采用斑马鱼做为模型的一个突出优点是对它们进行遗传操作非常方便，对它们的早期胚胎进行显微注射即可。通过这种技术，含有遗传物质或者沉默表达框架的的溶液会被注入囊胚细胞：它们是位于新受精卵卵黄上方的胚胎细胞。将遗传物质导入细胞质一般是通过直接注射囊胚细胞，或者先注入卵黄再通过细胞质的自然流动导入囊胚细胞。成功的遗传学操作会带来一定数量的胚胎表型，用于阐明其发育的遗传学机制。
本短片介绍了斑马鱼胚胎的显微注射。我们将先回顾该技术的基本工具，包括注射装置和显微注射器，它可以通过空气压力脉冲来控制液体的流动。然后会阐述重要的准备工作，例如灌制琼脂板，用以在注射过程中稳定胚胎，以及校正显微注射装置。接下来我们会展示显微注射过程包括什么时候在什么部位注射等实验技巧。最后，我们会讨论显微注射技术的应用，包括通过mRNA注射获得基因过量表达，通过注射反义吗啉环寡核苷酸来沉默基因，以及利于经过特殊基因工程操作的质粒DNA产生转基因斑马鱼。
斑马鱼胚胎的显微注射让研究者能将溶液直接注入到发育中的动物体内，从而研究基因的功能和动态发育。用于注射的胚胎通常选1－4 细胞期的胚胎。由于在这个胚胎发育早期没有膜分隔细胞和卵黄，注入一个细胞或卵黄的溶液将会均匀地扩散到整个有机体。
通过显微注射不同类型的物质，我们可以让合成蛋白的基因表达或者关闭。本短片将演示玻璃毛细移液管的备，胚胎的收集，显微注射过程，以及该技术目前在实验室中的一些应用。
首先，让我们来看看显微注射系统的主要构成：立体显微镜，显微注射器，持针器，和显微操作器。
显微注射器能利用可调节气压脉冲注入精确的体积 。持针器固定在注射过程中使用的注射针，并将它与注射器的空气管相连。
持针器通常装在显微操作器上。该装置使得研究人员能在注射过程中对注射针的位置进行细小和精确的调节。注射器还和一个脚踏板相连，使得研究人员在使用他们双手的同时还可以激活压力脉冲将实验材料注射入胚胎。最后，立体显微镜能让研究人员在显微注射过程中看到胚胎并对注射针所在的位置聚焦。
显微注射开始前，先要准备好玻璃针。显微注射用的玻璃针必须保持大小一致以便精确地注射实验材料。毛细管拉针器能保证每次制备的注射针又细又尖。毛细管拉针器在加热玻璃毛细管的同时牵拉毛细管，从而得到两根注射针。
在显微镜下用镊子将针头的尖端切断，既要保证定量液体的转移，又要使针头足够锋利以刺穿斑马鱼的胚胎。
酚红是用来帮助我们观察注射过程的一种染料，将酚红与要注射的溶液混合，就可以追踪溶液是否成功地注入了胚胎。
可通过针头的毛细管虹吸作用来上样注射液，也可使用微量加样注射器进行上样。一但注射针上样完毕，就可以把它插入显微操作器的持针器上固定。
注射针准备好后，要调节显微注射器的压力和时间设置来校准每个注射针，以保证每个胚胎中注射的液体容量一致。在立体显微镜下，将少量的溶液注入到玻片上的一滴矿物油中。通过测量液滴的大小来计算注射针释放的溶液容量。接下来可进一步调节显微注射器的压力并重复以上步骤直至液滴的大小满足要求。
一旦玻璃针备好并完成校准，就可以准备注射用的胚胎了。
胚胎必须小心放置并在整个注射过程中用显微注射室固定。显微注射室的制备方法是，先将模子置于培养皿，然后将熔化的琼脂糖倒入培养皿中使其凝固。
一旦琼脂糖凝固，去除模子，就可以倒入胚胎培养液了。可以用一根移液管将胚胎排列在模子的沟槽中。或者将他们沿着显微镜玻片的边缘排列，这样胚胎就被放置好准备显微注射了。
放置好胚胎 ，就可以开始注射了。
胚胎注射的部位可以是卵黄或者细胞质。卵黄注射较为容易，无需复杂的注射技术，而细胞质注射较难，但产量较高。
卵黄注射时，首先通过显微操作器移动玻璃针使其依次刺穿绒毛膜和卵黄。然后踩脚踏板将玻璃针内的液体注入卵黄。胞质环流和扩散会使注入的溶液流进细胞。
细胞质注射需要对胚胎进行小心定位，以使得细胞质能被有效注入。
注射完毕后，将胚胎转移到一个新的培养皿中置于摄氏28.5度培养。要经常检查以去掉死亡的胚胎。
如何确定注射是否成功，可以通过分析胚胎的总体外观，荧光标记物的存在与否，以及做基因型鉴定检查它们的基因组有没有发生变化来判断。
您已经知道如何注射斑马鱼胚胎了，那么现在我们来看看科学家是如何运用这个技术来了解基因功能的。
首先，科学家可以注射进合成的mRNA来过量表达某些基因并通过观察表型来确定这些基因的功能。同样的技术也可以用来表达某些蛋白来强化分子事件，比如在发育过程中发生的细胞骨架重排。
其次，我们可以通过注射反义吗啉环寡核苷酸morpholinos来关闭基因表达。反义吗啉环寡核苷酸Morpholinos 是稳定的合成的核酸类似物，它可以设计来结合特殊的mRNA序列来阻断蛋白翻译，这是通过标准的核酸碱基配对来实现的。与上面相反，这种方法导致mRNA蛋白产物的减少。研究者可以通过观察该蛋白减少时对发育的改变来理解某种特定基因在发育中所扮演的角色。
显微注射可以用来将外源DNA掺入到斑马鱼中。通过注射含有DNA修饰酶识别位点的序列，科学家们可以有效的产生修饰了基因组的转基因鱼，这意味着外源基因可以传递给下一代。根据序列设计的不同，基因能被控制在特定的组织中或特定的发育阶段表达。
您刚才观看的是JoVE关于早期斑马鱼胚胎显微注射的介绍。本短片介绍了显微注射的准备， 如何制备显微注射针，如何准备显微注射的胚胎， 显微注射技术以及显微注射的一些应用。感谢观看。
Microinjection of zebrafish embryos allows researchers to deliver solutions directly into the developing animal, in order to study gene function and developmental dynamics. Embryos from the 1 to 4-cell stage are frequently used for injection. Because there are no membranes separating the cells and the yolk at this early stage, solutions injected in either one cell or the yolk will evenly spread throughout the organism. By using microinjection, protein production genes can be expressed, or turned off, depending on the type of material injected. This video will demonstrate pipette preparation, embryo collection, the microinjection procedure, and discuss some of the ways this technique is used in labs today.
First, let’s cover the major components of the microinjection system: The stereoscope, microinjector, pipette holder, and micromanipulator.
The microinjector delivers precise volume through pressure pulses of air, which can be adjusted by the user. The pipette holder secures the pipette for use during the procedure and connects it to the airline of the injector. Typically, the pipette holder is placed in a micromanipulator. This instrument allows the researcher to make small and accurate adjustments to the pipette location during the injection procedure. A foot pedal is connected to the injector and allows the researcher to maintain use of their hands while simultaneously activating the pressure pulse for injection material delivery. Finally, the stereoscope allows the researcher to see the embryos and focus on the location of the pipette during the microinjection procedure.
Before microinjection can begin, glass needles must be prepared. Microinjection needles must be of a consistent size to allow for precise delivery of injection materials. A pipette puller helps ensure fine and sharp pipettes are prepared every time. The puller heats a glass capillary tube while exerting force that pulls on the tube, resulting in two pipette needles.
Under the microscope, using forceps, the tip is cut off in a manner that allows for a consistent volume of liquid to be delivered, yet maintains the sharpness of the needle for piercing the zebrafish embryo.
Phenol red, a dye used to help visualize the injection procedure, can be mixed with the injection solution in order to track successful injection into the embryo.
Needles can be loaded with injection solution from the tip side via capillary action, or they can be backfilled with a microloader syringe. Once the needle is loaded for injection, it can be inserted into the pipette holder on the micromanipulator.
After the injection needles are ready, the microinjector pressure and time settings are adjusted in order to calibrate each needle, which will ensure a consistent volume is delivered to each embryo. Under the stereoscope, a small amount of solution is injected into a drop of mineral oil on a slide. The size of the droplet is measured and the volume that the needle disperses can be calculated. Subsequently, the pressure of the microinjector can be further adjusted and the process repeated until a droplet of the desired size is regularly obtained.
Once needles are prepared and calibrated, embryos are obtained and arranged for injection.
Embryos must be positioned carefully and held stationary during injection by a microinjection chamber. To create a microinjection chamber, molds are placed in a petri dish and molten agarose is poured into the dish and allowed to harden. Once it has solidified, the mold is removed and the embryo medium is poured on top. The embryos can be lined up in troughs that were created by the mold using a transfer pipette. One alternative to arranging the embryos in agarose is to line them up along the edge of a microscope slide, so they are positioned in a column for microinjection
Once they are in position, the embryos are ready to be injected.
Embryos can be injected either into the yolk or cell cytoplasm. Injection into the yolk is simpler and requires less sophisticated injection technique, while injection into the cytoplasm is more difficult, but yields more robust results. To inject into the yolk, use the micromanipulator to move the needle so that it pierces the chorion and then the yolk. The foot pedal is then tapped to cause the contents of the needle to be expelled into yolk. Cytoplasmic flow and diffusion allows the injection solution to flow into the cell.
Injection into the cytoplasm requires careful positioning of the embryo, so that the cytoplasm can be effectively targeted.
Following injection, embryos are transferred to a new dish and incubated at 28.5 °C. They are frequently checked to remove dead embryos.
To determine the success of the injection, embryos can be analyzed based on their overall appearance, the presence of a fluorescent marker, and by looking for changes in their genome using genotyping.
Now that you understand how to inject zebrafish embryos, let’s look how scientists can use this technique to understand the function of genes.
First, scientists can inject synthesized mRNA to overexpress certain genes and determine their function by observing phenotype. This same technique can also be used to express proteins that highlight molecular events, such as the cytoskeletal rearrangements that occur during development.
Second, genes can be turned off by the injection of morpholinos. Morpholinos are stable synthetic nucleic acid analogs that can be designed to bind to specific mRNA sequences by standard nucleic acid base pairing, and block translation. In turn, this leads to loss of the protein produced by that mRNA. This effect allows researchers to understand the role of a particular gene in development by seeing how development is altered in its absence.
Injection can be used to incorporate foreign DNA into the zebrafish. By injecting sequences containing recognition sites for DNA modifying enzymes, scientists can efficiently generate “transgenic” fish with modified genomes, meaning that foreign genes will be passed on to future generations. Depending on the sequence design, gene expression can be confined to specific tissues or specific developmental timepoints.
You’ve just watched JoVE’s introduction to microinjection of early zebrafish embryos. This video has introduced the microinjection setup, demonstrated how to prepare microinjection needles, shown how to prepare embryos for microinjection, perform the microinjection technique, and some applications of microinjection. As always, thanks for watching!
