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Medaka-microinjection with an Upright Microscope
在正置显微镜下进行青鳉显微注射   

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参见作者原研究论文

本实验方案简略版
Zoological Letters
2017

Abstract

We described a simple method for microinjecting DNA/RNA/Protein solutions into medaka eggs under an upright microscope. Medaka is an excellent vertebrate model for reverse genetics, because of its daily spawning, short generation time, and egg transparency. These features enable us to efficiently perform functional genomic analyses of transgenic or genome edited fish. This protocol contains the initial steps necessary to create various types of genetically modified fish.

Keywords: Fish (鱼), Medaka (青鳉), Microinjection (显微注射), Upright microscope (正置显微镜), Genome editing (基因组编辑), Transgenesis (转基因)

Background

Medaka is a small freshwater teleost species and has desirable features for use as a vertebrate model, including daily spawning, complete genome sequence, and availability of a number of useful strains. Moreover, the transparency of its eggs enables precise introduction of DNA/RNA/Protein solutions into the cytoplasm of the eggs by microinjection. Microinjection is important for the functional genomic analysis; for example, DNA microinjection is an indispensable technique for generating transgenic fish, and helps us to understand the functions of introduced genes in vivo (Ozato et al., 1986). Furthermore, microinjection of CRISPR/Cas system is able to achieve targeted genome editings such as knockout and knock-in approaches (Ansai and Kinoshita, 2014; Murakami et al., 2017a).

Both of a stereoscopic microscope and an upright microscope can be used for microinjection into fish embryos. Although microinjection with an upright microscope requires attachment of some specific instruments including a micromanipulator and an injection needle holder to the microscope, this method may achieve more precise introduction of solutions into the cytoplasm than that with a stereoscopic microscope due to its high resolution property. In this paper, we described the outline of a microinjection method with an upright microscope. This method can be used together with other protocols, such as those for the generation of gene knockout or gene knock-in lines (Ansai and Kinoshita, 2014; Murakami et al., 2017b).

Materials and Reagents

  1. Spatula (SANSYO, catalog number: 93-4159 or equivalents)
  2. Cutter (SANSYO, catalog number: 97-0417 or equivalents)
  3. Glass capillary (NARISHIGE, catalog number: GD-1 )
  4. Egg holder plate (Figure 1)
  5. Silicone sealant; repair materials made of silicone (Konishi, catalog number: 04890 or equivalents)
  6. Micro-loading tip (Eppendorf, catalog number: 5242956003 )
  7. Disposable syringe (TERUMO, catalog number: 4987350396457 or equivalents)
  8. Disposable glass pipette (SANSYO, catalog number: 73-0102 or equivalents)
  9. Needle (Hamilton, catalog number: KF731 )
  10. Plastic dish (35 mm in diameter) (Corning, Falcon®, catalog number: 351008 )
  11. Male fish
  12. Female fish
  13. Mineral oil (NACALAI TESQUE, catalog number: 23306-84 )
  14. Methylene blue (NACALAI TESQUE, catalog number: 37125-95 )
  15. Phenol red (NACALAI TESQUE, catalog number: 26807-21 )
  16. Sodium chloride (NaCl)
  17. Potassium chloride (KCl)
  18. Calcium chloride dihydrate (CaCl2·2H2O)
  19. Magnesium sulfate heptahydrate (MgSO4·7H2O)
  20. 17 mM methylene blue (see Recipes)
  21. 100x embryo culture medium (see Recipes)
  22. Embryo culture medium (see Recipes)

Equipment

  1. Forceps (DUMONT, model: 91-3869 or equivalents)
  2. Micropipette puller (NARISHIGE, model: PC-100 )
  3. Microinjection system (NARISHIGE, models: M-152 and IM-6 )
  4. Stereoscopic microscope (Leica Microsystems, model: WILD MZ8 )
  5. Upright microscope modified to attach micro-manipulator system (Nikon, model: XF-PH-21 )

Procedure

  1. Acclimatization of fish for spawning
    Prepare 10-15 tanks with one pair of male and female adult fish, and acclimate each fish to a new environment for a week.
    Note: Fish are kept under a 14/10-h light/dark cycle at 26 °C to promote spawning.
  2. Preparation of an egg holder
    1. Prepare a commonly used slide glass (Figure 1A).
    2. Adhere glass chips on the slide glass to make a channel for setting a silicone sealant (Figure 1B).
    3. Fill the channel (enclosed by the red line in Figure 1C) of the egg holder plate with the silicone sealant, and flatten its surface with a spatula.
      Note: It will take overnight to completely solidify the sealant.
    4. Cut out the hardened sealant in parallel with a cutter, and make a channel (enclosed by the blue line in Figure 1D) of about 1.0 mm in width and about 2.0 mm in depth for fitting eggs.
      Note: Unlike an agarose plate, the silicone plate can be used permanently.


      Figure 1. Egg holder plate

  3. Preparation of microinjection in the evening before microinjection
    1. Pull the glass capillary by the micropipette puller to make several microinjection needles (according to the manufacturer’s instruction) (Kinoshita et al., 2009).
    2. Set the partition in the fish tank to separate a female from a male preventing from spawning.
  4. Microinjection procedure in the morning on the day of experiment
    1. Remove the partition in the fish tank to make the pairs begin spawning.
      Note: It will take a few minutes to finish spawning.
    2. Transfer the fertilized eggs to a plastic dish with a disposable glass pipette, and remove the long attaching filaments on the surface of the chorion using forceps under the stereoscopic microscope (Video 1).

      Video 1. The preparation of fertilized eggs for microinjection (Steps 3a-3b, 4a-4b)

    3. Load the solution of DNA/RNA or protein in the glass capillary needle using the micro-loading tip.
    4. Fill the glass capillary needle with mineral oils completely to the end using a disposable syringe fitted with the Hamilton needle.
    5. Apply pressure to the injector by turning the handle of the injector, and fill completely a line of tubing between injector and injection needle holder with mineral oils, to remove air in the line.
      Note: This work helps to prevent the end of the microinjection needle from clogging with air bubbles.
    6. Fix the glass capillary needle in the needle holder for injection, and attach the holder to the micromanipulator (Video 2).

      Video 2. The loading of the solution in the glass capillary needle, and the attachment of the needle to the micro-manipulator (Steps 4c-4f)

    7. Transfer the egg to the egg holder with a glass pipette, and place it on the stereoscopic microscope stage.
    8. Rotate the eggs with forceps to direct its cytoplasm toward the injection needle and fix the egg in the channel of the egg holder on the stereoscopic microscope stage (magnification 20x).
      Note: Although it may be difficult to find the cytoplasm just after fertilization, it can become easy to find it gradually, because it progressively accumulates toward the animal pole in the egg; it is easy to find the cytoplasm by observing the opposite side of the droplets that accumulate toward the vegetal pole.
    9. Place the holder on the upright microscope stage and place the cytoplasm at the center of the view by adjusting the stage of microscope (magnification 40x).
      Note: If the cytoplasm is not properly oriented toward the injection needle, it is necessary to adjust its position again under the stereoscopic microscope.
    10. Open the end of the needle by touching the end gently to the surface of the egg holder under the microscope.
    11. Bring the needle close to the cytoplasm by operating the manipulator.
    12. Just before to touch the egg envelope surface, apply pressure to the injector by turning the handle of the injector, and then penetrate the end of the glass needle into the cytoplasm through the egg envelope and cell membrane.
      Note: When the end is inserted into the cytoplasm, the injected solution automatically starts to flow into cytoplasm. If the solution doesn’t flow into the cytoplasm, apply additional pressure to the injector.
    13. Wait a second until outflow of the solution reaches the appropriate volume, and quickly withdraw the needle (Video 3).
      Note: The injection of the solution into the eggs should be stopped when the cytoplasm are expanded slightly, because over dose of the solution, especially DNA, may result in death or developmental disturbance of the eggs. The volume of the solution is not constant because the microinjection system cannot accurately control the injected volume.

      Video 3. The orientation of the cytoplasm toward the injection needle on the egg holder, and the microinjection of the solution into the egg (Steps 4g-4m)

    14. Stop giving pressure after the end of the glass needle is removed completely from the egg.
    15. Transfer the injected eggs from the egg holder to a plastic dish filled with embryo culture medium (see Recipes).
      Notes: To establish a knockout strain harboring the targeted mutagenesis, it is sufficient to inject the solution into at least 50 eggs. On the other hand, to establish a knock-in strain harboring the inserted gene, it is necessary to inject the solution into at least 100 eggs because of low efficiency in knock-in performance. It is recommended to inject the one- or two-cell stage eggs to prevent from reducing the efficiency of the injection (Video 4).
    16. Incubate the injected eggs in embryo culture medium at around 28 °C.

      Video 4. The reference movie of the microinjection into the two-cell stage eggs. Unlike the egg holder with a silicone channel described in this protocol, the egg is fitted a channel between glass plates in this movie. The first injection is performed with the one-cell stage egg, while the second injection is performed with the two-cell stage egg. It is recommended that the solution be injected into the one-cell stage eggs, because the two-cell stage eggs are required to be injected into each cytoplasm as shown in the second injection in this video. It should be noted that the microinjection into the eggs later than two-cell stage strongly reduces the efficiency of the injection, and leads to a high degree of mosaicism in the eggs. The injected solution gives a red color with a phenol red, so that can be visible in the egg in this movie, however, it is unnecessary for the practical experiment.

Recipes

  1. 17 mM methylene blue (methylene blue stock solution)
    Add 50 ml of purified water to 3.2 x 102 mg of methylene blue
    Note: Can be stored at RT for several months.
  2. 100x embryo culture medium
    1.7 M NaCl
    40 mM KCl
    27 mM CaCl2·2H2O
    65 mM MgSO4·7H2O
    Note: Can be stored at RT for several months.
  3. Embryo culture medium
    After dilution of 100x embryo culture medium with purified water, add 1 drop of the methylene blue solution per 1 L of the diluted solution

Acknowledgments

This protocol was adapted from our previous works (Murakami et al., 2017a). The authors declare no conflicts of interest or competing interests with this manuscript.

References

  1. Ansai, S. and Kinoshita, M. (2014). Targeted mutagenesis using CRISPR/Cas system in medaka. Biol Open 3(5): 362-371.
  2. Kinoshita, M., Murata, K., Naruse, K. and Tanaka, M. (2009). Medaka: Biology, management, and experimental protocols. Wiley-Blackwell 280-281.
  3. Murakami, Y., Ansai, S., Yonemura, A. and Kinoshita, M. (2017a). An efficient system for homology-dependent targeted gene integration in medaka (Oryzias latipes). Zoological Lett 3: 10.
  4. Murakami, Y., Ansai, S., Yonemura, A. and Kinoshita, M. (2017b). Genotyping-free selection of double allelic gene edited medaka using two different fluorescent proteins. Bio-protocol e2665.
  5. Ozato, K., Kondoh, H., Inohara, H., Iwamatsu, T., Wakamatsu, Y. and Okada, T. S. (1986). Production of transgenic fish: introduction and expression of chicken delta-crystallin gene in medaka embryos. Cell Differ 19(4): 237-244.

简介

我们已经描述了在直立显微镜下将DNA / RNA /蛋白质溶液显微注射到青eggs蛋中的简单方法。 青aka是一种优秀的反向遗传学脊椎动物模型,因为其每日产卵,短代时间和卵子透明度。 这些特征使我们能够有效地进行转基因或基因组编辑的鱼的功能基因组分析。 该协议包含了创建各种转基因鱼所需的初始步骤。

【背景】青aka是一种小型的淡水硬骨鱼物种,具有脊椎动物模型的特征,包括每日产卵,完整的基因组序列和一些有用菌株的可用性。此外,其蛋的透明度是通过显微注射将DNA / RNA /蛋白质溶液精确地引入蛋的细胞质中。显微注射对于功能基因组分析是重要的;例如,DNA显微注射是产生转基因鱼的不可缺少的技术,并且有助于理解体内基因的功能(Ozato等人,1986)。此外,CRISPR / Cas系统的显微注射能够实现靶向基因组编辑,如敲除和敲入方法(Ansai和Kinoshita,2014,Murakami等人,2017a)。

立体显微镜和直立显微镜均可用于鱼胚胎显微注射。虽然直立显微镜的显微注射正处于不稳定的状态。在本文中,我们描述了一个直立显微镜显微注射法的轮廓。该方法可以与其他方案一起使用,例如用于产生基因敲除或基因敲入系的方案(Ansai和Kinoshita,2014,Murakami等人,2017b)。

关键字:鱼, 青鳉, 显微注射, 正置显微镜, 基因组编辑, 转基因

材料和试剂

  1. 刮刀(SANSYO,产品目录号:93-4159或等同物)。
  2. 切割机(SANSYO,目录编号:97-0417或同等学历)
  3. 玻璃毛细管(NARISHED,目录号:GD-1)
  4. 蛋架板(图1)
  5. 硅酮密封胶;修理由硅树脂(Konishi,目录号:04890或等同)
    材料
  6. 微加载尖端(Eppendorf,目录号:5242956003)
  7. 一次性注射器(TERUMO,产品目录号:4987350396457或同等产品)
  8. 一次性玻璃吸管(SANSYO,产品目录号:73-0102或同等产品)。
  9. 针(汉密尔顿,目录号:KF731)
  10. 塑料盘(直径35毫米)(Corning,Falcon ,产品目录号:351008)
  11. 雄鱼
  12. 女鱼
  13. 矿物油(NACALAI TESQUE,目录号:23306-84)
  14. 亚甲蓝(NACALAI TESQUE,目录号:37125-95)
  15. 酚红(NACALAI TESQUE,目录号:26807-21)。
  16. 氯化钠(NaCl)
  17. 氯化钾(KCl)
  18. 氯化钙二水合物(CaCl 2)2H 2 O)。
  19. 七水硫酸镁(MgSO 4)7H 2 O)
  20. 17毫米亚甲蓝(见食谱)
  21. 100倍胚胎培养基(见食谱)
  22. 胚胎培养基(见食谱)

设备

  1. 镊子(DUMONT,型号:91-3869或等价物)
  2. 微管拉拔器(NARISHIGE,型号:PC-100)
  3. 微注射系统(NARISHIGE,型号:M-152和IM-6)
  4. 立体显微镜(徕卡显微系统,型号:WILD MZ8)
  5. 正置显微镜修改为附加微型机械手系统(尼康,型号:XF-PH-21)

程序

  1. 鱼类适应产卵。
    准备10-15坦克与一对男性和女性的成年鱼,适应每个鱼到一个新的环境一个星期。
    注:鱼在26°C下保持在14/10-h光照/黑暗周期以促进产卵。
  2. 准备一个蛋架
    1. 准备一个常用的幻灯片(图1A)。
    2. 在玻片上粘附玻璃芯片以形成硅酮密封剂的通道(图1B)。
    3. 将蛋架板的通道(图1C中的红色线所示)填充硅酮密封胶,并用抹刀将其表面弄平。
      注意:密封剂需要过夜才能完全固化。
    4. 用切刀平行切出硬化后的密封胶,制作宽度约1.0mm,深度约2.0mm的通道(图1D中的蓝色线),以便装配鸡蛋。
      评级:与琼脂糖板不同,硅胶板可永久使用。


      图1.蛋架板

  3. 在显微注射前的晚上制备显微注射
    1. (根据制造商的说明)(Kinoshita等人,2009)。通过微量吸管拉动玻璃毛细管以制造多个显微注射针。

    2. 在鱼缸中设置分隔区,与水槽中的人分开
  4. 实验当天早上的显微注射程序
    1. 去除鱼缸内的隔板,使鱼缸开始产卵。
      注意:完成产卵需要几分钟的时间。
    2. 将受精卵转移到一次性玻璃吸管中,在立体显微镜下使用镊子取出绒毛膜表面的长连接丝(视频1)。

      视频1


    3. 使用微量加样尖将DNA / RNA或蛋白质溶液加载到玻璃毛细管针头中。

    4. 用装有Hamilton针头的一次性注射器填充玻璃毛细管针,用矿物油完成
    5. 通过转动注射器手柄对注射器施加压力,并用注射器和注射针之间的一系列管线用矿物油完全填充,以除去管线中的空气。
      注意:这项工作有助于防止显微注射针的末端堵塞气泡。
    6. 将玻璃毛细管针头固定在注射针头上,并将其固定在显微操作器上(视频2)。

      视频2


    7. 用玻璃吸管将蛋转移到蛋架上,然后放在立体显微镜台上。
    8. 用镊子转动卵细胞,将其细胞质导入立体显微镜载物台上的卵孔(放大20倍)。
      注:尽管受精后可能很难找到细胞质,但它可能逐渐变得越来越可能,因为它逐渐积累在卵中的动物极;通过观察积累在植物杆上的液滴的另一侧很容易找到细胞质。
    9. 显微镜的阶段被放大了40倍。
      注意:如果细胞质没有正确朝向注射针,则需要在立体显微镜下再次调整其位置。
    10. 打开拿着玻璃末端的针头。
    11. 通过操作机械手将针头靠近细胞质。
    12. 就在接触蛋壳表面之前,通过转动注射器手柄对注射器施加压力,然后通过蛋壳和细胞膜将玻璃针的末端穿入细胞质。
      注:当末端插入细胞质时,注射的溶液自动开始流入细胞质。如果溶液不能流入细胞质,则向注射器施加额外的压力。
    13. 等待一会,直到流量达到适当的音量,然后迅速撤回针(视频3)。
      在细胞质扩大的情况下,由于溶液过量,特别是DNA过量,可能会导致鸡蛋死亡或发育障碍。由于显微注射系统,溶液的体积不是恒定的。

      视频3


    14. 在玻璃针的末端完全从蛋中取出后停止施压
    15. 将注射的鸡蛋从蛋架上转移到装满胚胎培养基的塑料培养皿中(见食谱)。
      注:为了建立一个具有定向诱变的敲除菌株,将溶液注入至少50个鸡蛋就足够了。由于低效率的敲击性能,100个鸡蛋。建议注射一个或两个细胞阶段的蛋,以降低注射效率(视频4)。

    16. 在28°C左右的胚胎培养基中孵育注射的鸡蛋
      s视频4

食谱

  1. 17毫米亚甲蓝(亚甲蓝储备液)。
    加入50毫升纯化水至3.2×10-2毫克亚甲蓝
    评级:可以在室温下保存数月。
  2. 100x胚胎培养基
    1.7M NaCl。
    40 mM KCl。
    27mM CaCl 2 2 / 2H 2 O 2 65mM MgSO 4 .7H 2。
    评级:可以在室温下保存数月。
  3. 胚胎培养基
    用纯化水稀释100倍胚胎培养基后,每1L稀释液加1滴亚甲蓝溶液。

确认

这个协议是从我们以前的作品(Murakami等人,2017a)改编的。作者声明与本手稿没有利益冲突或利益冲突。

参考

  1. Ansai,S.和Kinoshita,M.(2014)。 在青鳉使用CRISPR / CAS系统靶向诱变。 生物化学打开
  2. Kinoshita,M.,Murata,K.,Naruse,K。和Tanaka,M。(2009)。 青鳉:生物学,管理和实验方案的代码的 WILEY-布莱克韦尔 280-281。
  3. Murakami,Y.,Ansai,S.,Yonemura,A.和Kinoshita,M.(2017a)。 在青鳉用于同源依赖性靶向基因整合的有效系统(青鳉)。 Zoological Lett 3:10。
  4. 村上,Y.,安塞,S.,米村,A。和木下,M。(2017b)。双等位基因编辑青鳉的用两种不同的荧光蛋白游离基因分型选择。 生物协议 e2665。
  5. Ozato,K.,Kondoh,H.,井之原快,H.,岩松,T.若松,Y。和冈田,T. S.(1986)。转基因鱼的生产:介绍和在青鳉胚胎鸡Δ-晶状基因的表达 Cell Differ 19(4):237-244。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Murakami, Y. and Kinoshita, M. (2018). Medaka-microinjection with an Upright Microscope. Bio-protocol 8(3): e2716. DOI: 10.21769/BioProtoc.2716.
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