我们愿与收购在本视频中的整个海马低倍率图像的帮助，感谢马克Lucanic，郑怀钟。我们还要感谢莎拉帕里什帮助伴随视频的文本。

基因枪转染议定书“ 让子弹子弹长达6个月，但可能会减少2-3个月后开始转染效率。干燥剂颗粒的存在，子弹应保存在4 ° C。始终让闪烁瓶，存储在其中的子弹，温暖的室温打开小瓶之前。 在开始之前已经做好准备： <ul><li>亚精胺（0.05M） </li><li> 氯化钙 （1M） </li><li>乙醇（100％高档，未开封的一瓶） </li><li>蒸压H 2 0 </li><li> DNA转染</li><li>聚乙烯吡咯烷酮（PVP - 20毫克/毫升的股票） </li><li> 2 × 15 ml锥形（2/bullet设置） </li><li>管（X - 30英寸/子弹设置削减到1） </li><li>闪烁瓶（1/bullet集） </li><li>干燥颗粒</li><li> 10 ml注射器W /到底管</li></ul>准备管： <ol><li>干〜30英寸的管道，在管站W / N 2气体（0.4压力）至少20分钟（约2英寸扩展超越权利O形圈） 。 </li></ol>沉淀DNA的黄金珠： <ol><li>准备将在50μmL离心管总量（最高50微克总DNA）转染的DNA </li><li>称取6-8毫克的黄金和转让的离心管</li><li>加入100μL0.05M亚精胺的黄金和超声20秒</li><li>加入50微升的DNA的黄金/精胺</li><li>涡（周围设置5）W /盖打开</li><li>新增下降明智的100μL1M 氯化钙 </li><li>超声简要（&lt;5秒） </li><li>在室温为10分钟，允许沉淀</li><li>超声简要</li></ol>准备在超声PVP的解决方案： <ol><li>做稀PVP的解决方案，为每个子弹15 ml锥形，3.5mL稀释的PVP（100毫升3.5％的乙醇添加7.5μL20mg/ml PVP的股票） </li></ol>乙醇漂洗黄金珠： <ol><li>全速离心10秒中的微量黄金珠</li><li>弃上清，但保留的少量液体黄金珠上</li><li>洗净用1ml乙醇的3倍，超声简要每一次，如有必要</li></ol>悬浮金/ DNA在PVP的解决方案： <ol><li>重悬在3.5毫升稀释PVP /乙醇溶液200μL的黄金颗粒</li><li>涡街的黄金颗粒重悬（如果有必要简要超声） </li><li>金/ PVP溶液200μL转移到一个新的15 mL锥形</li><li>继续以这种方式，加入200μL的PVP一次，直到所有的黄金已转移至15 mL锥形，最终体积为3.2毫升</li></ol>涂层管： <ol><li>关闭管站N 2和取出干管</li><li>将干管10毫升注射器</li><li>大力摇晃15 mL锥形填充金/ PVP </li><li>放置在金/ PVP溶液管材年底和吸慢慢小心，以避免气泡</li><li>从油管两端保留1〜2英寸干</li><li>随着油管仍然附着在注射器和充满黄金/ PVP的解决方案，小心地进入车站的油管回</li><li>马克油管的解决方案坐在两端</li><li>让黄金与注射器连接5分钟解决</li><li>吸的解决方案非常缓慢拉动注射器使落户金留下（确保不反洗） </li><li>拔下注射器</li><li>旋转90 °，让我们坐2秒</li><li>旋转180 °，让坐在另2秒</li><li>旋转管，持续5秒</li><li>打开了N 2，使阀门读取0.4 - 0.5和旋转镀金管，而干燥5分钟。 </li></ol>切割管材： <ol><li>取出油管从油管准备站和切断两端的标志。 </li><li>把管斩波器的下一个标记的闪烁瓶中赶上削减墨盒</li><li>管放置在管斩波器和切割油管清洁剃须刀</li><li>闪烁瓶中放置在一个干燥颗粒</li><li>商店墨盒在4 ° C </li></ol>拍摄组织切片培养片拍摄时，重要的是聘请一个相对无菌的技术，以避免污染。请记住，子弹两套拍摄时，两个不同的结构，可以装到同一盒支架，但桶内衬和屏幕之间的结构应改变。 在开始之前已经做好准备： <ul><li> DNA的子弹（让闪烁瓶中的热烈开幕前室温） </li><li>组织切片</li><li>太阳神基因枪</li><li>氦气罐用基因枪软管连接&lt;/ LI&gt; <li>盒支架</li><li>在内衬塑料桶与O形圈</li><li>扩散器（修改） </li><li>漫射屏</li><li>镊子</li></ul>预备基因枪： <ol><li>使用镊子放入镀金塑料匣盒支架。 </li><li>先推回锁放置到基因枪盒支架</li><li>安全锁盒支架</li><li>获得一个O形环和安全的地方扩散屏幕每桶班轮</li><li>基因枪拧入每桶班轮</li></ol>用基因枪射击培养切片： <ol><li>戴上耳罩</li><li>基因枪插入软管连接氦气罐</li><li>打开水箱压力为180 PSI的</li><li>拍摄到空气中的空白，以消除从扩散器屏幕上的灰尘或碎片。要拍摄的枪，第一压低，直到枪鸣音安全联锁按钮，然后扣动扳机。 </li><li>拍摄后的空白，从孵化器中取出切片“ </li><li>预先枪首先构造</li><li>拍摄扩散屏幕约0.5 - 1英寸片</li><li>推进井间的墨盒。 </li><li>去年以及拍摄后，将切片放入培养箱</li><li>脱离氦软管枪之前，关闭气体和释放压力</li><li>拧开桶内衬，并取出硒鼓和使用的炮弹</li></ol>清洗墨盒持有人及桶内衬： <ol><li>放置盒，桶内衬，并用肥皂水在一个烧杯中的扩散屏幕</li><li>将烧杯中的浴缸sonicator和20分钟的超声</li><li>彻底用清水冲洗干净，肥皂残留物都将被删除，直到所有</li><li> 70％乙醇中浸泡了一个小时</li><li>干纸巾上放置过夜干</li><li>在随后的基因枪转染可以重复使用清洁盒，桶内衬和屏幕</li></ol>基因枪法转的故障排除技巧 基因枪法转，有时可能会导致不理想的转染条件 。 它可以导致过少或过多的脑细胞转染，过高或过低的表达水平，或可能会导致组织切片成为普遍不健康 。 往往是不健康片压力爆炸的结果。 这里有一些提示，以获得最佳的转染的组织切片。 如果片似乎是不健康的（或如果有太多的细胞转/片），请尝试： <ul><li>拍摄距离增加 </li><li>煤气罐上的压力下降</li><li> 减少的数量的黄金</li><li>切割出在Bio - Rad扩散的中心，并取代与扩散屏幕​​中心。 </li></ul>如果太少的细胞转染，尝试： <ul><li> 减少拍摄距离</li><li>煤气罐上的压力越来越大 </li><li> 增加黄金数量</li><li>组织切片的数目增加/ </li><li>确保在每桶班轮的O型圈是否完好。 </li></ul>如果你得到太多和太少的细胞在相同的拍摄 ，确保转片： <ul><li>你是拿着枪对称以上的</li><li>黄金是均匀地涂在管内。 （如果你已经看到了黄金线 ，从油管以更快的速度绘制出上清液，一旦黄金已落户和旋转，然后再打开氮气的黄金时间，另一方面， 如果你正在黄金裸奔这可能是由于过多的水分在子弹使曝光：确保您使用的是一瓶未开封的乙醇，管涂装前彻底干燥，和您封顶盖和及时准备的子弹）。 </li></ul>如果转染效率似乎最佳（＃细胞转/片），但表达水平似乎过高或过低： <ul><li>调整比例DNA黄金。 （例如，如果它太低保持的黄金数量， 但增加 DNA的量。） </li><li>调整拍摄和数据收集之间的时间量</li></ul>双转的情况下，如果你得到的太少，表达你的构造之一，以适当的方式调整两种结构的比例，并确保这两个构造相同的发起人，以确保一发起人不会出其他竞争。

<ol>
	<li>Klein, T. M., Fromm, M., Weissinger, A., Tomes, D., Schaaf, S., Sletten, M., Sanford, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+foreign+genes+into+intact+maize+cells+with+high-velocity+microprojectiles.">Transfer of foreign genes into intact maize cells with high-velocity microprojectiles.</a> Proc. Natl. Acad. Sci. USA. 85, 4305-4309 (1988).</li><li>Wellmann, H., Kaltschmidt, B., Kaltschmidt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+protocol+for+biolistic+transfection+of+brain+slices+and+dissociated+cultured+neurons+with+a+hand-held+gene+gun.">Optimized protocol for biolistic transfection of brain slices and dissociated cultured neurons with a hand-held gene gun.</a> J. Neurosci. Methods. 92, 55-64 (1999).</li><li>McAllister, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biolistic+transfection+of+neurons.">Biolistic transfection of neurons.</a> Sci. STKE. 51, 1-13 (2000).</li><li>Svoboda, K., Yasuda, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+two-photon+excitation+microscopy+and+its+applications+to+neuroscience.">Principles of two-photon excitation microscopy and its applications to neuroscience.</a> Neuron. 50, 823-839 (2006).</li></ol>

基因枪法转高速DNA涂金粒子轰击与活体组织细胞的转染通过物理手段。在粒子介导的基因转移，导致在一般的转染细胞时，在细胞核中的子弹来休息。尽管存在许多不同的转染方法，如显微注射法，脂质体转染，转染钙磷，电和病毒转染，基因枪转染可以提供劳动密集，转染细胞是不容易转用其他方法和有效的替代方案。事实上，这是首次在植物基因转移技术开发使用预先存在的方法1由于细胞壁的存在，所以很难作出转。同样，biolistics已经获得了在神经生物学领域的普及，因为有丝分裂后神经元是出了名的难以转染2,3。特别是，它是举世公认的原则，技术被用于实验旨在评估完整的脑切片中的单个神经元的罚款形态。这里所描述的方法提供了详尽和全面的解释如何使用基因枪举行的手（的Helios基因枪系统; Bio - Rad公司）进行基因枪转染培养的脑切片。 基因枪转染已成为转染片文化中的神经元的首选方法，因为它允许稀疏数量的整个大脑切片的细胞​​转染。这样，单个神经元可在隔离审查。由于这种技术特别适合转脑片神经元，它往往是使用双光子显微镜结合，使双光子激发荧光标记的细胞内光散射组织 4深的可视化以来。事实上，整个视频中的单锥体神经元的高倍率图像使用一个定制的双光子激光扫描显微镜被抓获。结论基因枪转是转染单个细胞内的一个细胞周围的高密度的背景下的有效手段，这种已被证明非常有用的荧光标签的单个神经元的神经切片文化。

虽然我们分别列出了所有产品，它是更经济的购买“赫利俄斯基因枪系统”，其中包括与Bio - Rad公司（CAT＃165-2431）上面列出的所有产品。

preparation of gene gun bullets and biolistic transfection of neurons in slice culture

基因枪法转的转高速DNA包覆颗粒轰击组织细胞的物质手段。我们提供了详细的基因枪转染大鼠海马脑片的协议，从DNA包覆子弹的前期准备工作，以最终拍摄使用基因枪的器官切片文化。基因枪转染，转染神经元的高效和便捷的手段和荧光标记的细胞组织切片中的一小部分是特别有用。在这段视频中，我们首先轮廓大衣金粒子与DNA所需的步骤。接下来我们演示如何行与金/ DNA子弹的塑料管内，以及如何削减油管获得加载到基因枪的塑料墨盒。最后，我们进行基因枪转染大鼠海马脑片培养，显示处理Bio - Rad公司赫利俄斯基因枪，并提供故障排除的意见，以获得健康的最佳转的组织切片。

Watch this Scientific Journal Video about Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture at JoVE.com

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

我们描述了一个准备DNA涂金子弹的方法，并展示了使用这种子弹biolistically转染培养的海马神经元。

基因枪子弹的制备和神经元的基因枪转染切片文化

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed ...

preparation-gene-gun-bullets-biolistic-transfection-neurons-slice

Research

JoVE Journal

Biology

52.2K 次观看.  University of California, Davis. 我们描述了一个准备DNA涂金子弹的方法，并展示了使用这种子弹biolistically转染培养的海马神经元。

视频: Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

Horizontal Slice Preparation of the Retina

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of retinal neurons, such as amacrine cells, expand their dendrites horizontally, so that the morphology of the cells is supposed to be severely damaged in the vertical slices. In the whole-mount preparation, especially for patch-clamp recordings, retinal neurons in the middle layer are not easily accessible due to the extensive coverage of glial cell (Mueller cell) s endfeets. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact. The slice was made horizontally at the inner layer of the retina using a vibratome slicer after the retina was embedded in the low-temperature melting agarose gel. In this horizontal slice preparation of the retina, we studied the function of retinal neurons compared with their morphology, by using patch-clamp recording, calcium imaging technique, immunocytochemistry, and single-cell RT-PCR.

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. Here, we describe the novel slicing method to preserve the dendritic morphology of retinal neurons intact.

视网膜水平切片制备

Traditionally the vertical slice and the whole-mount preparation of the retina have been used to study the function of retinal circuits. However, many of ...

科研

生物学

Gene-gun Transfection of Hippocampal Neurons

海马神经元的基因枪转

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

器官型E18大鼠脑切片文化

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

文化髓系树突状细胞从骨髓前体

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for neuronal survival and function. Neurotrophins are specifically required to maintain the survival and differentiation of innervating sensory neurons but the question of how these target-derived neurotrophins communicate to the cell body of innervating neurons has been an area of active research for over 30 years. The most commonly accepted model of how neurotrophin signals reach the cell body proposes that signaling endosomes carry this signal retrogradely along the axon. In order to study retrograde transport, a culture system was originally devised by Robert Campenot, in which cell bodies are isolated from their axons. The technique of preparing these compartmented chambers for culturing sensory neurons recapitulates the selective stimulation of neuron terminals that occurs in vivo following release of target-derived neurotrophins. Retrograde signaling events that require long-range microtubule dependent retrograde transport have important implications for the treatment of neurodegenerative disorders.

Here we describe the technique of preparing and maintaining compartmented chambers for culturing sensory neurons of the dorsal root ganglia.

隔间文化背根神经节神经元的制备及维修

Neurons extend axonal processes that are far removed from the cell body to innervate target tissues, where target-derived growth factors are required for ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

双分子荧光互补（附设）检测蛋白质相互作用的洋葱细胞使用的Helios基因枪

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

从小鼠胚胎肾肾区细胞的分离和培养

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

在细胞培养中缺氧诱导和测试

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.