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Ex utero Electroporation into Mouse Embryonic Neocortex
小鼠胚胎新皮质的宫外DNA电转移   

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Abstract

This technique allows highly efficient and reproducible transfer of DNA/RNA into the embryonic neocortex of rodents across multiple ages. Ex utero electroporation compliments the more technically difficult in utero electroporation technique by maximizing the number of embryos for available for a given experiment, as well as increasing the variety of constructs used in each experiment, thereby helping to reduce their overall numbers. Ex utero electroporation followed by short term organotypic slice culture of embryonic brain sections allows immediate access to multiple slices for choosing optimal ones for live-cell imaging experiments, and characterization of various NSC manipulations in the intact stem cell niche. (see also “In utero Electroporation of Mouse Embryo Brains” (Ge, 2012); “Organotypic Slice Culture of Embryonic Brain Sections” (Calderon de Anda, 2013). Additionally, ex utero electroporated neocortices can be used for in vitro primary cell cultures with further dissection, dissociation into single cells, and plating on cover slips or in multi-well dishes according to standard techniques: note this procedure can be performed immediately after electroporation, prior to the onset of ectopic gene expression, or after overnight slice culturing to collect just the region of electroporated cells.

Materials and Reagents

  1. Timed embryonic mice embryos: A wide range of developmental timepoints work very well for this procedure, from E10-18.5. Note however that earlier embryos (E10-12.5) are very delicate and may require modified vibratome slicing techniques and/or manual cutting with a micro-knife, which can be embedded in collagen 3D-gels (to help preserve tissue structure) and cultured on Millicell inserts, or floated as whole brain explants overnight in a multi-well plate on a nutator/rotator. Likewise, older embryos E17-18 will have more developed skin and craniums, requiring a bit more force to inject DNA solution into the ventricles, which are becoming smaller relative to the size of the animal.
  2. DNA prepared at (≥1 μg/μl): Qiagen Maxi Prep kit (QIAGEN, catalog number: 12663 ) or EndoFree Plasmid Kit (QIAGEN, catalog number: 12362 )
  3. Fast green FCF (Sigma-Aldrich, catalog number: F7252 )
  4. HBSS (Life Technologies, catalog number: 14025-126 )
  5. 4% Low-melting point agarose (in sterile 1x PBS or 1x HBSS)
  6. Low-Melting point agarose (GeneMate Sieve GQA Agarose, catalog number: E-3112 )
  7. Ice
  8. Kimwipe

Equipment

  1. 35 mm dishes
  2. Multi-well cell culture dish (12 to 24 wells)
  3. Electroporator (BTX The Electroporation Experts, model: ECM 830 Square Wave Electroporation System)
  4. 3 mm-paddle Platinum Tweezertrodes (BTX The Electroporation Experts, model: 45-0487 )
  5. Footswitch for ECM 830 (BTX The Electroporation Experts, model: 1250FS )
  6. Mouth pipette (Sigma-Aldrich, catalog number: P0799-1PAK )
  7. Micropipettes (Borosilicate with filament O.D.: 1 mm, I.D.: 0.78 mm, 10 cm length) (SUTTER INSTRUMENT, catalog number: BF100-78-10 )
  8. Micropipette puller P-97/IVF (SUTTER INSTRUMENT)
  9. Dissecting stereoscope with flexible lighting
  10. Dumont forceps (#5 and #55) (Fine Science Tools)
  11. Disposable plastic transfer pipettes
  12. Sterile scalpel
  13. Fluorescent dissecting stereoscope for checking transfections
  14. Digital camera (if necessary) (OLYMPUS, model: MVX10 )

Procedure

  1. Collect timed embryonic mice embryos according to approved protocols at your institution. Hold embryos in ice-cold HBSS filled dish until ready for use.
  2. Pour melted agarose into a 35 mm dish ~1/2 to 2/3 full, and let solidify over ice.
  3. Cut a small trapezoid out of the center of the dish with a scapel, discard (Figure 1A).
  4. Fill this dish with HBSS to almost full (Figure 1A).
  5. Transfer single embryo into the well of this dish using cut transfer pipette or forceps (Figure 1A).
  6. Fill micropipette with DNA solution mixed with enough Fast Green to just visualize.
  7. Position embryo in well, and hold in place with forceps while micro-injecting ~0.2-0.5 μl DNA into the lateral ventricle. Flip embryo over, reposition, and microinject DNA into the contralateral ventricle.
    Note: Each ventricle should be visualized due to colored solution (Figure 1A).
  8. Hold tweezertrodes over the head in manner to target current into the dorsal neocortex.
    Note: The targeted position is highly dependent on the orientation of the tweezertrodes. For example, holding the positive electrode over the dorsal neocortex on one side of the brain means that the negative electrode will be placed in a more ventral/diagonal position, slightly under the contralateral jaw. This is most easily accomplished by using another pair of forceps in the other hand (i.e. left), while holding tweezertrodes in the other hand (i.e. right).
  9. Electroporate embryo using the remote footswitch: 3–5 pulses, 35 mV, 100 ms intervals.
  10. Flip embryo over, reposition tweezertrodes accordingly, and re-electroporate the contralateral neocortex using the same procedure.
    Notes:
    1. Varying electrode placement can target other specific regions, including the hippocampus/Dentate, ventral telencephalon, as well as other regions that contain a lumen.
    2. During electroporation, it is best to not contact the embryo directly.
  11. Transfer electroporated embryo into multi-well dish placed on ice with HBSS to recover using cut transfer pipette/forceps.
  12. Clean electrodes after each embryo electroporation by wiping with dry Kimwipe.
  13. Repeat procedure until all embryos have been electroporated.
    Note: Multiple different DNA constructs and combinations can be used within this ex utero procedure.
  14. Dissect the brain from each embryo, and transfer into ice-cold HBSS in new multi-well plate until all brains have been removed.
    Note: In some cases the meninges could be removed to help vibratome sectioning depending on the actual age of embryos, younger embryos are more delicate (E10-12).
  15. Embed brains in 4% low-melting agarose and prepare for vibratome sectioning and organotypic brain slice culture as described in (see Organotypic Slice Culture of Embryonic Brain Sections (Calderon de Anda, 2013).
  16. Slices can be visualized the following day with a fluorescent stereoscope. With practice, multiple slices should contain fluorescent cells in each neocortex. For example, Figure 1B shows typical example of RFP expressing plasmid targeted in to each neocortex in a wild-type embryo. Figure 1C,D is example of RFP targeted into each neocortex of a transgenic GFP expressing animal (Nelson et al., 2013).


    Figure 1. Ex utero electroporation in embryonic mouse neocortex. A. Schematic of technique; B. Example of double electroporated RFP plasmid targeted to each neocortex in wild type E14.5 mice cultured overnight; C. Example of double electroporated RFP plasmid targeted to each neocortex in transgenic Tbr2GFP E14.5 neocortex; D. High power view. Black and red represent negative and positive electrodes, respectively, so keep in mind their orientation since it controls direction and targeting: green represents the Fast Green colored DNA solution that fills the lateral ventricles, revealing their shape, and indicates a successful fill.
    Note: Images were acquired with a fluorescent stereoscope equipped with GFP/RFP filter sets and digital camera (Olympus, MVX10)

Acknowledgments

This protocol was adapted from Nelson et al. (2013), and was supported by NIH Grants R21 MH087070 and RO1 MH080766-S to R.F.H.

References

  1. Calderon de Anda, F. (2013). Organotypic slice culture of embryonic brain sections. Bio-protocol, 3(3): http://www.bio-protocol.org/wenzhang.aspx?id=327.
  2. Ge, X. (2012). In utero electroporation of mouse embryo brains. Bio-protocol 2(13): e231.
  3. Nelson, B. R., Hodge, R. D., Bedogni, F. and Hevner, R. F. (2013). Dynamic interactions between intermediate neurogenic progenitors and radial glia in embryonic mouse neocortex: potential role in Dll1-Notch signaling. J Neurosci 33(21): 9122-9139.

简介

这种技术允许高效和可重复地将DNA / RNA转移到多个年龄的啮齿动物的胚胎新皮质中。通过最大化可用于给定实验的胚胎数量,以及增加每个实验中使用的构建体的多样性,从而有助于减少它们的总体数量,子宫内电穿孔在技术上更难以满足子宫电穿孔技术。胚胎电穿孔之后进行短期器官切片培养,可以立即进入多个切片,选择用于活细胞成像实验的最佳切片,以及在完整干细胞生态位中对各种NSC操作的表征。 (参见“子宫内电穿孔小鼠胚胎脑”(Ge,2012);“胚胎脑切片的器官切片培养”(Calderon de Anda,2013)。此外,子宫内电穿孔新生儿可用于体外原代细胞培养进一步解剖,分离成单个细胞,并根据标准技术在盖玻片或多孔培养皿中铺板:注意,该程序可以在电穿孔后立即进行,在异位基因表达发生之前,或在隔膜培养至仅收集电穿孔细胞的区域。

材料和试剂

  1. 定时胚胎小鼠胚胎:广泛的发展时间点对于该程序工作非常好,从E10-18.5。注意,早期的胚胎(E10-12.5)是非常精致,可能需要修改vibratome切片技术和/或手动切割与微刀,可以嵌入胶原3D凝胶(以帮助保存组织结构)和培养Millicell插入或作为全脑外植体漂浮在多孔板中的营养/旋转器上过夜。同样地,较老的胚胎E17-18将具有更发达的皮肤和颅骨,需要更多的力量将DNA溶液注射到心室,其相对于动物的尺寸变得更小。
  2. (≥1μg/μl)制备的DNA:Qiagen Maxi Prep试剂盒(QIAGEN,目录号:12663)或EndoFree质粒试剂盒(QIAGEN,目录号:12362)
  3. 快速绿FCF(Sigma-Aldrich,目录号:F7252)
  4. HBSS(Life Technologies,目录号:14025-126)
  5. 4%低熔点琼脂糖(无菌1×PBS或1×HBSS)
  6. 低熔点琼脂糖(GeneMate Sieve GQA琼脂糖,目录号:E-3112)

  7. Kimwipe

设备

  1. 35毫米培养皿
  2. 多孔细胞培养皿(12至24孔)
  3. 电穿孔仪(BTX The Electroporation Experts,型号:ECM 830方波电穿孔系统)
  4. 3 mm桨铂金Tweezertrodes(BTX电动专家,型号:45-0487)
  5. ECM 830脚踏开关(BTX The Electroporation Experts,型号:1250FS)
  6. 口腔移液管(Sigma-Aldrich,目录号:P0799-1PAK)
  7. 微量移液器(Borosilicate,长丝O.D .: 1mm,I.D .: 0.78mm,10cm长)(SUTTER INSTRUMENT,目录号:BF100-78-10)
  8. 微量拔管器P-97/IVF(SUTTER仪器)
  9. 解剖立体镜灵活的照明
  10. 杜蒙镊子(#5和#55)(Fine Science Tools)
  11. 一次性塑料移液管
  12. 无菌手术刀
  13. 用于检查转染的荧光解剖立体镜
  14. 数码相机(如有必要)(OLYMPUS,型号:MVX10)

程序

  1. 收集定时胚胎小鼠胚胎根据批准的协议在您的机构。 抓住胚胎在冰冷的HBSS填充盘,直到准备使用。
  2. 将融化的琼脂糖倒入35mm皿中〜1/2至2/3满,并在冰上固化。
  3. 用刀片从餐具中心切下一个小梯形,丢弃(图1A)。
  4. 用HBSS填充这个菜几乎完全(图1A)。
  5. 使用切割移液管或钳子将单个胚胎转移到这个皿的孔中(图1A)
  6. 填充微量移液器与DNA溶液混合足够的快速绿色只是可视化
  7. 定位胚胎在井,用钳子保持在位,同时微注射约0.2-0.5微升DNA到侧脑室。将胚胎翻转,重新定位和微注射DNA进入对侧脑室 注意:由于有色溶液,每个脑室应该可视化(图1A)。
  8. 保持镊子在头部以目标电流进入背部新皮层 注意:目标位置高度依赖于tweezertrodes的方向。例如,将正电极保持在脑的一侧的背侧新皮质上意味着负电极将被放置在更腹侧/对角位置,略微在对侧颌下方。这是最容易实现的,另一只手(即左)使用另一对钳子,而另一只手握住镊子(右)。
  9. 使用远程脚踏开关进行电穿孔胚胎:3-5个脉冲,35 mV,100 ms间隔。
  10. 翻转胚胎,相应地重新定位镊子,并使用相同的程序重新电穿孔对侧大脑皮层。
    注意:
    1. 不同的电极放置可以针对其他特定区域,包括海马/牙齿,腹侧端脑,以及其他包含腔的区域。
    2. 在电穿孔期间,最好不要直接接触胚胎。
  11. 转移电穿孔胚胎放置在冰上与HBSS的多孔皿,使用切割移液管/镊子恢复。
  12. 每次胚胎电穿孔后,用干燥的Kimwipe擦拭,清洁电极。
  13. 重复程序,直到所有胚胎都已电穿孔。
    注意:在此子程序中可以使用多种不同的DNA构建体和组合。
  14. 从每个胚胎解剖大脑,并转移到冰冷的HBSS在新的多孔板,直到所有的大脑被删除。
    注意:在某些情况下,根据胚胎的实际年龄,可以去除脑膜,以帮助振动切片,较年轻的胚胎更脆弱(E10-12)。
  15. 将大脑插入4%低熔点琼脂糖中,并准备振动切片和器官型脑切片培养,如(参见胚胎脑部的器官切片培养(Calderon de Anda,2013)。
  16. 第二天可以用荧光立体镜观察切片。实践中,多个切片应该在每个新皮层中包含荧光细胞。例如,图1B显示了在野生型胚胎中靶向每个新皮层的表达RFP的质粒的典型实例。图1C,D是靶向转基因GFP表达动物的每个新皮层的RFP的实例(Nelson等人,2013)。


    图1.胚胎小鼠新皮质中的 Ex utero 电穿孔。 A.技术示意图; B.靶向野生型E14.5小鼠中每个新皮层的双电穿孔RFP质粒的实例,所述野生型E14.5小鼠培养过夜; C.靶向转基因Tbr2GFP E14.5新皮层中的每个新皮层的双电穿孔RFP质粒的实例; D.大功率视图。黑色和红色分别代表负电极和正电极,因此记住它们的取向,因为它控制方向和靶向:绿色代表填充侧脑室的快速绿色DNA溶液,显示它们的形状,并指示成功的填充。 /> 注意:使用配备有GFP/RFP滤光片组和数码相机(Olympus,MVX10)的荧光立体镜获得图像。

致谢

该方案改编自Nelson等人(2013),并且由NIH Grants R21 MH087070和RO1 MH080766-S至R.F.H.支持。

参考文献

  1. Calderon de Anda,F。(2013)。 胚胎脑切片的器官切片培养。 生物方案, 3(3):http://www.bio-protocol.org/wenzhang.aspx?id=327。
  2. Ge,X。(2012)。 在子宫内小鼠胚胎脑的电穿孔。 生物协议 2(13):e231
  3. Nelson,B.R.,Hodge,R.D.,Bedogni,F.and Hevner,R.F。(2013)。 胚胎小鼠新皮层中间神经源性祖细胞和放射状胶质细胞之间的动态相互作用:Dll1-Notch信号传导中的潜在作用 。 J Neurosci 33(21):9122-9139。
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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: © 2013 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. Nelson, B. R. (2013). Ex utero Electroporation into Mouse Embryonic Neocortex. Bio-protocol 3(24): e998. DOI: 10.21769/BioProtoc.998.
  2. Nelson, B. R., Hodge, R. D., Bedogni, F. and Hevner, R. F. (2013). Dynamic interactions between intermediate neurogenic progenitors and radial glia in embryonic mouse neocortex: potential role in Dll1-Notch signaling. J Neurosci 33(21): 9122-9139.
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