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In utero Electroporation of the Embryonic Mouse Retina
小鼠子宫内胚胎视网膜DNA电转移

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Abstract

This protocol is useful to manipulate gene expression in the embryonic retina and compare the result with the contralateral non electroporated retina. In addition, the electroporation of a membrane or cytoplasmic tagged GFP allows to determine the effects of gene manipulation on the outgrowth of retinal ganglion cell axons (Garcia-Frigola et al., 2007) or simply to follow axon outgrowth in mutant embryos. DNA can be directed to different quadrants of the retina (ventral or dorsally) by modifying the position of the electrodes (Petros et al., 2009; Sánchez-Arrones et al., 2013). After the procedure, embryos are left developing to the desired stage, including postnatal stages.

Keywords: Gene Expression(基因的表达), Developmental Biology(发育生物学), Visual System(视觉系统), Retinal Ganglion Cells(视网膜神经节细胞), Neuroscience(神经科学)

Materials and Reagents

  1. E13 embryos from C57BL6J pregnant mice (2 - 4 months old) were electroporated as described below. Animals were collected and handled following the Spanish (RD 223/88), European (86/609/ECC), and American (National Research Council, 1996) regulations.
  2. Plasmid Midi Kit (Roche, catalog number: 0 3143414001 ) (e.g. Genopure Plasmid Midi Kit)
  3. Fast green FCF (Sigma-Aldrich, catalog number: F7252 )
  4. Sterile water
  5. Isofluorane (Abbott Laboratories, catalog number: 880393.4 )
  6. Sterile saline (see Recipes)
  7. 10x phosphate-buffered saline (PBS) (see Recipes)

Equipment

  1. Borosilicate glass capillaries (World Precision Instruments, catalog number: 1B 100 F-4)
  2. Micropipette puller (Sutter Instrument, model: P36 )
  3. Aspirator tube assembly (Sigma-Aldrich, catalog number: A5177-5EA )
  4. Black braided silk 3/0 (Lorca Marín S.A.Ctra, catalog number: 55159 )
  5. Sterile gauze and cotton swab (Aposan, catalog number:  343160.6 )
  6. Dissecting tools (Figure 1C)
    1. Ring forceps (Karl Hammacher GmbH, catalog number: HSC 703-96 )
    2. Serrated forceps (Fine Science Tools, catalog number: 11101-09 )
    3. Forceps (Fine Science Tools, catalog number: 91150-20
    4. Fine scissor (Fine Science Tools, catalog number: 14094-11 )
  7. Dissecting microscope (Leica Microsystems, model: MZ125 ) and fiber optic light (LEICA KL 2500 LCD)
  8. Squared Electroporator (BTX The Electroporation Experts, model: ECM830 )
  9. Generator Footswitch (BTX The Electroporation Experts, model: 1250FS )
  10. Platinum plate tweezers-type electrode (Nepa Gene, model: CUY650P5 )
  11. Isofluorane vaporizer (Surgivet®, Smiths Medical, model: 100)

Procedure

  1. DNA preparation
    1. Prepare plasmids using commercial kits (e.g. Genopure Plasmid Midi Kit).
    2. Dilute DNA in sterile water to final concentration of 1 μg/μl. Add 1/10 volume of 0.1% fast green solution in water to the DNA solution. A total volume of 5-10 μl will be sufficient to electroporated eight embryos.

  2. Preparation of micropipette
    1. Pull borosilicate glass capillaries with the Micropipette puller. Recommended Puller settings are the followings: 774 heat; 100 pull and 1 time (these parameters may vary according to the Puller).
    2. Cut the tip of the pulled pipette using forceps at 1 - 2 cm (Figure 1A).
      Note: The Figure 1 shows a new panel illustrating the initial and final shape of the pipette, left to right respectively.

  3. Electroporation.
    1. For E13 embryos and the indicated electroporator, the following settings are recommended: five pulses of 38 V, 50 ms each, at 950 ms intervals. For E14 embryos the recommended setting are five pulses of 48 V, 50 ms each, at 950 ms intervals.
      Note: The generation of the RGCs occurs at E13; therefore the electroporation was done at this stage.
    2. Aspirate the DNA mixture to fill the tip of the pulled micropipette (Figure 1B). Anesthetized pregnant mice with isofluorane.
    3. Place the anesthetized mice with the abdomen up on the bench top and place a mask with isofluorane vaporizer on the nose.
    4. Wash the abdomen with ethanol or povidone iodine. Perform a longitudinal incision of about 2 cm in the mid of the abdomen using a fine scissor (Figure 1C).


      Figure 1. Shape of the glass pipette before and after pulling A. Aspirator tube and glass capillary with DNA mixture B and dissecting tools C.  

    5. Place a cotton swab soaked in pre-warmed saline to 37 °C on the abdomen to maintain the tissue moist and avoid accidental damage of the organs, grab the two horns of the uterus with two cotton swab and gently place them on top of the sterile gauze (Figure 2A).
    6. Hold the embryos with a ring forceps in one hand making sure that you have a lateral view, so that the eye can be easily identified. The use of a pigmented mouse strain facilitates the procedure (as C57BL6J). Using the other hand to hold the mouth pipette. Poke the uterus with the pipette and introduce the tip of the prefilled pipette into the eye (being careful not through the lens) and gently blow the DNA solution into the eye (Figure 2B).
    7. Place the electrodes at the left and right side of the uterus so that the embryonic head is just in the middle. The positive electrode must be placed on the side of the injected eye and covered all it (see arrowhead in Figure 2C). Apply the electric pulses and perform the same procedure with the following embryo.  
    8. Once all embryos have been electroporated, place the uterine horns back into the mouse abdominal cavity.
    9. Suture the incision and replace the pregnant dam into the cage. Let embryos develop to the desired stage (Figure 2D).
    10. The electrodes can be placed in several angles of the eye for electroporated diverse retina domains (examples showed in Figure 2E).


      Figure 2. The anesthetized pregnant mice with embryos on top of the cotton swab A. Injection into the eye of the embryo B. Position of the electrodes on the uterine walls (arrowhead indicate the eye) C.  Suture the abdominal cavity D.  The diagram indicates several positions of the electrodes. The flat mounted retinas illustrate in green different electroporated domains, central, nasal and temporal E.

Representative data

  1. This protocol is useful to determine the effects of gene manipulation during embryonic retina (Figure 3).


    Figure 3. Overview of the flat-mount retina electroporated with a GFP plasmid A. Green labelled axons in the optic nerve (ON), optic chiasm (OC) and optic tract (OT) B. Magnification of the optic disc C and electroporated retinal cells D.

Recipes

  1. Saline solution
    Dissolve 9 g NaCl in 1 L of distilled water and autoclave

Acknowledgments

We would like to thank Paola Bovolenta for her support and critical reading. We are grateful to Eloisa Herrera and Cristina Garcia-Frigola for the adapted protocol.

References

  1. Garcia-Frigola, C., Carreres, M. I., Vegar, C. and Herrera, E. (2007). Gene delivery into mouse retinal ganglion cells by in utero electroporation. BMC Dev Biol 7(1): 103.
  2. Petros, T. J., Rebsam, A. and Mason, C. A. (2009). In utero and ex vivo electroporation for gene expression in mouse retinal ganglion cells. J Vis Exp (31), e1333.
  3. Sánchez-Arrones, L., Nieto-Lopez, F., Sánchez-Camacho, C., Carreres, M. I., Herrera, E., Okada, A. and Bovolenta, P. (2013). Shh/Boc signaling is required for sustained generation of ipsilateral projecting ganglion cells in the mouse retina. J Neurosci 33(20): 8596-8607.

简介

该方案可用于操纵胚胎视网膜中的基因表达,并将结果与对侧非电穿孔视网膜进行比较。 此外,膜或细胞质标记的GFP的电穿孔允许确定基因操作对视网膜神经节细胞轴突的生长的影响(Garcia-Frigola等人,2007)或简单地遵循突变体胚胎中的轴突生长。 通过修改电极的位置,DNA可以被引导到视网膜的不同象限(腹侧或背侧)(Petros等,2009;Sánchez-Arrones等,2013)。 手术后,胚胎发育到期望的阶段,包括产后阶段。

关键字:基因的表达, 发育生物学, 视觉系统, 视网膜神经节细胞, 神经科学

材料和试剂

  1. 如下所述对来自C57BL6J怀孕小鼠(2-4个月大)的E13胚胎进行电穿孔。 按照西班牙语(RD 223/88),欧洲(86/609/ECC)和美国(国家研究委员会,1996)规定收集和处理动物。
  2. 质粒Midi试剂盒(Roche,目录号:03143414001)(例如Genopure Plasmid Midi Kit)
  3. 快速绿FCF(Sigma-Aldrich,目录号:F7252)
  4. 无菌水
  5. 异氟烷(Abbott Laboratories,目录号:880393.4)
  6. 无菌盐水(见配方)
  7. 10x磷酸盐缓冲盐水(PBS)(见配方)

设备

  1. 硼硅玻璃毛细管(World Precision Instruments,目录号:1B100F-4)
  2. 微量吸管(Sutter Instrument,型号:P36)
  3. 抽吸管组件(Sigma-Aldrich,目录号:A5177-5EA)
  4. 黑色编织丝3/0(LorcaMarínS.A.Ctra,目录号:55159)
  5. 无菌纱布和棉签(Aposan,目录号:343160.6)
  6. 解剖工具(图1C)
    1. 环钳(Karl Hammacher GmbH,目录号:HSC703-96)
    2. 锯齿钳(Fine Science Tools,目录号:11101-09)
    3. 镊子(Fine Science Tools,目录号:91150-20
    4. 精细剪刀(Fine Science Tools,目录号:14094-11)
  7. 解剖显微镜(Leica Microsystems,型号:MZ125)和光纤灯(LEICA KL 2500 LCD)
  8. 平方电穿孔仪(BTX The Electroporation Experts,型号:ECM830)
  9. 发电机脚踏开关(BTX The Electroporation Experts,型号:1250FS)
  10. 铂板镊子型电极(Nepa基因,型号:CUY650P5)
  11. 异氟烷蒸发器(Surgivet ,Smiths Medical,型号:100)

程序

  1. DNA制备
    1. 使用商业试剂盒(例如 Genopure Plasmid Midi Kit)制备质粒。
    2. 在无菌水中稀释DNA至终浓度为1μg/μl。 加 1/10体积的0.1%快速绿色水溶液加入到DNA溶液中。 一个 总体积5-10微升将足以电穿孔八 胚胎。

  2. 微量移液器的制备
    1. 用Micropipette拉杆拉动硼硅玻璃毛细管。 推荐的拔轮器设置如下:774热; 100拉和1   时间(这些参数可根据拔出器而变化)。
    2. 使用镊子在1 - 2厘米(图1A)切割吸管的尖端。
      注意:图1显示了一个新面板,分别说明了移液器的初始和最终形状,从左到右。

  3. 电穿孔。
    1. 对于E13胚胎和指示的电穿孔仪,以下设置   推荐:38 V的五个脉冲,每个50 ms,间隔950 ms。 对于E14胚胎,推荐设置为48 V,50 ms的五个脉冲 每个,间隔950 ms。
      注意:RGC的产生发生在E13; 因此在此阶段进行电穿孔。
    2. 吸出DNA混合物,以填充提取的微量移液管的尖端 (图1B)。 用异氟烷麻醉怀孕小鼠。
    3. 将麻醉的小鼠腹部放在工作台上,并用异氟烷蒸发器在鼻子上放置一个面具。
    4. 用乙醇或聚维酮碘洗腹部。 执行a 纵向切口约2厘米,在腹部中部使用a 精细剪刀(图1C)。


      图1.玻璃移液管的形状 前后A。抽吸管和玻璃毛细管与DNA 混合物B和解剖工具C. 

    5. 放置棉签 浸泡在预温的盐水中37°C保持在腹部 组织湿润,避免器官意外损伤,抢两者 角的子宫与两个棉签,轻轻地将它们放在上面   无菌纱布(图2A)。
    6. 用一个戒指握住胚胎 镊子在一只手,确保你有一个侧视图,所以 可以容易地识别眼睛。 使用色素小鼠品系 方便程序(如C57BL6J)。 用另一只手握住   嘴移液器。 用移液管摘取子宫并引入尖端 的预填充移液器进入眼睛(小心不要通过 透镜),并将DNA溶液轻轻地吹入眼睛(图2B)。
    7. 将电极放置在子宫的左右两侧 胚胎头只是在中间。 正极必须   放置在注射的眼睛的侧面并覆盖所有它(见 箭头在图2C)。 应用电脉冲并执行相同操作 程序与以下胚胎。  
    8. 一旦所有的胚胎都被电穿孔,将子宫角放回小鼠腹腔。
    9. 缝合切口,并将怀孕的水坝更换为笼子。 让胚胎发育到所需的阶段(图2D)。
    10. 电极可以放置在眼睛的几个角度 电穿孔的不同视网膜结构域(实施例显示于图2E)

      图   2.麻醉的怀孕小鼠胚胎在棉花上面 拭子A。注射到胚胎的眼睛B.的位置 子宫壁上的电极(箭头指示眼睛)C. 缝合 腹腔D. 该图表示几个位置 电极。 平面视网膜以绿色不同的方式示出 电穿孔结构域,中枢,鼻和颞E。

代表数据

  1. 这个协议是有用的,以确定在胚胎视网膜基因操作的影响(图3)。


    图3.用GFP质粒A电穿孔的平顶视网膜概述。视神经(ON),视交叉(OC)和视神经中的绿色标记的轴突 (OT)B.视盘C和电穿孔的视网膜细胞D.的放大率

食谱

  1. 盐溶液
    将9g NaCl溶于1L蒸馏水和高压釜中

致谢

我们要感谢Paola Bovolenta的支持和批判性阅读。我们感谢Eloisa Herrera和Cristina Garcia-Frigola修改的协议。

参考文献

  1. Garcia-Frigola,C.,Carreres,M.I.,Vegar,C.and Herrera,E。(2007)。 通过子宫内 电穿孔将基因递送到小鼠视网膜神经节细胞中。 a> BMC Dev Biol 7(1):103.
  2. Petros,T.J.,Rebsam,A.and Mason,C.A。(2009)。 在子宫内和离体电穿孔小鼠视网膜神经节细胞中的基因表达。 (31),e1333。
  3. Sánchez-Arrones,L.,Nieto-Lopez,F.,Sánchez-Camacho,C.,Carreres,M.I。,Herrera,E.,Okada,A.and Bovolenta,P.(2013)。 Shh/Boc信号传导是在小鼠视网膜中持续产生同侧突出的神经节细胞所必需的。 a> J Neurosci 33(20):8596-8607。
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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Nieto-Lopez, F. and Sanchez-Arrones, L. (2014). In utero Electroporation of the Embryonic Mouse Retina. Bio-protocol 4(19): e1255. DOI: 10.21769/BioProtoc.1255.
  2. Sánchez-Arrones, L., Nieto-Lopez, F., Sánchez-Camacho, C., Carreres, M. I., Herrera, E., Okada, A. and Bovolenta, P. (2013). Shh/Boc signaling is required for sustained generation of ipsilateral projecting ganglion cells in the mouse retina. J Neurosci 33(20): 8596-8607.
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