作者感谢Sharmin侯赛因时间推移电影捕获一个不成熟的神经元和德里克Dunfield增长标准煤micromicropipette提示的电子显微镜图像。

设备设置： <ol><li>这种技术所需的电气设备是一个刺激器，示波器和微量架配有一个银色的线插入到微。我们通常使用一个轴突文书Axoporator 800A刺激，但如草文书等共同刺激SD9刺激，也适用于单细胞电穿孔。刺激器的探头，放在里面的玻璃内部进行解决方案含有微量的银线电极接口连接。从刺激导致连接到示波器的输入端，还接收来自外部的蝌蚪地面的输入。蝌蚪外部接地电紧张的蝌蚪在电接触仍然是一个简单的银线。该电路是完整的，一旦微量接触蝌蚪。 </li><li>单细胞电需要一个良好的工作距离（至少5厘米）的显微镜，因为这给了回旋的余地，并允许在约30-45度的角度带来的微量。 </li></ol>制备微量： 适当微量的制备是在这个协议中的一个关键的一步。困难在于，一个狭窄的尖端（直径小于1微米）平衡，不会轻易打破穿刺组织时。我们还发现，一个大于10度的锥度角的秘诀是可取的。 <ol><li>准备一把拉住玻璃微吸管尖端直径小于1μm的，和一个大于10度的尖端锥度。 <ol><li>硼硅玻璃与内部灯丝外径1.5mm和内在0.75毫米直径非常适合用于这一目的。 <ol><li>比较厚的玻璃提供了一定程度的刚性给小费。 </li><li>在制订对微量尖回填流体内部的灯丝是很重要的。 </li></ol></li></ol></li><li>制造适当的提示，往往需要若干修改的基础上观察到的结果。不同的组织可能需要一个稍微不同的形状，或尖端直径的提示。 </li></ol>单细胞电协议： 对于新用户，最好是开始与右旋糖酐荧光染料，不像荧光蛋白基因编码的，因为他们可以立即根据epifluorescence看到。使用荧光染料，使用户能够直接看到组织内的微管尖端的位置，并经电提供设备是否已正确设置和微量提示是否是合适的即时反馈。 <ol><li>填充微量的复合解决方案，通过使用装载注射器或回填电穿孔。 <ol><li>如果使用质粒DNA，确保样品无内毒素，并在浓度之间1-2μg/ml，在水中准备。 </li><li>荧光葡聚糖染料的浓度应凭经验确定。我们经常使用的荧光染料，加水稀释至约300μM。 </li><li>通常情况下，0.5 -1μl进卷就足够了。 </li><li>装货前，短暂离心高速的解决方案，以减少颗粒碎片绘制成微管，这可能会导致堵塞。 </li><li>气泡内提示，往往会影响电流的流动，应大力弹的微量探头安装前脱落。 </li></ol></li><li>插入持有微量或探头上安装一个3轴显微。确保，银线电极插入微管，电紧张与内部的解决方案接触。 </li><li>麻醉浸泡在0.02％蝌蚪饲养中期（Steinberg的溶液，pH 7.4）编写的MS - 222（3 - 氨基甲酸乙酯）蝌蚪。 <ol><li>我们通常使用在我们的实验阶段44-48白化非洲爪蟾蝌蚪。 </li><li>蝌蚪通常大约5分钟后完全麻醉。 </li><li>然而，几个蝌蚪，可能是麻醉的同时，以减少等待时间，避免接触超过1小时，期间到MS - 222。 </li></ol></li><li>一个单一的麻醉蝌蚪电室使用塑料转移微量。 <ol><li>电室，可以很容易地在内部雕刻一个蝌蚪形的空腔，在一个小Sylgard ®硅块和插入一个银色的接地线腔等，将在电紧张的联系与蝌蚪在会议厅内。 </li></ol></li><li>蘸位置蝌蚪了背侧使用画笔。 </li><li>下的微管，直到它与皮肤接触，直接相邻地区的利益。 <ol><li>针对密密麻麻的细胞机构的地区，将大大增加成功的电击机会。通常，我们的目标蝌蚪视神经顶盖。 </li><li>在微量带来30至45度角。较浅的轨迹使皮肤穿刺比较困难，而陡峭的角度往往阻碍一个人的看法。 </li></ol></li><li>继续降低微量最初将酒窝的皮肤，然后通过到底层的组织。 <ol><li>表层细胞在活体成像的首选，并可以通过有针对性的确保慢慢降低后的皮肤凹陷，微管尖端。 </li></ol></li><li>一是要不断监察一旦组织内放置的微量阻力。如果使用Axoporator 800A（Axon仪器），约10 -40MΩ的电阻是最优的。阻力是尖端直径是否适合于单细胞电穿孔的良好指标。 <ol><li>高微量电阻（&gt;100MΩ）堵塞提示或技巧，过于狭窄的微量指示。 </li><li>低微量电阻（&lt;10MΩ）是一个尖端直径太大，往往是一个破碎的一角的结果的指示。 </li><li>如果使用的是一个刺激，不提供直接的电阻测量，微管阻力，可以间接地通过观察示波器测量的电压脉冲（下面讨论）的振幅来测量。 </li></ol></li><li>应用电压列车。 <ol><li>质粒DNA，脉冲序列的发现，最有效的负方波脉冲持续时间为1ms，在300Hz脉冲500ms的火车时间交付，包括。 <ol><li>凭经验确定示波器测得的脉冲幅度介于0.75和1.5μA的脉冲电压。 </li></ol></li><li>对于葡聚糖荧光染料，提供积极的方波脉冲持续时间为300μs，在300Hz的频率与脉冲持续时间为10ms的火车。 <ol><li>至于协议的DNA，脉冲电压是凭经验确定示波器测得的脉冲幅度介于0.75和1.5μA。 </li><li>下epifluorescence电观测结果的基础上，可进一步调整刺激参数。 <ol><li>如果电穿孔细胞出现暗淡，脉冲参数应逐步增加，直到细胞显得更亮。 </li><li>另一方面，如果被标记的细胞群，脉冲参数应减少直至单个细胞标记。 </li></ol></li></ol></li></ol></li><li>缩回微管，重新插入到一个组织内的不同的网站，并施加电压的列车。 <ol><li>为了提高产量，我们通常electroporate在每个半球有足够的间距蝌蚪视神经顶盖多个站点，标记的细胞，以确保不重叠。 </li></ol></li><li>与Steinberg的饲养解决方案的容器转移到电穿孔蝌蚪。 <ol><li>蝌蚪会迅速恢复在几分钟内麻醉。 </li></ol></li><li>相同的微管可用于许多蝌蚪。重要的是要不断观察示波器上观察到的脉冲幅度。 <ol><li>如果脉冲幅度显着提高脉冲强度后仍然偏低，这可能是一个被堵塞的一角，电穿孔许多蝌蚪在一次会议时，经常看到的结果。 <ol><li>撞出一个小的提示阻塞的一种方法是申请一个正脉冲，后插入的微管组织。 </li><li>如果堵塞不能脱落，或堵塞频繁复发，更换微管。 </li></ol></li><li>后，大大减少了脉冲参数，如果脉冲幅度仍然非常大，这是一个提示已损坏，需要更换的迹象。 </li></ol></li></ol>成功的电穿孔细胞的筛选： <ol><li>电击后，蝌蚪使用一个堂堂正正的epifluorescence显微镜标记的细胞筛选。 </li><li>在荧光染料的情况下，蝌蚪可筛选后短短30分钟后电。 <ol><li>然而，我们发现较长的时间间隔与背景荧光的水平降低有关。 </li></ol></li><li>对于荧光蛋白基因编码，筛选通常是进行电后至少12小时。 <ol><li>荧光蛋白的表达，将继续得到美好的时间，因此昏暗的细胞可能会重新筛选后12-24小时的额外时间间隔。 </li></ol></li><li>蝌蚪麻醉如上所述，放置在一个Sylgard ®厅和coverslipped。个别蝌蚪，然后根据epifluorescence检查。 <ol><li>请注意，过度暴露于epifluorescence光会导致光毒性，可能会杀死标记细胞。因此，最大限度地减少，细胞暴露在荧光灯的时间量。 </li></ol></li><li>返回蝌蚪容器与Steinberg的饲养解决方案。 </li></ol>

<ol>
	<li>Bestman, J. E., Ewald, R. C., Chiu, S. L., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+single-cell+electroporation+for+transfer+of+DNA+and+macromolecules.">In vivo single-cell electroporation for transfer of DNA and macromolecules.</a> Nature Protocols. 1, 1267-1272 (2006).</li><li>Dunfield, D., Haas, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+electroporation.">Single cell electroporation.</a> Encyclopedia of Neuroscience (4th Ed.). , .</li><li>Haas, K., Jensen, K., Sin, W. C., Foa, L., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+electroporation+in+Xenopus+tadpoles+in+vivo--from+single+cells+to+the+entire+brain.">Targeted electroporation in Xenopus tadpoles in vivo--from single cells to the entire brain.</a> Differentiation. 70, 148-154 (2002).</li><li>Haas, K., Sin, W. C., Javaherian, A., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+electroporation+for+gene+transfer+in+vivo.">Single-cell electroporation for gene transfer in vivo.</a> Neuron. 29, 583-591 (2001).</li></ol>

单细胞电（SCE）是一个强大的工具用于确定基因功能，并进行有针对性的基因操纵。白化蝌蚪的透明度和大脑的辅助功能，使这个模型系统，非常适合用于可视化的神经细胞生长和细胞内事件，在活的有机体。 SCE允许可视化的单个神经元的生长，并执行一个不变的大脑内的细胞自主操作。虽然这种视频文章演示了单细胞电在爪蟾蝌蚪的过程，这种技术已被用于在其他生物中，也一直在海马脑片和游离细胞培养使...

single cell electroporation in vivo within the intact developing brain

单细胞电（SCE），是一家专业的技术，使DNA或其他大分子成单个细胞内完整的组织，在体内的准备工作，包括交付。这项技术的独特优势是，实验操作，可对单个细胞，同时使周围组织保持不变，从而区别于那些导致全球治疗的细胞自主的效果。拥有先进的体内成像技术相结合时，荧光标记标准煤许可证的细胞形态，细胞生长，细胞内事件从几秒钟到几天不等的时间尺度，直接可视化。虽然这种技术是使用各种在体内和体外的准备，我们在爪蟾蝌蚪的使用这种技术进行了优化。在这个视频文章中，我们详细标准煤荧光染料或白化非洲爪蟾蝌蚪的完整的大脑内的神经细胞的质粒DNA的过程。我们还讨论了方法，以优化产量，并显示由SCE荧光标记的神经元的活的双光子荧光成像的例子。

Watch this Scientific Journal Video about Single Cell Electroporation in vivo within the Intact Developing Brain at JoVE.com

Single Cell Electroporation in vivo within the Intact Developing Brain

单细胞电（SCE）是一个专门的技术，允许交付到单个细胞内完整的组织，在体内的准备工作，包括的DNA或其他大分子。在这里，我们标准煤荧光染料或成爪蟾蝌蚪的完整的大脑内的神经细胞的质粒DNA的详细过程。

在体内的单细胞电穿孔内完整的大脑发育

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

single-cell-electroporation-in-vivo-within-the-intact-developing-brain

Research

JoVE Journal

Biology

13.2K 次观看.  University of British Columbia - UBC. 单细胞电（SCE）是一个专门的技术，允许交付到单个细胞内完整的组织，在体内的准备工作，包括的DNA或其他大分子。在这里，我们标准煤荧光染料或成爪蟾蝌蚪的完整的大脑内的神经细胞的质粒DNA的详细过程。

视频: Single Cell Electroporation in vivo within the Intact Developing Brain

Electroporation of Mycobacteria

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall, but is compounded by the fact that most molecular techniques have been developed for distantly-related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified and DNA transformation of many mycobacterial species have now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the  conditions for the electroporation pulse. Optimized methods for the transformation of both slow- and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

Mycobacterial pathogenic strategies remain poorly understood. The slow growth rate of most species, the impenetrable nature of the cell-wall, and the hazards of working with pathogens make mycobacteria difficult to study and are largely responsible for our poor understanding of these organisms. In this video we will demonstrate the technique of electroporation, which involves subjecting cells to a brief high electrical impulse to allow the entry of DNA. It is the most widely used method for introducing DNA into mycobacterial cells.

电穿孔分枝杆菌

High efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly ...

科研

生物学

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

在子宫内和前体内的基因表达在小鼠视网膜神经节细胞的电穿孔

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

使用哺乳动物骨骼肌的DNA转染在体内电穿孔

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

单从小鼠锥感光细胞吸录音

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

在完整的果蝇幼虫体内的亚细胞分辨率成像

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

在体内电穿孔Morpholinos到成年斑马鱼视网膜

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

再生的成年斑马鱼尾鳍在吗啉代入体内电穿孔

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

单线粒体超氧闪烁在完整心脏或共聚焦成像<em&gt;在体内</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

钙火花的评估在正常肌肉纤维

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

In Vivo Microinjection and Electroporation of Mouse Testis

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis.
The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV).
For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular&#160;cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red&#160;fluorescence, illustrating transfection success.
Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques.

In Vivo Microinjection and Electroporation of Mouse Testis

This article describes microinjection and electroporation of mouse testis in&#160;vivo as a&#160;transfection technique for testicular mouse cells to study unique processes of spermatogenesis. The presented protocol involves steps of glass capillary preparation, microinjection via the efferent duct, and transfection by electroporation.

在小鼠睾丸活体显微注射和电穿孔

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...