作者要感谢来自日本科学技术振兴机构的探索性研究的先进技术（JST-ERATO）中，新能源产业技术综合开发机构（NEDO）和跨部级战略创新促进计划（SIP），日本的资金。

1.基于肽制剂的制备<ol><li>制备的每种肽的储备溶液如下:（KH）9 -BP100（1毫克/毫升或800纳米），Cytcox-（KH）9（1毫克/毫升），BP100（1毫克/毫升）和（BP100）2 K 8（70微米）。称重所需的每种肽的量在1.5ml微量离心管中，并添加蒸压超纯水以溶解肽。直到得到澄清的溶液反复吹打拌匀。 </li><li>扩增并纯化质粒DNA（质粒DNA），双链DNA（dsDNA）的和根据标准分子方法的双链RNA（dsRNA）的。在1.5ml微量离心管，使储备溶液具有1毫克/毫升（质粒DNA和双链DNA）或400nM的（dsRNA）的浓度。 </li><li>制备蛋白原液以7μm的浓度，以1 mg蛋白质（ 例如 ，醇脱氢酶，ADH）粉末溶解在1毫升碳酸钠溶液（0.1M，pH为9）。标签使用f蛋白质根据标准方法luorescent探针如若丹明B（罗丹明B），以使递送到细胞中的蛋白质的微观可视化。 </li><li>结合在1.5ml微量离心管中的各个部件。 <ol><li>对于肽的pDNA制剂靶向细胞质中，添加6.4微升（KH）9 -BP100（1毫克/毫升），以20微升的pDNA（1毫克/毫升），并通过移液混合均匀。使混合物在25℃下稳定15分钟。添加773.6微升灭菌超纯水稀释至800微升的最终体积。使用质粒DNA构建体设计用于核表达（P35S-与Rluc-TNOS， 表1）。 </li><li>对于肽的pDNA制剂靶向线粒体，添加6.6微升Cytcox-的（KH）9（1毫克/毫升），以20微升的pDNA（1毫克/毫升），并通过移液混合均匀。使混合物在25℃下稳定15分钟，然后添加BP100 2.4微升（1毫克/毫升）。使混合物在25稳定15分钟6，下再15分钟。添加771微升灭菌超纯水稀释至800微升的最终体积。使用质粒DNA构建体设计用于线粒体表达（PDONR-COX2:与Rluc， 表1）。 </li><li>对于肽双链DNA的配方，添加5.1微升（KH）9 -BP100（1毫克/毫升），以8微升双链DNA（1毫克/毫升），并通过移液拌匀。使混合物在25℃下稳定15分钟。添加786.9微升灭菌超纯水稀释至800微升的最终体积。 </li><li>对于肽dsRNA的配方，加入50微升（KH）9 -BP100（800纳米）至50μl双链RNA（400纳米）和吹打拌匀。使混合物在25℃下稳定15分钟。加入700微升的无RNase水稀释该溶液到800微升的最终体积。 </li><li>对于肽蛋白制剂，加入16微升（BP100）2 K 8（70微米）到ADH的16微升（7微米），混合深受移液。使混合物在25℃下稳定15分钟。添加768微升灭菌超纯水稀释至800微升的最终体积。 </li></ol></li><li>允许制剂在25℃下稳定15分钟。 </li></ol> 2.基于肽制剂的表征<ol><li>每个转移溶液（800微升）转化为动态光散射（DLS）分析比色皿。确定形成的复合物的流体动力学直径和多分散性指数用的ζNanosizer测，使用633纳米的氦氖激光器，在25℃下的173°的反向散射检测角度。 </li><li>以下大小的测量，将各溶液（800微升）在介电常数，折射率和水粘度的默认参数，在25℃转移到折叠毛细管单元用于zeta电位测量。 </li><li>观察小体积络合物溶液，用于DLS分析，通过原子力显微镜（AFM）。存10微升络合物溶液到云母片的新暴露的切割表面和离开该云母，以空气中的有盖的塑料培养皿干燥过夜。 </li><li>在轻敲模式27,28使用硅悬臂具有1.3牛顿/米的弹簧常数在25℃获得在空气中的复合物的图像。 </li></ol> 3.植物叶片渗透<ol><li>用3周龄的土壤中生长的植物（ 拟南芥 24， 本生烟 24或26杨树）。转染至少3片叶，作为一式三份的基因表达或蛋白质输送的量化。 </li><li>装载1毫升的无针注射器，塑料用100微升一个叶转复杂的解决方案。叶子的背面表面上的注射器的尖端的位置。 </li><li>按压叶注射器尖端稍慢慢压低注射器活塞，而施加反压的我胶乳手套的手的手指ndex从相对侧。成功的浸润，可以观察到在叶的水浸泡部的扩散。为便于识别标签的渗透叶子。 </li><li>孵育优化持续时间的转叶以下用肽的pDNA（12小时），肽的双链DNA（12小时），肽的dsRNA浸润 - 在下述条件下（9 48小时）和肽 - 蛋白（6小时）的配方:16小时光/在29℃，22℃， 拟南芥和杨树，或24小时恒亮的烟草本塞姆氏 8小时黑暗。 </li></ol> 4.评价转染效率<ol><li>切除小型工厂的整体转叶（ 例如 ， 拟南芥 ）或渗透区域的大型水泥厂1厘米2部分（ 例如 ， 烟草本塞姆氏 ）。 </li><li>对于使用Renilla荧光素酶（与Rluc）报告载体转染实验，确定transfectio氮效率定量使用它们间检测试剂盒。 <ol><li>放置在1.5ml微量离心管各切叶或叶节。加入100微升1×它们间测定裂解液每管。以相同的方式，制备非转染的对照叶（一式三份）的裂解物。 </li><li>磨用均质杵的叶和孵育在25℃下将所得溶胞产物为6 - 10小时。 </li><li>离心在12470×g下的裂解物中为1分钟的离心。转移20微升澄清的裂解液的一个井在96孔微量培养板，以及用于使用根据制造商的协议的BCA蛋白质测定试剂盒总蛋白质浓度的定量剩余体积。 </li><li>加入100微升1×它们间检测底物（使用它们间试验缓冲液）的入井，并缓慢吹打混合。放置在一个多模酶标仪微孔板并启动测量。 </li><li>减去背景发光（指的是没有正转从每个实验样品的发光式三份）。计算的光致发光，以蛋白（毫克）的量的比率（相对光单位，RLU）。 </li></ol></li><li>对于使用绿色荧光蛋白（GFP）的报告载体或荧光标记的蛋白质的转染实验（ 例如 ，ADH-罗丹明B），使用共聚焦激光扫描显微镜观察荧光。 <ol><li>切全叶（以有助于除去空气空间）的边缘，而可使用一个切片叶原样。从10毫升注射器卸下柱塞，并放置在注射器的每个切叶或叶节。 </li><li>更换柱塞，轻轻推到注射器的底部，但不会破碎的叶子。打水入注射器，直到它被大约一半填充。 </li><li>指向上的注射器和柱塞推到从通过尖端注射器除去空气。覆盖注射器的尖端并拔出柱塞向下慢慢从LEA排出空气F。重复此过程数次，直到叶出现半透明。 </li><li>覆盖胶带显微镜载玻片的表面上。切割胶带大到足以容纳使用刀片叶样品上的正方形区域，和剥离的正方形片胶带的关闭用镊子以创建一个样品室。磁带将作为幻灯片和盖玻片之间的间隔物。 </li><li>放置叶朝上的背面室和装满水的剩余室面积。密封用玻璃盖玻片的腔室中的叶片和用胶带固定盖玻片的边缘。 </li><li>检查使用下一个40倍物镜的共聚焦激光扫描显微镜或63X水浸物镜的叶样品。 GFP或罗丹明荧光可以在分别488纳米或555纳米，激发波长可视化。 </li></ol></li></ol>

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S., Serino, G., Chaudhuri, S., Maliga, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plastid+transformation.">Plastid transformation.</a> Arabidopsis thaliana Plant Cell Rep. 18, 20-24 (1998).</li><li>Svab, Z., Hajdukiewicz, P., Maliga, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+transformation+of+plastids+in+higher+plants.">Stable transformation of plastids in higher plants.</a> Proc. Natl. Acad. Sci. U. S. A. 87, 8526-8530 (1990).</li><li>Liu, Y. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementation+of+plant+mutants+with+large+genomic+DNA+fragments+by+a+transformation-competent+artificial+chromosome+vector+accelerates+positional+cloning.">Complementation of plant mutants with large genomic DNA fragments by a transformation-competent artificial chromosome vector accelerates positional cloning.</a> Proc. Natl. Acad. Sci. U. S. A. 96, 6535-6540 (1999).</li><li>Park, H. S., Lee, B. M., Salas, G. M., Srivatanakul, M., Smith, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shorter+T-DNA+or+additional+virulence+genes+improve+Agrobactrium-mediated+transformation.">Shorter T-DNA or additional virulence genes improve Agrobactrium-mediated transformation.</a> Theor. Appl. Genet. 101, 1015-1020 (2000).</li><li>Frary, A., Hamilton, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficiency+and+stability+of+high+molecular+weight+DNA+transformation:+an+analysis+in+tomato.">Efficiency and stability of high molecular weight DNA transformation: an analysis in tomato.</a> Transgenic Res. 10, 121-132 (2001).</li><li>Que, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maize+transformation+technology+development+for+commercial+event+generation.">Maize transformation technology development for commercial event generation.</a> Front. Plant Sci. 5, 379 (2014).</li><li>Miranda, A., Janssen, G., Hodges, L., Peralta, E. G., Ream, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium+tumefaciens+transfers+extremely+long+T-DNAs+by+a+unidirectional+mechanism.">Agrobacterium tumefaciens transfers extremely long T-DNAs by a unidirectional mechanism.</a> J. Bacteriol. 174, 2288-2297 (1992).</li><li>Loeb, T. A., Spring, L. M., Steck, T. R., Reynolds, T. L., Bajaj, Y. P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+wheat+(Triticum+spp.).">Transgenic wheat (Triticum spp.).</a> Transgenic Crops I. , 14-36 (2000).</li></ol>

作者什么都没有透露。

已经凭经验确定的协议中的关键步骤进行了讨论。通过注射器渗入，基于肽的制剂引入到通过气孔植物叶内的空域。以确保溶液的最大吸收，渗透过程应进行时的植物有利于气孔开口即条件下，提供足够的水，并在光周期下。至于转染复合物的制备，对涉及两个肽（线粒体靶向DNA递送）的组合制剂，用于添加各成分的顺序是至关重要的，不应该被反转。 有许多可行的修改的过程。对于植物细胞的浸润另一种选择是使用真空浸润，这是能够在复杂的溶液引入整个植株和/或部分组织，包括顶端分生组织。对于此替代过程，苗浸没在转染溶液，并基于由真空产生的压力，所述肽 - 货物复合物通过气孔和进植物细胞（吉住，T。，未发表的数据）被迫。 瞬时基因表达的基础上，使用动物细胞的研究中，已被证明能增加与更高的DNA浓度29-31。要注意的是肽的DNA的比例会影响复合物的生物物理性质（大小，表面电荷）是非常重要的（ 图4），从而影响转染效率;因此，最佳的比率需要甚至具有增加的DNA浓度被维持。 之一的在使用肽作为基因/蛋白输送剂中的主要优点是，它的顺序是适合于调谐到满足所希望的功能。例如，所述载体肽的线粒体靶向域在该螺柱描述y可能与本地化这些细胞器叶绿体或过氧化物酶体靶向序列所取代。而叶绿体转化使用基因枪法32-34在几种植物是可能的，既不是农杆菌也不是生物射弹方法能够将基因导入线粒体或其它细胞器除了核（ 表2）。 有用于基于肽的转染没有刚性宿主范围限制，不像农杆菌基于方法（ 表2）。到目前为止，对于拟南芥 ， 烟草本塞姆氏和杨树的转染制剂已被优化（ 表1），但也可以适用于烟草的方法中， 番茄品种微汤姆，稻（基于预备实验），和其它的单 - 和双子叶植物。 迄今为止，有在转基因大小没有已知的限制时肽用于一小号转染载体。与此相反，已发现大量的DNA分子，以减少的土壤杆菌介导的方法35,36的转化效率，以及用于已报道37-39的约200kb的转基因的大小上限。使用基因枪，在另一方面，大的DNA片段可以制备或输送到植物中38中剪切。虽然没有上限被确定为生物射弹转化，到目前为止，已发现物理约束来限制可被转移到远小于150 kb的40 DNA的大小。使用相比于采用任何农杆菌或微粒轰击现有方法，以上所讨论的用于基因转移的基于肽的系统的主要优点，归纳在表2中 。 一些限制这种方法存在，因为它主张。首先，有针对性的pDNA的递送至特定的细胞器，如MITochondria，虽然效率较低已被证实可以通过DNA结合，细胞穿透和细胞器转运序列的简单组合。转基因表达可以被检测到，通过共聚焦显微术，仅在细胞内的线粒体的一个小口。因此，进一步的改进是必要的:（ⅰ）提高整个小区/细胞器膜更配合物的易位，以及（ii）提高的解离和从载体肽的pDNA转移到靶细胞器中表达。其次，使用该DNA递送系统中，外源报道基因的瞬时表达，成功在细胞的胞质和线粒体隔室来实现的。在植物核/细胞器基因组中导入的基因的稳定掺入和表达都尚未建立，但是，由于缺乏合适的选择策略。 虽然承认有改善或再经过D区才有发展，这里所描述的肽衍生战略仍然是一个简单而通用的技术已经铺平了交付各种货物到不同植物类型的方式。

Plant genetic engineering is conventionally used for transferring beneficial traits to plants. In recent years, this technology has been applied to convert plants into bio-factories for the production of pharmaceutically important and commercially valuable proteins, many of which cannot be chemically synthesized and are very costly to produce using animal or microbial systems1. By the introduction of new genes, the plant's own metabolism can be manipulated for the production of various biopharmaceuticals like antibodies, metabolic enzymes, hormones, antigens or vaccine2.
Established gene transfer technologies for plants are the Agrobacterium-mediated delivery3, bombardment with DNA-coated microprojectiles (biolistics)4, and electroporation5 or polyethylene glycol6 treatment of protoplasts. Techniques requiring protoplasts are generally avoided because they are time-consuming, cumbersome and yield inconsistent results7. As a result, virtually all plant modification work at present utilizes either Agrobacterium or microprojectiles for gene transfer. The Agrobacterium method is more extensively used but not applicable to many economically important plant species. Meanwhile, microprojectile bombardment is more versatile due to a broad range of susceptible plants but requires specialized equipment and often causes severe tissue damage. Furthermore, these methods involve either a random (biolistics) or complex (Agrobacterium) delivery mechanism and have variable transformation rates8. Hence, a novel plant transformation technology that is simple yet effective, and applicable to different plant types is required.
Currently, plants are subjected to genetic modification primarily by the delivery of exogenous DNA encoding a desired trait, rather than by direct delivery of a target protein. The higher stability of DNA over proteins is a prime advantage; nevertheless, potential problems associated with DNA delivery include the random insertion of exogenous DNA into the plant genome and unintended transmission of antibiotic resistance genes to pathogenic bacteria via horizontal gene transfer9. For genome-editing purposes, the ability to edit plant genomes without introducing foreign DNA into cells may circumvent regulatory concerns related to genetically modified plants. Thus, an alternative DNA-free strategy for the modification of plants by direct delivery of protein will be able to cater to these needs.
Here we introduce a peptide-based system, originally developed for human gene therapy10-14, for the targeted delivery of exogenous genes or proteins in intact plants. Peptides are able to protect DNA from nuclease degradation and can mediate gene transfer across cell as well as organellar membranes15-17. They also have diverse and tunable properties besides being non-cytotoxic18-20. More importantly, with the use of peptides, genes can be precisely targeted to intracellular organelles such as the mitochondria21 or plastids (chloroplasts)22 for expression-a task not achievable by biolistic or Agrobacterium-mediated transformation. Depending on the cargo type, this new plant modification technology can be exploited to deliver proteins23 and either express (plasmid21,24 or double-stranded DNA25) or down-regulate (double-stranded RNA26) specific genes within the plant, throughout its cytosolic space24-26 or within a specific organellar compartment21. The designed carrier peptides consist of a cationic domain in the form of either polylysine (K8) or polylysine-co-histidine (KH)9 for binding and/or condensation of negatively-charged cargoes, which is conjugated to cell penetrating (BP100 peptide) or mitochondria transit (Cytcox peptide) sequences.

<table border="0" cellpadding="0" cellspacing="0" width="2358">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:453px;">(KH)9-BP100 peptide</td>
			<td style="width:355px;">Custom-synthesized by RIKEN Brain Science Institute</td>
			<td style="width:315px;">N/A</td>
			<td style="width:1235px;">Sequence: KHKHKHKHKHKHKHKH- 
			KHKKLFKKILKYL</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:453px;">(BP100)2-K8 peptide</td>
			<td>Custom-synthesized by RIKEN Brain Science Institute</td>
			<td style="width:315px;">N/A</td>
			<td>Sequence: KKLFKKILKYLKKLFKKIL- 
			KYLKKKKKKKK</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BP100 peptide</td>
			<td>Custom-synthesized by RIKEN Brain Science Institute</td>
			<td style="width:315px;">N/A</td>
			<td>Sequence: KKLFKKILKYL</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Cytcox-(KH)9 peptide</td>
			<td>Custom-synthesized by RIKEN Brain Science Institute</td>
			<td style="width:315px;">N/A</td>
			<td>Sequence: MLSLRQSIRFFKKHKHKH- 
			KHKHKHKHKHKH</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">P35S-GFP(S65T)-TNOS and P35S-RLuc-TNOS</td>
			<td style="width:355px;">N/A</td>
			<td style="width:315px;">N/A</td>
			<td style="width:1235px;">Encodes green fluorescent protein and Renilla luciferase genes, respectively, under the control of CaMV 35S constitutive promoter (Ref: Lakshmanan et al. Biomacromolecules 2013, 14, 10)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pDONR-cox2:gfp and pDONR-cox2:rluc</td>
			<td style="width:355px;">N/A</td>
			<td style="width:315px;">N/A</td>
			<td style="width:1235px;">Encodes green fluorescent protein and Renilla luciferase genes, respectively, under the control of cox2 mitochondrial-specific promoter (Ref: Chuah et al. Sci Rep 2015, 5, 7751)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">dsDNA (PCR-amplified from pBI221-P35S-Rluc-TNOS)</td>
			<td style="width:355px;">N/A</td>
			<td style="width:315px;">N/A</td>
			<td style="width:1235px;">Encodes green fluorescent protein and Renilla luciferase genes, respectively, under the control of CaMV 35S constitutive promoter (Ref: Lakshmanan et al. Plant Biotechnol 2015, 32, 39)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GFP5 siRNA</td>
			<td style="width:355px;">N/A</td>
			<td style="width:315px;">N/A</td>
			<td style="width:1235px;">For RNA interference-mediated silencing of green fluorescent protein (Ref: Numata et al. Plant Biotechnol J 2014, 12, 1027)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CHS siRNA</td>
			<td style="width:355px;">N/A</td>
			<td style="width:315px;">N/A</td>
			<td style="width:1235px;">For RNA interference-mediated silencing of chalcone synthase (Ref: Numata et al. Plant Biotechnol J 2014, 12, 1027)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">1 mL and 10 mL Plastic Syringes</td>
			<td>TERUMO Corporation</td>
			<td>SS-01T, SS-10ESZ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">1.5 mL Microcentrifuge Tube</td>
			<td>AS ONE Corporation</td>
			<td>151212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">96-Well Flat-Bottom Plate</td>
			<td>Asahi Glass Co., Ltd.</td>
			<td>3860-096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Adhesive Tape</td>
			<td>Sekisui Chemical Co., Ltd.</td>
			<td>No. 835</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Alcohol Dehydrogenase</td>
			<td style="width:355px;">Sigma-Aldrich Co., LLC.</td>
			<td style="width:315px;">A-7011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Atomic Force Microscope</td>
			<td>Seiko Instruments Inc.</td>
			<td>SPI3800, SPA 300HV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Atomic Force Microscope</td>
			<td>Hitachi High-Tech Science Corporation</td>
			<td>AFM5300E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">BCA Protein Assay Kit</td>
			<td>Thermo Fisher Scientific Inc.</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Cantilever</td>
			<td>Hitachi High-Tech Science Corporation</td>
			<td>K-A102001593</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Confocal Laser Scanning Microscope</td>
			<td>Carl Zeiss</td>
			<td>LSM700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Cork Borer</td>
			<td style="width:355px;">Sigma-Aldrich Co., LLC.</td>
			<td>Z165220</td>
			<td>For excision of leaves into 1-cm diameter disks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Coverslip</td>
			<td style="width:355px;">Matsunami Glass Ind., Ltd.</td>
			<td style="width:315px;">C02261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Cuvette</td>
			<td style="width:355px;">Sarstedt</td>
			<td style="width:315px;">759116</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Folded Capillary Cell</td>
			<td style="width:355px;">Malvern Instruments, Ltd.</td>
			<td style="width:315px;">DTS1070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Forceps</td>
			<td style="width:355px;">Shimizu Akira Inc.</td>
			<td style="width:315px;">Stainless pincet 150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Homogenization Pestle</td>
			<td>Ieda Trading Corp.</td>
			<td style="width:315px;">9993</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Mica</td>
			<td>Nisshin EM Co., Ltd.</td>
			<td style="width:315px;">LC23Z</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Microcentrifuge</td>
			<td>Beckman Coulter</td>
			<td style="width:315px;">BKA46472</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microplate Reader</td>
			<td>Molecular Devices Corporation</td>
			<td style="width:315px;">Spectra MAX M3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Microscope Slide</td>
			<td style="width:355px;">Matsunami Glass Ind., Ltd.</td>
			<td>S011120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Pipette</td>
			<td style="width:355px;">Eppendorf Research&#174; plus</td>
			<td>3120000909</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Pipette Tips</td>
			<td>AS ONE Corporation</td>
			<td>2-5138-01, 2-5138-02, 2-5138-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Plastic Petri Dish</td>
			<td>AS ONE Corporation</td>
			<td>1-7484-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Renilla Luciferase Assay Kit</td>
			<td>Promega corporation</td>
			<td>E2810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Rhodamine B Isothiocyanate</td>
			<td style="width:355px;">Sigma-Aldrich Co., LLC.</td>
			<td style="width:315px;">283924</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">RNase-Free Water</td>
			<td style="width:355px;">Qiagen</td>
			<td style="width:315px;">129112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Sodium Carbonate</td>
			<td>Wako Pure Chemical Industries, Ltd.</td>
			<td>199-01585</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Surgical Blade and Handle</td>
			<td>FEATHER Safety Razor Co., Ltd.</td>
			<td>Stainless steel No. 14 (blade), No. 3L (handle)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Syringe Tip Cap</td>
			<td>Musashi Engineering Inc.</td>
			<td>NC-3E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Weighing Balance</td>
			<td>Sartorius</td>
			<td>CPA225D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Zeta Potentiometer</td>
			<td>Malvern Instruments, Ltd.</td>
			<td>Zetasizer Nano-ZS</td>
			<td style="width:1235px;"></td>
		</tr>
	</tbody>
</table>

peptide-derived method to transport genes and proteins across cellular and organellar barriers in plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

核酸和蛋白质的货物的阵列被成功地引入到使用设计的肽作为递送载体的各种植物。阳离子肽和带负电荷的货物之间的静电相互作用导致转染复合物的形成，可直接渗入植物使用无针注射器（ 图1）叶。优化制剂的植物细胞的转染（在这些研究中21,23-26经验确定）列在表1中 ，其中的大范围递送货物（质粒DNA，双链DNA，双链RNA和蛋白质）的各类型的代表。所有基于肽的制剂的平均直径为在150的大致范围内 - 300纳米。根据DLS分析，所有制剂显示相对低的大小多分散性，这表明形成肽 - 货物聚集体具有均匀的尺寸分布。形貌肽和质粒DNA（ 图2A）或蛋白质（ 图2B）之间的复合物，在云母，由原子力显微镜成像。观察均匀的球状物为肽的pDNA和肽 - 蛋白质组合，与从DLS测量数据的协议。在配合物（ 表1）的ζ电位而言，pDNA-和双链DNA衍生的复合物具有净负表面电荷，而基于双链RNA复合物具有一个接近中性表面电荷。肽 - 蛋白质复合物，在另一方面，被带正电。 肽质粒DNA，并在调解拟南芥或烟草本塞姆氏作为模式植物系统的转肽dsDNA抗体制剂的效率进行了定量评价以及定性。在与Rluc基因表达测定法用于基因表达水平的定量（ 表1），因此，对于这个实验的pDNA或双链DNA编码与Rluc基因必须用于络合与各自的载体肽。使用（KH）9 -BP100 /质粒DNA制剂，核靶向递送和质粒DNA的表达，可以实现以下12小时的潜伏期，以大约1×10 5的估计RLU /毫克值。对于质粒DNA，肽的组合的线粒体靶向递送和表达，Cytcox-（KH）9和BP100，需要为复合物形成。用12小时的相同的优化潜伏期，然而，获得转染（约1×10 3 RLU /毫克）的低得多的水平。同时，需要类似潜伏期（12小时）和基因表达水平（约1×10 3 RLU /毫克）/记录基于双链DNA复合物，使用（KH）9 -BP100肽还配制。基因表达的定性评估，通过复合prepar处理的叶片直接显微镜观察下进行ED使用质粒DNA的双链DNA或编码GFP报告基因。与非靶向肽pDNA复合转染的细胞，弥散性对应与GFP表达清楚地观察，发现在胞质溶胶（ 图3A）来本地化绿色荧光。在绿色荧光的本地化模式截然不同的差异与线粒体靶向肽pDNA复合浸润细胞明显。这里，点状的绿色荧光与线粒体染色共定位是可见的，证实基因表达的特异性只在细胞的线粒体隔室（ 图3B）。 在肽 - 蛋白制剂的情况下，蛋白质的货物（ADH）以荧光团（罗丹明B）的共轭将使在胞内区室的递送蛋白可视化。在6小时的潜伏期短，ADH-蛋白罗丹明B（蓝色）被发现在整个分发细胞质和浸润细胞的液泡（ 图3C）。与此同时，基因表达的快速和有效的下调可以使用肽的dsRNA制剂各种植物来实现。在第一个实验中， 拟南芥叶用肽的dsRNA渗透络合物沉默的查耳酮合成酶基因（CHS）负责在干旱条件下花青素（红色颜料）的生物合成。在拟南芥的外观差异叶在正常（ 图3D，a）和干旱条件（ 图3D，B）提供了一种简单的方法来评价社区卫生服务利用优化肽双链RNA沉默配方（箭头1指示渗透区域）。在第二个实验中，肽的dsRNA复合物渗入转基因拟南芥的表达黄色荧光蛋白（YFP）的叶子。在YFP表达明显减少可能会在表皮细胞9小时P为可见OST转染（ 图3E，F）。在下调的基因表达的制剂在不同的植物系统（杨树，12小时后转染）的效力也进行了验证（ 图3G中的H）。 <img alt="图1" src="/files/ftp_upload/54972/54972fig1.jpg" /> 图 1: 核酸和蛋白质的货物交付到活体植物肽基配方。 所设计的载体肽包括结合到细胞穿透或细胞器转运序列聚阳离子。聚阳离子启用从内涵体隔室下面的内化到细胞结合和/或带负电荷的货物的缩合以及逃逸。货物递送到细胞中，随后对特定的细胞器是通过细胞穿透序列和细胞器转运序列分别介导。各种货物，可能是事业有成LLY输送到植物包括核酸如质粒DNA，双链DNA和双链RNA，以及诸如牛血清白蛋白（BSA），醇脱氢酶（ADH）和黄晶（黄色荧光蛋白的变体）的模式蛋白。生物活性分子能够形成与通过静电相互作用，其被引入到植物的叶通过注射器浸润肽缀合物转染复合物。 <a href="http://ecsource.jove.com/files/ftp_upload/54972/54972fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54972/54972fig2.jpg" /> 图2: 基于肽制剂的形态。 （A）AFM幅度（KH）在N / P 0.5 9 -BP100 /质粒DNA配方的形象。 （B）（BP100）在摩尔慧慧2 K 8 / ADH配方AFM高度图像Ø10.转载许可已公布的资料23,24。 <a href="http://ecsource.jove.com/files/ftp_upload/54972/54972fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54972/54972fig3.jpg" /> 图3: 使用优化的基于肽制剂转染效率的微观评价。 （A）的细胞溶质的GFP表达（绿色），来自叶绿体自发荧光（红色）明确区分，在拟南芥的海绵组织细胞中观察到叶渗入带（KH）9 -BP100 /质粒DNA制剂（N / P 0.5; 12小时）。 （B）GFP表达（绿色）与Cytcox-（KH）渗透拟南芥叶9 / BP100 /质粒DNA制剂（N / P 0.5的每个肽类表皮细胞线粒体（红色）检测Ë; 12小时）。与GFP表达线粒体的放大图像显示在最右侧的面板中。 ADH-罗丹明B（蓝色）的（C）的传递到拟南芥的海绵叶肉细胞叶由（BP100）2 K 8以10的肽/蛋白质的摩尔比介导的，可视化6小时后的渗透。 （D）的前拟南芥叶（a）和之后（b）与（KH）9 -BP100 /双链RNA制剂（摩尔比2; 48小时）处理，这导致了花青素生物合成途径的抑制。含有双链RNA GFP5了类似表述渗入到叶为阴性对照组（C）。箭头1和3表示的熔渗区域，而箭头2和4表示的叶内非渗入区。 （E） 拟南芥叶表达黄色荧光蛋白（YFP）和（F）在与浸润减少YFP荧光<html> （KH）9 -BP100 / dsRNA的配方（摩尔比2; 9小时）。 （G）的转基因杨树的叶子表达黄色荧光蛋白（YFP）和（H）的减弱YFP荧光下浸润（KH）9 -BP100 / dsRNA的配方（摩尔比2; 12小时）。从公布的资料来源21,23,24,26转载许可。 <a href="http://ecsource.jove.com/files/ftp_upload/54972/54972fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54972/54972fig4.jpg" /> 图4: 变异肽对DNA（N / P）比率及配合生物物理特性的影响。 随着肽DNA比，肽基配方的尺寸减小，而他们的ζ电位值过渡由负转正。/files/ftp_upload/54972/54972fig4large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="表格1" src="/files/ftp_upload/54972/54972table1.jpg" /> 表1: 表征及各种基于肽配方评价。 <a href="http://ecsource.jove.com/files/ftp_upload/54972/54972table1large.jpg" target="_blank">请点击此处查看该表的放大版本。</a> <img alt="表2" src="/files/ftp_upload/54972/54972table2.jpg" /> 表2:完整植物为基础的肽与其他现有的DNA传递技术的比较。 <a href="http://ecsource.jove.com/files/ftp_upload/54972/54972table2large.jpg" target="_blank">请点击此处查看该表的放大版本。</a>

Watch this Scientific Journal Video about Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants at JoVE.com

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

肽衍生的方法在运输跨越植物细胞的基因和蛋白质和细胞器障碍

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

peptide-derived-method-to-transport-genes-proteins-across-cellular

Research

JoVE Journal

Genetics

10.5K 次观看.  RIKEN Center for Sustainable Resource Science. Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

视频: Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation of gene functions. Their therapeutic potential in the heart can also be determined. There are limited approaches for in vivo molecular intervention in the mouse heart. Recombinant adeno-associated virus (rAAV)-based genome engineering has been utilized as an essential tool for in vivo cardiac gene manipulation. The specific advantages of this technology include high efficiency, high specificity, low genomic integration rate, minimal immunogenicity, and minimal pathogenicity. Here, a detailed procedure to construct, package, and purify the rAAV9 vectors is described. Subcutaneous injection of rAAV9 into neonatal pups results in robust expression or efficient knockdown of the gene(s) of interest in the mouse heart, but not in the liver and other tissues. Using the cardiac-specific TnnT2 promoter, high expression of GFP gene in the heart was obtained. Additionally, target mRNA was inhibited in the heart when a rAAV9-U6-shRNA was utilized. Working knowledge of rAAV9 technology may be useful for cardiovascular investigations.

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

在小鼠心脏rAAV9的制备过表达或敲除基因

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation ...

科研

遗传学

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

光遗传学随机突变用组蛋白miniSOG在<em&gt;℃。线虫</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

暴露在生理条件下的氧气在人体细胞中帽结合蛋白质分析

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic fragments. Organellar genomes also exist in admixtures within a given cell or tissue type (heteroplasmy), and an abundance of subtypes may change throughout development or when under stress (sub-stoichiometric shifting). Next-generation sequencing (NGS) technologies are required to obtain deeper understanding of organellar genome structure and function. Traditional sequencing studies use several methods to obtain organellar DNA: (1) If a large amount of starting tissue is used, it is homogenized and subjected to differential centrifugation and/or gradient purification. (2) If a smaller amount of tissue is used (i.e., if seeds, material, or space is limited), the same process is performed as in (1), followed by whole-genome amplification to obtain sufficient DNA. (3) Bioinformatics analysis can be used to sequence the total genomic DNA and to parse out organellar reads. All these methods have inherent challenges and tradeoffs. In (1), it may be difficult to obtain such a large amount of starting tissue; in (2), whole-genome amplification could introduce a sequencing bias; and in (3), homology between nuclear and organellar genomes could interfere with assembly and analysis. In plants with large nuclear genomes, it is advantageous to enrich for organellar DNA to reduce sequencing costs and sequence complexity for bioinformatics analyses. Here, we compare a traditional differential centrifugation method with a fourth method, an adapted CpG-methyl pulldown approach, to separate the total genomic DNA into nuclear and organellar fractions. Both methods yield sufficient DNA for NGS, DNA that is highly enriched for organellar sequences, albeit at different ratios in mitochondria and chloroplasts. We present the optimization of these methods for wheat leaf tissue and discuss major advantages and disadvantages of each approach in the context of sample input, protocol ease, and downstream application.

The comparison and optimization of two plant organellar DNA enrichment methods are presented: traditional differential centrifugation and fractionation of the total gDNA based on methylation status. We assess the resulting DNA quantity and quality, demonstrate performance in short-read next-generation sequencing, and discuss the potential for use in long-read single-molecule sequencing.

适用于下一代测序的植物细菌DNA富集方法的优化与比较分析

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic ...

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

激光 Microirradiation 研究体内细胞对简单复杂 DNA 损伤的反应

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can potentially increase risk of sudden unexpected death in epilepsy (SUDEP). Electroencephalography (EEG) and electrocardiography (ECG) are widely used clinical diagnostic tools to monitor for abnormal brain and cardiac rhythms in patients. Here, a technique to simultaneously record video, EEG, and ECG in mice to measure behavior, brain, and cardiac activities, respectively, is described. The technique described herein utilizes a tethered (i.e., wired) recording configuration in which the implanted electrode on the head of the mouse is hard-wired to the recording equipment. Compared to wireless telemetry recording systems, the tethered arrangement possesses several technical advantages such as a greater possible number of channels for recording EEG or other biopotentials; lower electrode costs; and greater frequency bandwidth (i.e., sampling rate) of recordings. The basics of this technique can also be easily modified to accommodate recording other biosignals, such as electromyography (EMG) or plethysmography for assessment of muscle and respiratory activity, respectively. In addition to describing how to perform the EEG-ECG recordings, we also detail methods to quantify the resulting data for seizures, EEG spectral power, cardiac function, and heart rate variability, which we demonstrate in an example experiment using a mouse with epilepsy due to Kcna1 gene deletion. Video-EEG-ECG monitoring in mouse models of epilepsy or other neurological disease provides a powerful tool to identify dysfunction at the level of the brain, heart, or brain-heart interactions.

Here, we present a protocol to record brain and heart bio signals in mice using simultaneous video, electroencephalography (EEG), and electrocardiography (ECG). We also describe methods to analyze the resulting EEG-ECG recordings for seizures, EEG spectral power, cardiac function, and heart rate variability.

同步视频脑电图-心电图监测对小鼠癫痫模型 Neurocardiac 功能障碍的鉴别

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can ...

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including cancers. These small regulatory RNAs mainly interact with the 3&#8217; untranslated regions (3&#8217; UTR) of their target messenger RNAs (mRNAs) ultimately resulting in the inhibition of decoding processes of mRNAs and the augmentation of target mRNA degradations. Based on the expression levels and intracellular functions, miRNAs are able to serve as regulatory factors of oncogenic and tumor-suppressive mRNAs. Identification of bona fide target genes of a miRNA among hundreds or even thousands of computationally predicted targets is a crucial step to discern the roles and basic molecular mechanisms of a miRNA of interest. Various miRNA target prediction programs are available to search possible miRNA-mRNA interactions. However, the most challenging question is how to validate direct target genes of a miRNA of interest. This protocol describes a reproducible strategy of key methods on how to identify miRNA targets related to the function of a miRNA. This protocol presents a practical guide on step-by-step procedures to uncover miRNA levels, functions, and related target mRNAs using the probe-based real-time polymerase chain reaction (PCR), sulforhodamine B (SRB) assay following a miRNA mimic transfection, dose-response curve generation, and luciferase assay along with the cloning of 3&#8217; UTR of a gene, which is necessary for proper understanding of the roles of individual miRNAs.

This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3&#8217; untranslated regions (3&#8217; UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of miRNAs.

用于评估肿瘤细胞中的微RNA水平、功能和相关靶基因的体外协议

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...

Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational steps are used to regulate specific genes. Sequence-specific nucleic acid-binding proteins target specific sequences to control spatial or temporal gene expression. The binding sites in nucleic acids are typically characterized by mutational analysis. However, numerous proteins of interest have no known binding site for such characterization. Here we describe an approach to identify previously unknown binding sites for RNA-binding proteins. It involves iterative selection and amplification of sequences starting with a randomized sequence pool. Following several rounds of these steps-transcription, binding, and amplification-the enriched sequences are sequenced to identify a preferred binding site(s). Success of this approach is monitored using in vitro binding assays. Subsequently, in vitro and in vivo functional assays can be used to assess the biological relevance of the selected sequences. This approach allows identification and characterization of a previously unknown binding site(s) for any RNA-binding protein for which an assay to separate protein-bound and unbound RNAs exists.

Sequence specificity is critical for gene regulation. Regulatory proteins that recognize specific sequences are important for gene regulation. Defining functional binding sites for such proteins is a challenging biological problem. An iterative approach for identification of a binding site for an RNA-binding protein is described here and is applicable to all RNA-binding proteins.

探索序列空间,识别调节RNA结合蛋白的结合位点

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational ...

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic screening using chemical mutagens is a useful strategy for mapping mutant phenotypes to genes among Drosophila and other model organisms that share conserved cellular pathways with humans. If the mutated gene of interest is not lethal in early developmental stages of flies, a climbing assay can be conducted to screen for phenotypic indicators of decreased brain functioning, such as low climbing rates. Subsequently, secondary histological analysis of brain tissue can be performed in order to verify the neuroprotective function of the gene by scoring neurodegeneration phenotypes. Gene mapping strategies include meiotic and deficiency mapping that rely on these same assays can be followed by DNA sequencing to identify possible nucleotide changes in the gene of interest.

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection.

在Vivo前向遗传屏幕识别新的神经保护基因在果蝇黑色素

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic ...

A Bioinformatics Pipeline to Accurately and Efficiently Analyze the MicroRNA Transcriptomes in Plants

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in regulating gene expression at the post-transcriptional level. Sequencing sRNA libraries by Next Generation Sequencing (NGS) methods has been widely employed to identify and analyze miRNA transcriptomes in the last decade, resulting in a rapid increase of miRNA discovery. However, two major challenges arise in plant miRNA annotation due to increasing depth of sequenced sRNA libraries as well as the size and complexity of plant genomes. First, many other types of sRNAs, in particular, short interfering RNAs (siRNAs) from sRNA libraries, are erroneously annotated as miRNAs by many computational tools. Second, it becomes an extremely time-consuming process for analyzing miRNA transcriptomes in plant species with large and complex genomes. To overcome these challenges, we recently upgraded miRDeep-P (a popular tool for miRNA transcriptome analyses) to miRDeep-P2 (miRDP2 for short) by employing a new filtering strategy, overhauling the scoring algorithm and incorporating newly updated plant miRNA annotation criteria. We tested miRDP2 against sequenced sRNA populations in five representative plants with increasing genomic complexity, including Arabidopsis, rice, tomato, maize and wheat. The results indicate that miRDP2 processed these tasks with very high efficiency. In addition, miRDP2 outperformed other prediction tools regarding sensitivity and accuracy. Taken together, our results demonstrate miRDP2 as a fast and accurate tool for analyzing plant miRNA transcriptomes, therefore a useful tool in helping the community better annotate miRNAs in plants.

A bioinformatics pipeline, namely miRDeep-P2 (miRDP2 for short), with updated plant miRNA criteria and an overhauled algorithm, could accurately and efficiently analyze microRNA transcriptomes in plants, especially for species with complex and large genomes.

生物信息学管道，用于准确、高效地分析植物中的微RNA转录

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in ...