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[Bio101] Injecting Zebrafish Embryos
[Bio101] 斑马鱼胚胎的显微注射技术   

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Abstract

Zebrafish is convenient for transient genetic manipulations such as morpholino-mediated gene knockdown, DNA/mRNA-based genetic overexpression. These genetic approaches do not involve the challenge of making transgenic animals, and their effect can last up to a week. This protocol describes the detailed steps of injecting to zebrafish embryos.

Materials and Reagents

  1. Glass capillary tubing (Fisher)
  2. Zebrafish mating boxes (Aquatics)
  3. Mineral oil (Sigma)

Equipment

  1. Micropipette puller (Sutter instrument)
  2. Microinjection system Pico-injector (Harvard apparatus)
  3. Microscope (Leica)
  4. Micrometer slide (American 3B Scientific)
  5. Benchtop microcentrifuge (Biotool)

Procedure

  1. The night before:
    Set up 10-15 pairs of fish, keeping the male and female separated from one another. By doing this, the fish won’t mate until you are ready for them, but they will still be amenable to mating!
    Pull the needle using the needle puller according to the manufacture’s instructions.
  2. The morning of injections:
    1. Putting fish together (in the fish room)
      1. In each of 3-5 single boxes, combine the male and female fish so that the female will begin to lay eggs (egg laying will begin in 15-45 min, and the same fish may lay eggs more than one time).
    2. Filling Needles (in the injection room)
      1. Hang needle above bench
      2. Spin RNA/cDNA/dye sample in benchtop microfuge (30 sec) so that any debris will go to bottom of the tube
      3. Using gel loading tip, remove 1-1.5 μl of sample from tube and load into back end of needle
      4. Wait for capillary action to carry sample to needle tip
  3. Meanwhile…
    1. Collect eggs from the fish you have put together, labeling the dish with the date and type of fish and take them to the injection room
    2. Add embryos to injection dish and use forceps to push them into the individual rows
    3. Using forceps, rotate embryos so that the yolk is facing the future direction of the needle
    4. Remove enough E3 so that the embryos do not float, but be careful not to let them dry out!
    5. Set this dish aside while you break the needle.
  4. Breaking Needles
    1. Turn on air supply.
    2. Turn on injection apparatus and microscope light source.
    3. Set the Pinj to 25-30 kPa (turn "inject" knob to change)- the higher the P, the better for breaking the needle
    4. Make sure the Pout and Pbal are slightly positive (0.1-0.2) (turn "balance" knob, if needed)
    5. Place a drop of oil on micrometer slide, set this aside
    6. Insert filled needle into micromanipulator
    7. Find the end of the needle under the microscope (this will likely require you to move the needle with the micromanipulator until it comes into the field of view)
    8. Adjust the microscope to the highest magnification, keeping the end of the needle in view
    9. Using forceps, clip end of needle (This takes practice to clip the needle in the correct place!)
    10. Put micrometer slide in field of view
    11. Put tip of needle in oil on slide so that it is positioned over markings. Do not let the tip of the needle touch the slide!
    12. Adjust the injection time to 6-8 msec
    13. Step on the injection pedal->INJECT into oil and look at droplet size
    14. Adjust Pinj and injection time and continue to inject into the oil until the droplet is 0.12-0.15 mm (see chart on wall to calculate drop volume).
    15. The drop size can be larger, but it is more likely to be toxic to the embryo
    16. You are now ready to inject embryos!

      **Do not leave the broken needle tip exposed to the air for too long, or the RNA/cDNA/dye may dry and clog the tip of the needle. You may put the tip of the needle into a dish with water/E3 to keep it wet, but be sure to check that the water is not going into the needle, or that your sample is not leaking into the water!

      If the tip of the needle does clog, put the needle tip in some water/E3 and push the "clear" button. This will sometimes remove the clog.

简介

斑马鱼方便用于瞬时遗传操作,例如吗啉基介导的基因敲低,基于DNA/mRNA的遗传过表达。 这些遗传方法不涉及制造转基因动物的挑战,它们的效果可持续长达一周。 这个协议描述注射到斑马鱼胚胎的详细步骤。

材料和试剂

  1. 玻璃毛细管(Fisher)
  2. 斑马鱼配套箱(Aquatics)
  3. 矿物油(Sigma)

设备

  1. 微量吸管(Sutter仪器)
  2. 显微注射系统Pico-injector(Harvard装置)
  3. 显微镜(Leica)
  4. 千分表(美国3B Scientific)
  5. 台式微量离心机(Biotool)

程序

  1. 前一天晚上:
    设置10-15对鱼,保持男性和女性彼此分离。 通过这样做,鱼不会交配,直到你准备好他们,但他们仍然会顺从交配!
    根据制造商的说明用针拔器拉出针。
  2. 注射的早晨:
    1. 把鱼放在一起(在鱼室里)
      1. 在每个3-5个单箱中,结合雄鱼和雌鱼,使雌鱼开始产卵(产卵将在15-45分钟开始,同一鱼可能产卵超过一次)。
    2. 灌装针(在注射室)
      1. 吊针上面
      2. 在台式微量离心机中旋转RNA/cDNA /染料样品(30秒),使任何碎片进入管底部
      3. 使用凝胶加载尖端,从管中取出1-1.5μl样品,并加载到针的后端
      4. 等待毛细管作用将样品带到针尖
  3. 与此同时…
    1. 从鱼类中收集鸡蛋,将鱼类的日期和类型标记在盘子上,并将它们送到注射室。
    2. 将胚胎添加到注射皿中,并使用镊子将其推入单独的行
    3. 使用镊子,旋转胚胎,使蛋黄面向未来的针头方向
    4. 删除足够的E3,以便胚胎不浮动,但要小心不要让它们干掉!
    5. 将这个菜放在一边,而你打破了针。
  4. 打破针
    1. 打开气源。
    2. 打开注射装置和显微镜光源。
    3. 将Pinj设置为25-30 kPa(转动"注射"旋钮更改) - P越高,打破针头越好
    4. 确保Pout和Pbal略微为正(0.1-0.2)(如果需要,转动"平衡"旋钮)
    5. 将一滴油放在千分尺载玻片上,将其放在一边
    6. 将填充针插入微操纵器
    7. 在显微镜下找到针的末端(这可能需要您使用显微操纵器移动针直到它进入视野)
    8. 将显微镜调整到最高放大倍数,保持针的末端在视图中
    9. 使用镊子,针的夹端(这需要练习把针夹在正确的地方!)
    10. 将测微计滑块置于视野
    11. 将针尖放在滑块上的油中,使其位于标记上方。 不要让针尖碰到滑块!
    12. 将注射时间调整为6-8毫秒
    13. 踏上注油踏板 - > INJECT进油,看看液滴大小
    14. 调整Pinj和注射时间,并继续注入油中,直到液滴为0.12-0.15 mm(参见墙上的图表来计算液滴体积)。
    15. 液滴尺寸可以更大,但更可能对胚胎有毒
    16. 您现在可以注射胚胎!

      **不要将破裂的针尖暴露在空气中过长,否则RNA/cDNA /染料可能会干燥并堵塞针头。 您可以将针头放入带有水/E3的盘子中,以保持其湿润,但请务必检查水是否不会进入针头,或者样品是否漏入水中!

      如果针的尖端堵塞,将针尖放在一些水/E3中,并按下"清除"按钮。 这有时会去除堵塞。
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:Jing, L. (2011). Injecting Zebrafish Embryos. Bio-protocol Bio101: e22. DOI: 10.21769/BioProtoc.22;
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景 枫
协和医学院
10/17/2012 11:37:14 PM Reply
Lili Jing
Department of Cell and Molecular Biology, University of Pennsylvania, USA

E3 是斑马鱼胚胎培养液。可以让其不脱水和保持活性。做显微注射的时候不要加太多E3,胚胎就不会悬浮。
配方见下。
60X E3 stock
(makes 2 liters)

34.4 g NaCl
1.52 g KCl
5.8 g CaCl2.2H2O
9.8 g MgSO4.7H2O
add double distilled water up to 2000 ml

Store in refrigerator. To dilute to 1X for rearing zebrafish, use 160 ml of stock and fill to 10 liters with ddH2O.

10/25/2012 1:34:58 PM