缩短身体的纵轴,坏死,misshaped体节(三),和独眼畸形(四)。 (A)和(C)是4X DIC的图像;(B)和(d)为20倍DIC的图像。

Discussion

斑马鱼模型系统的优势之一是与特定的基因产物可以添加或从胚胎显微注射所淘汰的缓解。无所不在的过度表达一种特定的蛋白质,mRNA编码是注入蛋黄1细胞阶段。在胚胎的后续发展,RNA是分布在整个生物体和翻译。相反,要消除一种特定的蛋白质,morpholinos使用。 Morpholinos是与特定的RNA的反义互补性,设计合成寡核苷酸。一样的mRNA,morpholinos注入蛋黄1细胞阶段。在胚胎里,他们结合自己的靶RNA,防止翻译。

主要的修改,需要为每个啉或mRNA注射材料的浓度。过高的浓度啉或表达可能导致非特异性的毒性,而每个啉必须的实证检验,以确定其最佳浓度,通常啉浓度200微米,500微米之间的基因的活动有效地击倒,而不会造成非特异性缺陷。针开放精确的尺寸并不重要。针头大小的范围内,注射压力和注射时间内可以进行调整,以产生正确的卷丸。

Morpholinos有许多应用,包括域范围内的一种蛋白质的功能夹层。设计针对特定的外显子 - 内含子路口时,morpholinos防止发生有拼接。我们研究了两个morpholinos的影响,结合外显子-内含子在mRNA前体 HEG的交界处。啉heg_e3i3_egfr1结合的第3外显子和内含子3之间的交界处,防止纳入成熟笔录第3外显子剪接机器。吗啉heg_e4i4_egfr2同样删除第4外显子。外显子3和4都含有EGF样重复编码域。有些胚胎注入heg_e4i4_egfr2中等型模拟突变,将需要进一步的实验,以了解HEG第4外显子在心脏发育中的作用。令人惊讶的是,与heg_e3i3_egfr1注射的胚胎水肿,HEG突变体的功能。这可能是由于morpholinos能力结合产妇的成绩单将在合子突变体的影响。

Acknowledgments

我们承认他们的技术援助马布利实验室和Narie斯托勒成员,和美国心脏协会(NCRP的科学家开发格兰特0635363N)和国家心脏,肺和血液研究所(批准SCCOR射频消融HL02 - 027)提供资金。

Materials

Name Type Company Catalog Number Comments
Microinjector Tool Harvard Apparatus PLI 100
Micromanipulator Tool Narishige International MN 153
Needle Puller Tool Sutter Instrument Co. P 97
Glass Capillaries Tool World Precision Instruments, Inc. TW100 F6
Microloader Pipettes Tool Eppendorf 5242956.003
Needle Holder Tool World Precision Instruments, Inc. MPH310

DOWNLOAD MATERIALS LIST

References

  1. Mably, J. D., Mohideen, M. P. K., Burns, C. G., Chen, J., Fishman, M. C. heart of glass regulates the concentric growth of the heart in zebrafish. Curr. Biol. 13, 2138-2147 (2003).

Comments

19 Comments

  1. Request:           Respested sir/ madam                                Please can you send the protocol of DNA isolation, RAPD, and Procedure, culture media composition etc for protoplast culture. thanks.

    Reply
    Posted by: atanu d.
    April 2, 2009 - 5:09 AM
  2. You must have posted on the wrong protocol.  Ours is for zebrafish embryo microinjection.  Good luck finding the information you need!

    Reply
    Posted by: Anonymous
    April 2, 2009 - 3:24 PM
  3. My translation-blocking morpholino is carboxy-fluoresecin tagged but I don't see the fluorescence under the microscope after injection? Can the concentration be too low? How dŒs the tag work? Thank you 

    Reply
    Posted by: Anonymous
    April 17, 2009 - 10:05 PM
  4. Because the fluorescence of carboxyfluorescein dŒs not require any morpholino-related mechanism, I would verify that the injection material alone is fluorescing under your microscope. The tag is simply attached to the morpholino during manufacturing to ensure the presence of the morpholino in the injected tissue. The final concentration within the embryo would have to be extremely low to not be visible at all.    

    Reply
    Posted by: Michael S.
    April 21, 2009 - 10:47 AM
  5. Hey guys do you see any intergration events when injecting into the yolk? Would you expect to see a greater number intergration events if you injected directly into the cell? Have you seen yolk glowers when injecting reporter genes (e.g RXP/GFP) and if so what do you think is occuring here? Intergaration?

    Regards

    Giles

    Reply
    Posted by: Anonymous
    June 22, 2009 - 3:40 AM
  6. Hi Giles,
    When you inject DNA into the yolk, you get some integration, but not as much as when you inject into the cell. If you use the tol² system and inject DNA with particular flanking sequences and transposase RNA, you get a huge amount of integration. We do see yolk glowers; we're not sure whether this is due to maintenance of the plasmid as an episome, or whether integration has occured.

    Reply
    Posted by: Anonymous
    June 24, 2009 - 1:11 PM
  7. Hi, Jonathan Rosen
    First of all thanks for such an informative presentation. I really liked it a lot and I am highly impressed with the techniques which has been used by your lab.Could you send the protocol for the microinjection of zebrafish embryo to analyse gene function. Also can you please let me know which equipment you use to displace embryos from one place to another inside the petridish or in agarose gel.
    I shall be thankful to you
    Kind Regards
    Avdesh
    chaudharyavdesh@gmail.com

    Reply
    Posted by: Anonymous
    August 23, 2009 - 10:18 AM
  8. dŒs the orientation of the embryo matter prior to injection? i always thought that the needle should pierce opposite to the cell, through the yolk sac. also, can a thick, firm needle damage the embryo, as opposed to a thin unsupported one?
    thanks

    Reply
    Posted by: Anonymous
    August 12, 2010 - 9:16 PM
  9. I find it easiest to inject without going through the cell. Too thick a needle can definitely damage the embryo; if you're seeing a lot of yolk leak out after you inject, the needle is too thick.

    Reply
    Posted by: Anonymous
    August 17, 2010 - 6:06 PM
  10. Sir i would like to know a simple study using wild type embryos of the Zebra fish with out using GFP

    Reply
    Posted by: Anonymous
    August 25, 2010 - 2:20 AM
  11. Sir i would like know a simple invitro study on the wild type embryos of Zebrafish with out GFP

    Reply
    Posted by: Anonymous
    August 25, 2010 - 2:22 AM
  12. Hi, Thanks for this video! Other protocols suggest injecting DNA/RNA/morpholino dissolved in KCl or Danieau's. Do you use one of these or just miliQ water, and do you think the solution makes a difference for survival? Thanks!

    Reply
    Posted by: Anonymous
    January 24, 2012 - 10:21 AM
  13. Hi. Glad the video was helpful. Although KCl or Danieau's solution are often used, we haven't found that either improves embryo survival over just water. I believe Gene-Tools recommends dissolving the stock morpholino in water now, rather than Danieau's solution as they recommended in the past. They may have done a more thorough comparison of the different solutions and their influence on embryo development.

    Good luck with your studies.

    Reply
    Posted by: Anonymous
    January 25, 2012 - 4:26 PM
  14. I am trying micro injection of EGFP-N3 vector to standardize the injection protocol. But in all my attempts, embryos are dying. what might be the reason. I am using methylene blue as a tracker. Please help me out.

    Reply
    Posted by: Anonymous
    February 7, 2012 - 7:23 AM
  15. I have never tried it with methylene blue. I would use phenol red. I have also found that I get more lethality from DNA injection than from RNA injection. If you want to globally over-express a gene early in embryonic development, RNA is better anyway because it will go into every cell, whereas DNA will be mosaic.

    Reply
    Posted by: Anonymous
    February 7, 2012 - 10:55 AM
  16. I WAS WONDERIN IF U HAVE EXPERIENCE USING TH PARASITE BLASTOCYST HOMINIS AS THE SHELL TO INJECTED OTHER PARASITES INTO AS MEANS OF PROTEIN PRODUCTION IF SO IWAS INTERESTED IN UR FININDINDS TO COMPARE TO THE THE VERY STRANG FINDINS FROM MY PROJECT

    Reply
    Posted by: Anonymous
    March 27, 2012 - 8:07 PM
  17. How to prepare egg water

    Reply
    Posted by: Yumei C.
    July 22, 2012 - 11:25 PM
  18. http://staff.missouriwestern.edu/users/daggett/PR1D.ZF.E3.pdf

    Reply
    Posted by: Michael S.
    November 6, 2012 - 6:38 PM
  19. Hello,
    I am currently using a Hsp70I promoter in the Tol² system for generating transgenic zebrafish. Any suggestions for the best time and condition to do heat shock after microinjecton ? Is Hsp70I promoter leaky after recovery from heat shock? Your suggestions and comments will be highly appreciated.

    Reply
    Posted by: Hsu k.
    March 6, 2013 - 3:19 AM

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