作者想感谢简·约翰逊的MATH1，Ngn1，的Ptf1a抗体和康妮Cepko为“pCAG :: GFP的质粒。这项工作是由NIH的R15的1R15HD059922-01。

1。以电穿孔前的准备<ol><li>由马克西准备（总理或Qiagen公司）的DNA扩增为电。 DNA的浓度应该是最低的1毫克/毫升的高效吸收。 </li><li> 5微升0.01％快速绿色1×PBS（磷酸盐缓冲液）在离心管取出495μLDNA和混合。 </li></ol> 2。胚胎丰收<ol><li> CD-1小鼠（哈伦）建立定时交配。检查阴道塞的存在，并把阴道塞观察胚胎一天（五）0.5的日期。胚胎将可视化插件（E11.5）后11天收获。 </li><li>将在70％乙醇消毒工具。层流罩与紫外线灯治疗至少一小时前使用。喷雾罩，托盘解剖，解剖用70％乙醇的范围。预热1×PBS至37°C。 </li><li>在E11.5安乐死的女性，根据研究所批准的条件护理和使用动物（ICCUA）的成分委员会。放置在层流罩的解剖托盘。喷用70％乙醇对女性的腹部。 </li><li>打开腹腔，针城墙解剖的托盘。拉出子宫角，以便它的腹腔内休息。 </li><li>仔细剖开的子宫角的墙上。使用20毫米的勺子（精细的科学工具）轻轻取出胚胎卵黄囊远离胎盘。预填充10毫升无菌1×PBS 2.2预热到100毫米组织培养皿的地方胚胎。 </li><li>出席会议的所有胚胎重复。 </li></ol> 3。 E11.5胚胎的电（ 图1） <ol><li>使用20毫米的勺子，第一胚胎转移到与预先填写10 mL无菌1XPBS预热至37中一个新的100毫米菜°C。 </li><li>使用1 0.05×0.02毫米的小费（精细的科学工具）11厘米镊子仔细重新移动和丢弃卵黄囊。 </li><li>胚胎的位置，因此，其背侧朝上，以便它类似于图1A卡通。使用7毫米的电极桨（哈佛器械）轻轻按住胚胎。电极应定位在任的神经管在一侧的后脑第四脑室水平。脑室是肉眼可见的，但使用解剖显微镜可以方便准确桨安置。桨的定位是，以确定该地区接收的电穿孔DNA的关键。如果是理想的，较低的菱形唇（LRL）背胚胎后脑桨必须是这样，它们直接侧翼第四脑室开放的最广泛的部分。 </li><li>用1毫升注射器制订混有0.01％的快速绿色的质粒DNA。 500μL混合物应为至少8-10胚胎（见3.5）的电足够。 </li><li>轻轻穿刺roofplate OVerlying与25G 5/8结核菌素1毫升注射器针第四脑室及脑室注入DNA染料混合物。成功的注射填补了整个脑室系统（ 图1C）由DNA染料混合物的特点。通常注射DNA的染料混合物的数量少于50μL。确切数额之间胚胎变量的混合物的一部分，往往泄漏到周围的胚胎PBS心室。将通过访问软腭上覆的脑穿刺脑室系统提供的DNA染料混合物的替代手段。再次，成功的注射DNA染料混合物填充整个脑室系统的特点。 </li><li>交付使用电脉冲发生器（BTX）和7毫米的电极桨5平方脉冲。每个脉冲50 V的持久5毫秒500毫秒之间的每个脉冲，每脉冲。组织最接近的带正电荷的电极，然后德电子商务支撑的载体。 </li></ol> 4。文化的胚胎<ol><li>在层流罩，填写2毫升辅以10％小牛血清，5％马血清，1％谷氨酰胺，1％青霉素/链霉素预热的DMEM/F12媒体外井12井培养皿到37°C。培养条件进行了改编自15 de地亚哥和他的同事们。 </li><li>在层流罩在中段（以下心）用钳子捏胚胎并取出胚胎的后部。放入前部的12孔培养皿中充满井之一。 </li><li>重复所有的胚胎。只填写12孔板外井文化，以避免污染。 </li><li>在37℃培养箱培养胚胎用5％的CO 2。电穿孔质粒表达应观察24小时内。 </li><li>应该更长文化理想，填写一个新的12孔板出井，2毫升媒体4.1。用消毒勺转移胚胎以及在新板。放回37℃培养箱。 48小时的文化是可能的。 </li><li>当达到所需的培养时间，解决胚胎进行分析（见下文）。 </li></ol> 5。用于分析胚胎的制备<ol><li>在4°C冲洗胚胎五分钟，1×PBS。重复。 </li><li>修正了2％多聚甲醛分析2小时1XPBS（PFA）的胚胎在4°C </li><li>在4°C冲洗胚胎五分钟，1×PBS。重复。 </li><li>在30％蔗糖1×PBS平衡胚胎一夜之间在4°C。 </li><li>嵌入最佳切削温度（OCT）复合使用干冰/乙醇浴中的胚胎。胚胎可以保存在-20°C </li><li>低温恒温器（徕卡）到30微米的部分幻灯片上装载（厂商VWR，Superfrost加），第胚胎。贮存于-20°C </li></ol> 6。免疫nohistochemistry分析<ol><li> 16所述进行免疫组化。用于这项研究的主要抗体稀释包括兔的α-GFP（Invitrogen公司）1:2500;小鼠α-，MASH1（BD Biosciences公司）1:100;兔α-Ngn1，（简·约翰逊）1:5000;兔α-Ptf1a（简·约翰逊）1:2500;兔MATH1（简·约翰逊）1:100。孵育平面幻灯片，标本方在4°C过夜染色托盘。 </li><li>幻灯片分析了复合显微镜（奥林巴斯BX51）。 </li></ol> 7。代表结果 图1A中的示意图描绘了电实验。 图1B显示了一个E11.5胚胎矢状前操纵的。相同的胚胎，含造影:: GFP质粒注射0.01％快速绿色显示在图1C和有代表性的非固定胚胎参展单方面的GFP显示在图1D背后脑24小时以下文化的表达。 LRL的，是成功的电穿孔面积的范围是可变的，似乎是高度依赖的电极定位。在我们的研究结果发现，52 65（80％）的电穿孔胚胎成功表达GFP。组织被认为是成功的电穿孔，如果它是固定和免疫组化分析（见下文）后的几个部分，在局部地区的GFP阳性。胚胎未能满足此条件，取得了在电不成功的尝试。 电效率的进一步评估，确定对GFP电穿孔胚胎在第四脑室水平的横截面进行免疫组化。 图2显示了从代表显示单方面胚胎的连续切片GFP的表达。 图2A显示了一个理想化的原理，说明胚胎左侧是朝正电极。横截面（ 图2A中的卡通所代表的水平），揭示了完全本地化的GFP表达左侧后脑组织（ 图2C和2D）。电穿孔的LRL的面积没有扩大整个前后轴的神经管延髓300微米或更大，以图2C显示，部分考试不表达GFP（ 图2B）。 此法对基因表达操纵的工具是依赖于内源性蛋白的表达域的稳定。 LRL的特点是原神经转录因子的表达差异（审查1）具有独特的祖域已被定性为。这些因素中的一个子集（MASH1，MATH1，Ngn1，Ptf1a），被选择的分析，由于他们的建议和/或规范的LRL的，今后的研究课题16-18 precerebellar神经亚型特点的角色。所有四个蛋白质有高度在在E11.5 16-18尾后脑特征表达域。我们观察到，置于文化的胚胎未能扩大规模，也未能启动生产的脉络丛上皮内陷LRL的和roofplate，E11.5和E12.5 5之间发生的形态学事件。根据这些意见，它被确定在这些胚胎的正常发育停止或严重延迟和培养的胚胎相媲美的控制应该是落后的胚胎在E11.5。 为了确保文化和电不打扰内源性蛋白的水平，我们分析了四个不同的蛋白质由immunohistoch我 emistry化（IHC），电穿孔，然后培养24小时，至少在34个不同的胚胎。 表显示每个标记和分析，保留了正常的蛋白质水平。 图3免疫组数据显示代表胚胎的百分比分析胚胎数从蛋白质分析，MASH1（ 图3A和3B）和MATH1（ 图3C和3D）。我们观察到，大部分胚胎电和文化表达（ 图3B和3D）保留正常水平相比，控制胚胎E11.5（ 图3A和3C）。重要的是，这些蛋白质的特征表达域，不慌乱。 <img src="/files/ftp_upload/3983/3983fig1.jpg" alt="图1"/> 图1。在E11.5胚胎的电。日（一）示意图ê电实验。 E11.5胚胎分离和表达质粒在0.01％的快速绿色到第四脑室注射。胚胎，然后两侧电极桨和50 V脉冲之前被放置到文化（二）注射前的E11.5胚胎矢状。 （三）同一E11.5胚胎后，在0.01％的快速绿色注射质粒。 （四）单方面后脑表达GFP的观察E11.5胚胎后24小时的文化。 MB-脑; 4V第四脑室。 <img src="/files/ftp_upload/3983/3983fig2.jpg" alt="图2"/> 图2。电穿孔组织中的绿色荧光蛋白表达的影响。（一）在左边的卡通描绘电极周围的E11.5胚胎的位置。中东卡通描绘胚胎左侧质粒编码绿色荧光蛋白的摄取和表达。右边的卡通是一个理想化的横向部分通过胚胎在l原理evels由中间卡通黑线表示。通过电穿孔E11.5胚胎培养24小时后的横截面的绿色荧光蛋白的免疫组织化学（的B-D）。箭头指示的roofplate（RP）的陷阱二级抗体。图像是在10X的放大倍率。显示部分的相对水平描述（一）通过中间卡通水平线。 MB-脑，L 4V第四脑室; RP-roofplate。 <img src="/files/ftp_upload/3983/3983fig3.jpg" alt="图3"/> 图3。菱形唇下的内源性蛋白的免疫组织化学，MASH1（A和B）或比较E11.5胚胎的横截面，培养与电穿孔与CAG的胚胎（C）MATH1（C和D）的表达。 GFP和培养24小时（B和D）。在10倍放大倍率拍摄的图像。 <table border="1"><tbody><tr><td> 原神经Transcripti分析因子</td><td> 胚胎数分析 </td><td> 挡土墙的正常表达模式的百分比 </td></tr><tr><td> MATH1 </td><td> 15 </td><td> 86.7％ </td></tr><tr><td> MASH1 </td><td> 12 </td><td> 83.3％ </td></tr><tr><td> ngn1 </td><td> 7 </td><td> 71.4％ </td></tr><tr><td> ptf1a </td><td> 6 </td><td> 100％ </td></tr></tbody></table> 表一百分比电穿孔和培养胚胎，保留原神经正常的转录因子在LRL的域。

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没有利益冲突的声明。

在这项研究中提出的技术在体外电击是一种新颖的方法，可以有效地利用胚胎年龄小于12天的妊娠期，操纵基因表达。胚胎放置到文化允许引入基因的表达和规避杀伤力观察电穿孔胚胎时，被允许留在体内 。这种技术允许基于电的研究，这在以前无法进入的胚胎祖细胞的基因表达操纵。 电技术，导致在引入80％的胚胎基因的高效表达分析。观察图2中...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>低温恒温器</td><td>徕卡</td><td> CM-1850 </td><td></td></tr><tr><td> biologie尖Dumoxel治疗杜蒙镊子</td><td>精细的科学工具。 </td><td> 11252-30 </td><td></td></tr><tr><td> 20毫米莫里亚穿孔勺</td><td>精细的科学工具。 </td><td> 10370-17 </td><td></td></tr><tr><td>流脑830方波电脉冲发生器</td><td> A型肉毒毒素（厂商VWR） </td><td> 47745-928 </td><td></td></tr><tr><td>哈佛仪器7 *电极毫米Tweezertrodes </td><td> A型肉毒毒素（雪） </td><td> BTX450165 </td><td></td></tr><tr><td>费舍尔Isotemp 二氧化碳培养箱</td><td>费舍尔</td><td> 1325525 </td><td></td></tr><tr><td>新机场工程统筹署的CO 2气体调节器</td><td>费舍尔</td><td> 15497020 </td><td></td></tr><tr><td> 12孔培养板</td><td> BD猎鹰（费舍尔） </td><td> 877229 </td><td></td></tr><tr><td>液体介质DMEM/F-12胎牛血清（1:1）;与L-谷氨酰胺和肝素钠; 500毫升</td><td>赛默飞世尔科技（费舍尔） </td><td> SH3002301 </td><td></td></tr><tr><td>胎牛血清*捐助马血清</td><td>赛默飞世尔科技（费舍尔） </td><td> SH3007402 </td><td></td></tr><tr><td>胎牛血清，合格，热灭活</td><td> Invitrogen公司</td><td> 16140-063 </td><td></td></tr><tr><td> cellgro * 10,000 IU青霉素，10,000μg/ mL链霉素</td><td>敏达（费舍尔） </td><td> MT-30-002-CI </td><td></td></tr><tr><td>胎牛血清·L-谷氨酰胺L-谷氨酰胺; 200MM在0.85％的NaCl </td><td>赛默飞世尔科技（费舍尔） </td><td> SH3003401 </td><td></td></tr><tr><td>快绿</td><td>费舍尔</td><td> AC41053-0250 </td><td> 0.01％ </td></tr></tbody></table>

in vitro electroporation of the lower rhombic lip of midgestation mouse embryos

菱形唇是位于后脑的胚胎神经上皮之间的神经管及第四脑室（1检讨）roofplate交界。可分为菱形嘴唇上的菱形唇（URL），其中包括1（R1）和菱产生小脑的神经元和低菱形唇（LRL的），而产生不同的神经脑干谱系2-4。 LRL的衍生工具包括耳蜗核的听觉神经细胞和那些的原子核参与调节平衡和电机控制5-8 precerebellar的。神经再生的LRL的发生在一个大的时间窗口，包括胚胎（五）9.5-16.5 5天，9。不同的神经细胞谱系出现分裂后的细胞在不同发育天，在此期间神经窗口（或出生）LRL的。 基因表达结构的电可以用来操纵LRL的祖细胞的基因表达，并可能改变命运10-12从本地区产生的神经元。通过在子宫内的电LRL的祖细胞在小鼠的基因表达改变已经非常成功地操纵胚胎一天E12.5或以后10 12-14出生的谱系。 在宫内 electroporations前到E12.5已经失败主要是由于穿刺第四脑室roofplate的必要步骤，在提供外源DNA进入LRL的电穿孔相关的杀伤力。然而，许多LRL的派生谱系来自LRL的早于E12.5 9。这些以前出生的谱系，包括外侧网状神经元组成，外部的楔形，下橄榄核的precerebellar系统的功能连接脊髓和大脑皮质的投入小脑5。为了操作的LRL的表达胚胎E12.5年轻，我们开发了在体外系统在胚胎放置到文化电。 这项研究提出了一个在E11.5 LRL的祖细胞的基因表达操纵的高效率和有效的方法。重复性表示培养24小时后的GFP绿色荧光蛋白（GFP）的广泛积极的CAG启动驱动与电穿孔的胚胎。此法的一个重要方面，是基因表达的改变只因为外源基因的表达，并没有因为二次效应，从电和栽培技术的结果。它被确定内源性基因表达模式保持原状电穿孔和培养胚胎。此法可以用来改变命运的细胞从胚胎E12.5年轻的过度表达质粒引进LRL的新兴或击倒（通过RNA干扰）不同亲神经转录因子。

Watch this Scientific Journal Video about In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos at JoVE.com

In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

这项研究描述了一个发展<em&gt;在体外</em&gt;电穿孔技术，允许在较低的菱形唇midgestation胚胎的基因表达操纵。

菱形唇下的Midgestation小鼠胚胎在体外电穿孔

The rhombic lip is an embryonic neuroepithelium located in the hindbrain at the junction between the neural tube and the roofplate of the fourth ventricle ...

in-vitro-electroporation-lower-rhombic-lip-midgestation-mouse

Research

JoVE Journal

Neuroscience

In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

10.3K 次观看.  University of Illinois at Springfield. 这项研究描述了一个发展在体外电穿孔技术，允许在较低的菱形唇midgestation胚胎的基因表达操纵。

视频: In vitro Electroporation of the Lower Rhombic Lip of Midgestation Mouse Embryos

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

科研

神经科学

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

量化的活动顺</em在小鼠视网膜>调控元件外植体电穿孔

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

在子宫内电穿孔：鼠标控制时空基因Transefection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and the organ of Corti in the coiled cochlea. The cristae and maculae contain vestibular hair cells that transduce mechanical stimuli to subserve the special sense of balance, while auditory hair cells in the organ of Corti are the primary transducers for hearing 1. Cell fate specification in these sensory epithelia and morphogenesis of the semicircular canals and cochlea take place during the second week of gestation in the mouse and are largely completed before birth 2,3. Developmental studies of the mouse inner ear are routinely conducted by harvesting transgenic embryos at different embryonic or postnatal stages to gain insight into the molecular basis of cellular and/or morphological phenotypes 4,5. We hypothesize that gene transfer to the developing mouse inner ear in utero in the context of gain- and loss-of-function studies represents a complimentary approach to traditional mouse transgenesis for the interrogation of the genetic mechanisms underlying mammalian inner ear development6.
The experimental paradigm to conduct gene misexpression studies in the developing mouse inner ear demonstrated here resolves into three general steps: 1) ventral laparotomy; 2) transuterine microinjection; and 3) in vivo electroporation. Ventral laparotomy is a mouse survival surgical technique that permits externalization of the uterus to gain experimental access to the implanted embryos7. Transuterine microinjection is the use of beveled, glass capillary micropipettes to introduce expression plasmid into the lumen of the otic vesicle or otocyst. In vivo electroporation is the application of square wave, direct current pulses to drive expression plasmid into progenitor cells8-10. 
We previously described this electroporation-based gene transfer technique and included detailed notes on each step of the protocol11. Mouse experimental embryological techniques can be difficult to learn from prose and still images alone. In the present work, we demonstrate the 3 steps in the gene transfer procedure. Most critically, we deploy digital video microscopy to show precisely how to: 1) identify embryo orientation in utero; 2) reorient embryos for targeting injections to the otocyst; 3) microinject DNA mixed with tracer dye solution into the otocyst at embryonic days 11.5 and 12.5; 4) electroporate the injected otocyst; and 5) label electroporated embryos for postnatal selection at birth. We provide representative examples of successfully transfected inner ears; a pictorial guide to the most common causes of otocyst mistargeting; discuss how to avoid common methodological errors; and present guidelines for writing an in utero gene transfer animal care protocol.

Gene Transfer to the Developing Mouse Inner Ear by In Vivo Electroporation

The mouse inner ear is a placode-derived sensory organ whose developmental program is elaborated during gestation. We define an in utero gene transfer technique consisting of three steps: mouse ventral laparotomy, transuterine microinjection, and in vivo electroporation. We use digital video microscopy to demonstrate the critical experimental embryological techniques.

转基因小鼠内耳的发展<em&gt;在体内</em&gt;电穿孔

The mammalian inner ear has 6 distinct sensory epithelia: 3 cristae in the ampullae of the semicircular canals; maculae in the utricle and saccule; and ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

可视化和遗传操作在小鼠大脑皮层和使用海马树突和棘<em&gt;在子宫内</em&gt;电穿孔

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.

How neuronal networks are established in the embryonic brain is a fundamental question in developmental neurobiology. Here we combined an electroporation technique with novel genetic tools, such as Cre/Lox&#x2013;plasmids and PiggyBac-mediated DNA transposition system in the avian hindbrain to label dorsal interneurons and track their axonal projections and synaptic targets at various developmental stages.

电穿孔轴突轨迹跟踪和突触目标的后脑在鸡胚

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

遗传操纵鼠标的发展下丘脑通过<em&gt;在子宫内</em&gt;电穿孔

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

一个<em在体外</em从成年小鼠的肌间神经丛制备隔离肠道神经元和神经胶质

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.