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Labeling of Precursor Granule Cells in the Cerebellum by ex vivo Electroporation
通过体外电穿孔转染对小脑颗粒细胞前体进行标记   

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参见作者原研究论文

本实验方案简略版
Neuron
Nov 2012

Abstract

This protocol will be useful to introduce the genes of interest into the cerebellar granule cells at early stages of development. Since the granule cell precursors are localized in the external granule layer before migration, DNA plasmids can be specifically incorporated into the granule cells by injecting DNA solution into the cerebellar fissures followed by application of electric pulses. This technique can be performed prior to the preparation of either dissociated or organotypic culture, which can be used to study the molecular mechanisms of cell migration, axon elongation and synapstogenesis during development.

Keywords: Electroporation (电穿孔), Cerebellum (小脑), Granule cell (颗粒细胞), Neuron (神经元)

Materials and Reagents

  1. Plasmid Maxi Kit (QIAGEN)
  2. 10x HEPES buffered saline (HBS)
  3. Fast green FCF (Sigma-Aldrich, catalog number: F7252 )
  4. 1x Phosphate buffered saline (PBS)
  5. 10x HBS (see Recipes)

Equipment

  1. Capillary glass (Harvard Apparatus, catalog number: 30-0066 )
  2. Micropipette puller P-97/ IVF (Sutter Instrument)
  3. Aspirator Tube Assembly (Drummond, catalog number: 2-000-000 )
  4. Square pulse electroporator and a foot switch (Nepagene, catalog number: CUY21 )
  5. Platinum plate tweezers-type electrode (Protech International, catalog number: CUY650-P5 )
  6. Dissecting microscope
  7. Dissecting tools: Forceps and some fine scissors
  8. 10 cm-petri dish

Procedure

A.   Preparation of micropipettes for DNA injection

  1. Pull the capillary glass using the puller with a single pull. Adjust the puller setting to make the capillary taper approximately 10-20 mm. When using P-97, start with the following setting: Heat equal of the Ramp value, Pull 0, Vel 40 and Time 200.
  2. Cut out 1/3 – 1/2 of the tips of the pulled pipettes using the forceps.

B.  DNA preparation

  1. Prepare plasmids using the Plasmid Maxi Kit and elute DNA with distilled water. The concentration of the DNA should be 4-5 μg/μl for storage.
  2. For injection, the final concentration of DNA should be 2 μg/μl. Add 1/10 volume of 10x HBS, 1/10 volume 1% fast green (final 0.1%) and water to the DNA solution to adjust the concentration.

C. Electroporation

  1. Attach a micropipette to an aspirator tube assembly and draw 10 μl of DNA -solution into the pipette (Figure 1).


    Figure 1. An aspirator tube and a glass capillary

  2. Remove a whole-brain from a mouse (postnatal days 7-9) and put it into an ice-cold PBS in a 10-cm petri dish.
  3. Remove the forebrain and the brain stem using the forceps. If the cerebellum is planned to be used for organotypic culture, the meninges should not be removed in order to keep the cellular layers intact. If the cerebellum is to be further processed for dissociated culture, the meninges should be removed to avoid glial cell overgrowth.
  4. Observe the cerebellum under a dissecting microscope. Inject the DNA solution (2 -3 μl/fissure) into the cerebellar fissures by mouth pipetting (Figure 2). Inject into at least 2 or 3 fissures, starting from those in the vermis. If high transfection efficiency is required, inject into as many fissures as possible.


    Figure 2. Injection of plasmid DNA solution into cerebellar fissures

  5. Place the forceps-type electrodes so that cerebellum is in between the two electrode planes. The two planes should be positioned parallel to the fissures of the cerebellar vermis. They should be held about 3-5 mm apart from the cerebellum surface (Figure 3).


    Figure 3. Placement of electrodes

  6. Apply electric pulses; 99.9 V, ON 50 msec, OFF 450 msec, 5 pulses. Adjust the voltage value depending on the outcome. When the transfection efficiency is too low, the voltage should be increased up 120 V. When the transfection efficacy is too high or when the number of dying cells is too high, the voltage setting should be decreased.
  7. The cerebellum can be further processed for either organotypic slice or dissociated cultures (please see reference 1 for the images showing the labeled granule cells).

Recipes

  1. 10x HBS

    10x conc. (mM)
    g/100 ml H2O
    NaCl
    1,400
    8.2
    Na2HPO4.2H2O
    15
    0.3
    HEPES
    500
    11.9

Acknowledgments

This protocol is adapted from Yang et al. (2004) and Ito-Ishida et al. (2012).

References

  1. Ito-Ishida, A., Miyazaki, T., Miura, E., Matsuda, K., Watanabe, M., Yuzaki, M. and Okabe, S. (2012). Presynaptically released Cbln1 induces dynamic axonal structural changes by interacting with GluD2 during cerebellar synapse formation. Neuron 76(3): 549-564.
  2. Yang, Z. J., Appleby, V. J., Coyle, B., Chan, W. I., Tahmaseb, M., Wigmore, P. M. and Scotting, P. J. (2004). Novel strategy to study gene expression and function in developing cerebellar granule cells. J Neurosci Methods 132(2): 149-160.

简介

该方案可用于在发育的早期阶段将感兴趣的基因引入小脑颗粒细胞。 由于颗粒细胞前体在迁移前位于外部颗粒层中,因此通过将DNA溶液注射到小脑裂隙中,然后施加电脉冲,可将DNA质粒特异性地掺入颗粒细胞中。 该技术可以在制备解离或器官型培养物之前进行,其可以用于研究在发育期间细胞迁移,轴突伸长和突触发生的分子机制。

关键字:电穿孔, 小脑, 颗粒细胞, 神经元

材料和试剂

  1. 质粒Maxi Kit(QIAGEN)
  2. 10x HEPES缓冲盐水(HBS)
  3. 快速绿FCF(Sigma-Aldrich,目录号:F7252)
  4. 1x磷酸盐缓冲盐水(PBS)
  5. 10x HBS(请参阅配方)

设备

  1. 毛细管玻璃(Harvard Apparatus,目录号:30-0066)
  2. 微量移液器P-97/IVF(Sutter仪器)
  3. 抽吸管组件(Drummond,目录号:2-000-000)
  4. 方形脉冲电穿孔仪和脚踏开关(Nepagene,目录号:CUY21)
  5. 铂板镊子型电极(Protech International,目录号:CUY650-P5)
  6. 解剖显微镜
  7. 解剖工具:镊子和一些精美的剪刀
  8. 10厘米培养皿

程序

A.  制备用于DNA注射的微量移液管

  1. 用单拉拉拔器拉出毛细管玻璃。 调整拉杆设置,使毛细管锥度约为10-20 mm。 使用P-97时,从以下设置开始:热量等于斜坡值,拉0,Vel 40和时间200.
  2. 使用镊子切出被吸移的吸管尖端的1/3 - 1/2

B.  DNA制备

  1. 使用质粒Maxi试剂盒准备质粒,用蒸馏水洗脱DNA。 DNA的浓度应为4-5μg/μl用于储存。
  2. 对于注射,DNA的终浓度应为2μg/μl。 向DNA溶液中加入1/10体积的10x HBS,1/10体积1%快速绿(最终0.1%)和水以调节浓度。

C.电穿孔

  1. 将微量吸管连接到吸气管组件,并吸取10微升的DNA溶液到吸量管(图1)。


    图1.吸气管和玻璃毛细管

  2. 从小鼠(出生后7-9天)删除全脑,并将其放入冰冷的PBS在10厘米培养皿。
  3. 使用镊子去除前脑和脑干。 如果小脑计划用于器官型培养,则不应该除去脑膜以保持细胞层完整。 如果小脑进一步加工解离培养,应该去除脑膜以避免胶质细胞过度生长。
  4. 在解剖显微镜下观察小脑。通过嘴移液(图2)注入DNA溶液(2-3微升/裂解)到小脑裂隙。注入至少2或3个裂隙,从在真皮中的那些。如果需要高转染效率,注入尽可能多的裂缝

    图2.将质粒DNA溶液注射到小脑裂隙

  5. 放置镊子型电极,使小脑在两个电极平面之间。两个平面应平行于小脑蚓部的裂缝定位。它们应该保持距离小脑表面约3-5mm(图3)

    图3.电极的放置

  6. 施加电脉冲; 99.9V,ON 50msec,OFF 450msec,5脉冲。根据结果​​调整电压值。当转染效率太低时,电压应升高120 V. 转染效率太高或死亡细胞数量太高时,电压设置应降低
  7. 小脑可以进一步处理器官型切片或解离的培养物(对于显示标记的颗粒细胞的图像,请参见参考文献1)。

食谱

  1. 10x HBS

    10x浓。 (mM)
    g/100ml H 2 O 2 /
    NaCl
    1,400
    8.2
    2
    15
    0.3
    HEPES
    500
    11.9

致谢

该协议改编自Yang等人(2004)和Ito-Ishida等人(2012)。

参考文献

  1. Ito-Ishida,A.,Miyazaki,T.,Miura,E.,Matsuda,K.,Watanabe,M.,Yuzaki,M.and Okabe,S。(2012)。 突触前释放的Cbln1通过与小鼠突触形成过程中GluD2相互作用诱导动态轴突结构变化。 神经元 76(3):549-564。
  2. Yang,Z.J.,Appleby,V.J.,Coyle,B.,Chan,W.I.,Tahmaseb,M.,Wigmore,P.M.and Scotting,P.J。(2004)。 研究小脑颗粒细胞基因表达和功能的新策略。 J Neurosci Methods 132(2):149-160。
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引用:Ito-Ishida, A. (2013). Labeling of Precursor Granule Cells in the Cerebellum by ex vivo Electroporation. Bio-protocol 3(12): e778. DOI: 10.21769/BioProtoc.778.
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Mariana Silveira
Instituto de Biofísica Carlos Chagas Filho, UFRJ.
Dear Dr Ito-Ishida,
I am interested in obtaining details on your ex vivo electroporation protocol for cerebellar progenitors.
I would really appreciate if you could answer some of my doubts. Please feel free to send me any other detail you consider relevant.
- we had a hard time to build the capillary device you use. Have you ever tried to use Hamilton syringe? Do you believe this could work?
- How do you restrict the diffusion to occur after the use of the capillary?
Sorry to bother you and thank you in advance for your help.
Regards,
Mariana Silveira
silveira@biof.ufrj.br
2/7/2014 10:04:27 AM Reply
Aya Ito-Ishida
Department of Cellular Neurobiology, Graduate School of Medicine, University of Tokyo, Japan

Dear Dr. Silveira,
1. I have never used Hamilton syringe for this purpose myself, but I think it is possible. You may need to practice several times so that the plasmid-containing liquid will go into the fissures slowly. It may be easier to try it with another person's help: one person could hold the cerebellum and another person could manipulate the syringe.
2. When injecting the plasmid-containing liquid, the tip of the capillary has to be within the cerebellar fissures. The two cerebellar lobules will make walls on both sides of the capillarly tip and prevent the liquid from diffusing. In addition, the electric pulses should be applied right after the injection.
_ I hope this helps. Please let me know if you have any further questions.
Best wishes,
Aya Ito-Ishida

2/7/2014 8:29:14 PM