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In utero Electroporation of Mouse Cerebellar Purkinje Cells
子宫内电穿孔法检测小鼠小脑蒲肯野细胞   

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Abstract

In utero electroporation (IUE) of mouse cerebellar Purkinje cells allows high expression levels of transgenes without toxicity (Nishiyama et al., 2012). This technique is suitable for co-transfection of multiple plasmid genes. Therefore, it is useful to express various sets of genes such as drug-inducible Cre/loxP constructs and CRISPR/Cas9 genome editing constructs (Takeo et al., 2015). Murine Purkinje cells arise from subventricular zone of fourth ventricle at embryonic day (E) 10-12. IUE at E11.5 into fourth ventricle results the most efficient transfection into Purkinje cells.

Keywords: Purkinje cell(浦肯野细胞), In utero electroporation(子宫穿孔), Cerebellum(小脑)

Materials and Reagents

  1. Glass capillary [(Sutter Instrument Company, catalog number: BF100-50-10 ) for microinjector or (World Precision Instruments, catalog number: 1B150F-3 ) for mouth pipette]
  2. Sterile gauze (KAWAMOTO CORPORATION, catalog number: 7164 )
  3. Surgical tape (3M, catalog number: 1527SP-0 )
  4. Surgical scalpel blade (Swann Morton, model: No.11 )
  5. Suture needle (Alfresa pharma, catalog number: HT1605 NA75-KF2 )
  6. E11.5 (or E10~12) pregnant mouse
  7. QIAFilter plasmid maxi kit (QIAGEN, catalog number: 12263 )
  8. Fast green (Sigma-Aldrich, catalog number: F7258 )
  9. Sodium pentobarbital (Somnopentyl) (KYORITSU SEIYAKU CORPORATION, catalog number: 4992945014418 )
  10. Ethanol
  11. Ritodrine hydrochloride (Sigma-Aldrich, catalog number: R0758 )
  12. (Optional) EEG conductive paste (NIHON KOHDEN CORPORATION, catalog number: Z181JE )
  13. HEPES (Sigma-Aldrich, catalog number: H4034 )
  14. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  15. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333 )
  16. Disodium hydrogenphosphate 12-water (Na2HPO4・12H2O) (Wako Pure Chemical Industries, catalog number: 196-02835 )
  17. Potassium dihydrogen phosphate (KH2PO4) (Wako Pure Chemical Industries, catalog number: 169-04245 )
  18. HEPES-buffered saline (HBS) (see Recipes)
  19. Phosphate-buffered saline (PBS) (see Recipes)

Equipment

  1. Micropipette puller (Sutter Instrument Company, model: P-87 )
  2. Microscope with 40x objective (Nikon Instruments Inc., model: Eclipse E100 )
  3. Scissors, fine forceps and ring forceps
  4. Square wave electroporator (Nepa Gene Co., model: CUY21SC )
  5. Tweezers-type electrodes (Nepa Gene Co., model: CUY650P3 )
  6. Microinjector (Eppendorf AG, model: 5242 ) or mouth pipette
  7. LED light source with flexible light guide (Kenko Tokina, model: KTX-20L )
  8. Reflex 7 mm wound clip applier (Cellpoint Scientific Inc., catalog number: 204-1000 )

Procedure

  1. DNA preparation
    1. Purify plasmids using the QIAGEN plasmid maxi kit. Dissolve plasmids into 1x HBS to make plasmid stock solutions. 4 mg/ml or higher concentration of plasmid stock is recommended. Plasmid stock solutions can be stored at 4 °C for several months, though fresh plasmids bring good results.
    2. Dilute plasmids into 30~60 μl 1x HBS at a final concentration of 2-5 mg/ml. Add 1/100 volume of 1% fast green solution.

  2. Preparation of glass micropipettes for DNA injection
    1. Pull glass capillaries with the micropipette puller.
    2. Cut the tip of a pulled pipette at 30-45 μm in diameter (Figure 1B) using a surgical scalpel blade. The cut edge must be sharpened, not a clean cut shape (Figure 1A-B). Check the cut edge under microscope. Make 3~5 glass micropipettes.


      Figure 1. Glass micropipette with sharp cut edge. A. Representative image of the tip of glass micropipette (Scale bar, 10 μm). B. Schematic of the cut edge.

  3. Electroporation
    1. Anesthetize the pregnant mouse via an intraperitoneal injection of sodium pentobarbital (50-60 μg/g body weight). All procedures related to animal care and treatment were performed in accordance with the guidelines set by the Animal Resource Committee of Keio University.
    2. Fill the plasmid solution into glass micropipettes.
    3. Place the anesthetized mouse with the abdomen upside and fix the tail and limbs gently on the operation board with a surgical tape. Wipe the skin of the abdomen with 70% ethanol.
    4. Incise with scissors the midline of the abdomen skin, and then the abdomen wall.
    5. Place sterile gauze around the incision. Wet the gauze with sterile warm PBS. Apply 100-200 μl ritodrine hydrochloride solution (0.1 mg/ml) into intraperitoneal space to relax uterine muscle.
    6. Pinch the abdomen wall with a fine forceps at one side of the incision, insert a ring forceps into abdomen and carefully pull out one of the two uterine horns. Drop sterile warm PBS throughout the exposed uterine horn. Keep the uterus wet with PBS during surgery.
    7. Inject DNA solution into the fourth ventricle using the mouth pipette or microinjector (Video 1). To visualize the fourth ventricle, illuminate an embryo with the LED light from the side or underneath. Hold the embryo between the light and your fingers. Gently push the embryo so that the fourth ventricle is fixed in superficial portion of the uterus (Figure 2A-B). Insert the glass micropipette into the fourth ventricle through the uterus wall, and inject DNA by air pressure until the rostral part of fourth ventricle is filled with DNA (Figure 2C). For an embryo, take 5 seconds or longer to inject DNA solution.


      Figure 2. Schematics of DNA injection into the fourth ventricle and electroporation. A. Schematic of embryo held through uterus; B. Light illumination visualize the forth ventricle; C. Schematic of fourth ventricle filled with injected DNA solution (blue color); D. Schematic of electroporation.

    8. Wet the uterus and electrodes with warm PBS. Attach the plus end of electrodes onto the rostral edge of 4th ventricle (Video 2). Minus end is located on the back or the neck. If it is difficult to contact electrodes tightly on the uterus, put a small amount of EEG paste on the electrodes to fill the gaps between electrodes and uterus. Apply electrical pulses (33 V, with a duration of 30 ms, at intervals of 970 ms per pulse, 5 cycles). 45~50 mA current is delivered (Figure 2D). For an embryo at E10-12, apply two to four sets of electrical pulses. Too much electrical pulses might be harmful.
    9. Move on to the next embryo and repeat steps C7-8. When finished with one of the two uterine horns, gently put them back into abdomen using your fingers.
    10. Pull out the other uterine horn, just like step C7. If the uterine wall is not relaxed, drop 50 μl ritodrine hydrochloride (0.1 mg/ml) directly on the exposed uterine horn. Do steps C7-9. It is important to finish steps 6-10 within 40 min. Prolonged surgery can lead to death of the embryos or the pregnant mouse.
    11. After electroporated uterus are returned into abdomen, suture the wound of abdomen wall.
    12. Close the skin with wound clips.
    13. Recover the mouse under warm (~37 °C) condition. Return the mouse to the home cage.
    14. Electroporated mice can be analyzed at embryonic or postnatal ages (Figure 3). In the cerebellar vermis which was electroporated at E11.5, 95% of the transfected cells are Purkinje cell.


      Figure 3. Representative result of IUE. E11.5 mouse was electroporated with EGFP-encoding plasmids and analyzed at P33. A. Mosaic labeling of Purkinje cells by EGFP is observed in right half of the cerebellum (Scale bar, 2 mm). In this example, the plus end of the electrodes was placed on the right side of the subventricular zone during electroporation; B. Sagittal slice of the electroporated cerebellum shown in A (Scale bar, 1 mm).

Representative data

Video 1. DNA injection into the fourth ventricle


Video 2. Electroporation

Recipes

  1. HEPES-buffered saline (HBS)
    21 M HEPES
    137 mM NaCl
    5 mM KCl
    0.7 mM Na2HPO4
  2. Phosphate-buffered saline (PBS) [Sterile and pre-warmed (37 °C)]
    137 mM NaCl
    2.7 mM KCl
    8.1 mM Na2HPO4
    1.47 mM KH2PO4

Acknowledgments

This protocol was adapted from Nishiyama et al. (2012). This work was supported by Japan Society for the Promotion of Science.

References

  1. Nishiyama, J., Hayashi, Y., Nomura, T., Miura, E., Kakegawa, W. and Yuzaki, M. (2012). Selective and regulated gene expression in murine Purkinje cells by in utero electroporation. Eur J Neurosci 36(7): 2867-2876.
  2. Takeo, Y. H., Kakegawa, W., Miura, E. and Yuzaki, M. (2015). RORalpha regulates multiple aspects of dendrite development in cerebellar purkinje cells in vivo. J Neurosci 35(36): 12518-12534.

简介

在小鼠小脑浦肯野细胞的子宫电穿孔(IUE)中,允许没有毒性的转基因的高表达水平(Nishiyama等,2012)。 该技术适用于多重质粒基因的共转染。 因此,表达各种基因如药物诱导型Cre / loxP构建体和CRISPR / Cas9基因组编辑构建体是有用的(Takeo等,2015)。 小鼠浦肯野细胞起因于胚胎期(E)10-12的第四脑室下室。 IUE在E11.5进入第四脑室导致最有效的转染到浦肯野细胞。

关键字:浦肯野细胞, 子宫穿孔, 小脑

材料和试剂

  1. 玻璃毛细管[用于微量注射器的(Sutter Instrument Company,目录号:BF100-50-10)或用于口腔移液管的(World Precision Instruments,目录号:1B150F-3)
  2. 无菌纱布(KAWAMOTO CORPORATION,目录号:7164)
  3. 手术带(3M,目录号:1527SP-0)
  4. 外科手术刀刀片(Swann Morton,型号:No.11)
  5. 缝合针(Alfresa pharma,目录号:HT1605NA75-KF2)
  6. E11.5(或E10〜12)怀孕小鼠
  7. QIAFilter质粒大量试剂盒(QIAGEN,目录号:12263)
  8. Fast green(Sigma-Aldrich,目录号:F7258)
  9. 戊巴比妥钠(三新戊基)(KYORITSU SEIYAKU CORPORATION,目录号:4992945014418)
  10. 乙醇
  11. 盐酸利托君(Sigma-Aldrich,目录号:R0758)
  12. (可选)EEG导电糊(NIHON KOHDEN CORPORATION,目录号:Z181JE)
  13. HEPES(Sigma-Aldrich,目录号:H4034)
  14. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  15. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333)
  16. 磷酸氢二钠12水(Na 2 HPO 4·12H 2 O)(Wako Pure Chemical Industries,目录号:196-02835) br />
  17. 磷酸二氢钾(KH 2 PO 4)(Wako Pure Chemical Industries,目录号:169-04245)
  18. HEPES缓冲盐水(HBS)(见Recipes)
  19. 磷酸盐缓冲盐水(PBS)(见Recipes)

设备

  1. Micropipette puller(Sutter Instrument Company,型号:P-87)
  2. 具有40x物镜的显微镜(Nikon Instruments Inc.,型号:Eclipse E100)
  3. 剪刀,细钳和环钳
  4. 方波电穿孔仪(Nepa Gene Co.,型号:CUY21SC)
  5. 镊子型电极(Nepa Gene Co.,型号:CUY650P3)
  6. 微量注射器(Eppendorf AG,型号:5242)或口腔移液器
  7. 带柔性光导的LED光源(Kenko Tokina,型号:KTX-20L)
  8. 反射7mm伤口夹子施放器(Cellpoint Scientific Inc.,目录号:204-1000)

程序

  1. DNA制备
    1. 使用QIAGEN质粒maxi试剂盒纯化质粒。 将质粒溶解到1×HBS中以制备质粒储备溶液。 建议4 mg/ml或更高浓度的质粒储备液。 质粒储备溶液可以在4℃保存几个月,但新鲜质粒带来好的结果
    2. 将质粒稀释到30〜60μl1×HBS中,终浓度为2-5 mg/ml。 加入1/100体积的1%快速绿色溶液
  2. 制备用于DNA注射的玻璃微量移液管
    1. 用微量吸液管拉出玻璃毛细管。
    2. 使用外科手术刀刀片切割直径为30-45μm的拉拔移液管的尖端(图1B)。 切割边缘必须锐化,而不是干净的切割形状(图1A-B)。 在显微镜下检查切边。 制作3〜5个玻璃微量移液器

      图1.具有锐利边缘的玻璃微量移液管。A.玻璃微量移液管尖端的代表图像(比例尺,10μm)。 B.切割边的示意图。

  3. 电穿孔
    1. 通过腹膜内注射戊巴比妥钠(50-60μg/g体重)麻醉怀孕的小鼠。 所有与动物护理和治疗相关的程序都按照庆应义io大学动物资源委员会规定的指南进行
    2. 将质粒溶液填充到玻璃微量移液管中
    3. 将麻醉的鼠标与腹部上面,用手术胶带轻轻地固定在操作板上的尾巴和四肢。用70%乙醇擦拭腹部皮肤。
    4. 用剪刀剪下腹部皮肤的中线,然后腹壁。
    5. 在切口周围放置无菌纱布。用无菌温PBS浸湿纱布。将100-200μl盐酸利托君盐溶液(0.1 mg/ml)加入腹腔内以放松子宫肌肉。
    6. 在切口的一侧用细镊子夹住腹壁,将环镊子插入腹部,并小心地拉出两个子宫角中的一个。滴无菌温暖PBS整个暴露的子宫角。手术期间用PBS保持子宫湿润。
    7. 使用嘴移液管或微量注射器(视频1)注入DNA溶液到第四脑室。为了可视化第四脑室,用侧面或下面的LED灯照亮胚胎。抓住胚胎之间的光和你的手指。轻轻推动胚胎,使第四脑室固定在子宫的表面部分(图2A-B)。将玻璃微量吸管通过子宫壁插入第四脑室,并通过空气压力注射DNA,直到第四脑室的嘴部填充DNA(图2C)。对于胚胎,注射DNA溶液需要5秒或更长时间

      图2. DNA注射到第四脑室和电穿孔的示意图。 A。通过子宫保持胚胎的示意图; B.光照显示第四脑室;第四脑室充满注射的DNA溶液(蓝色)的示意图;电穿孔示意图。

    8. 用温热的PBS湿润子宫和电极。将电极的正端连接到第4脑室的顶端边缘(视频2)。负端位于背部或颈部。如果难以在子宫上紧密接触电极,则在电极上放入少量EEG糊剂以填充电极和子宫之间的间隙。施加电脉冲(33 V,持续时间为30 ms,每个脉冲间隔970 ms,5个周期)。输送45〜50mA电流(图2D)。对于E10-12处的胚胎,应用两到四组电脉冲。太多的电脉冲可能是有害的。
    9. 移动到下一个胚胎,并重复步骤C7-8。当完成了两个子宫角之一,用你的手指轻轻地把它们放回腹部。
    10. 拉出另一个子宫角,就像步骤C7一样。如果子宫壁不放松,直接滴露50微升盐酸利托君盐(0.1毫克/毫升)暴露的子宫角。执行步骤C7-9。重要的是在40分钟内完成步骤6-10。长期手术可导致胚胎或怀孕老鼠死亡。
    11. 电穿孔子宫返回腹部后,缝合腹壁的伤口
    12. 用伤口夹闭合皮肤。
    13. 在温暖(〜37°C)条件下恢复小鼠。 将鼠标返回到主笼。
    14. 可以在胚胎或出生后年龄分析电穿孔小鼠(图3)。 在E11.5电穿孔的小脑蚓部中,95%的转染细胞是浦肯野细胞。


      图3. IUE的代表性结果。 E11.5小鼠用编码EGFP的质粒电穿孔并在P33分析。 A。 在小脑的右半部分(比例尺,2mm)中观察到通过EGFP的Purkinje细胞的镶嵌标记。 在该实施例中,在电穿孔期间将电极的正端放置在脑室下区的右侧; B. A(比例尺,1mm)中显示的电穿孔小脑的矢状切片。

代表数据

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视频1. DNA注射到第四脑室
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视频2.电穿孔
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食谱

  1. HEPES缓冲盐水(HBS)
    21 M HEPES
    137 mM NaCl 5 mM KCl
    0.7mM Na 2 HPO 4
  2. 磷酸盐缓冲盐水(PBS)[无菌并预热(37℃)]
    137 mM NaCl 2.7 mM KCl
    8.1mM Na 2 HPO 4
    1.47mM KH 2 PO 4 sub/

致谢

该协议改编自Nishiyama em al。(2012)。 这项工作是由日本促进科学协会支持。

参考文献

  1. Nishiyama,J.,Hayashi,Y.,Nomura,T.,Miura,E.,Kakegawa,W.and Yuzaki,M。(2012)。 通过子宫内电穿孔在小鼠浦肯野细胞中选择性和受调节的基因表达。 Eur J Neurosci 36(7):2867-2876。
  2. Takeo,Y.H.,Kakegawa,W.,Miura,E。和Yuzaki,M.(2015)。 RORalpha调节小脑浦肯野细胞中树突发育的多个方面 体内。 J Neurosci 35(36):12518-12534。
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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。
Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Takeo, Y. H. (2016). In utero Electroporation of Mouse Cerebellar Purkinje Cells. Bio-protocol 6(11): e1835. DOI: 10.21769/BioProtoc.1835.
  2. Takeo, Y. H., Kakegawa, W., Miura, E. and Yuzaki, M. (2015). RORalpha regulates multiple aspects of dendrite development in cerebellar purkinje cells in vivo. J Neurosci 35(36): 12518-12534.
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