Retroviral Gene Transfer and Expression User Manual (PT3132-1)_061113

核心岩藻糖基转移酶fut8基因敲除小鼠肠道菌群特征下载提示：该文档是本店铺精心编制而成的，希望大家下载后，能够帮助大家解决实际问题。

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嗅觉信号转导通路中重要蛋白质的结构和功能。

嗅觉信号转导通路是人类嗅觉感知的基础，它的建立和转导离不开许多关键蛋白质的作用。

这些蛋白质通过特定的结构和功能，完成了嗅觉信号从外界到视网膜，再到大脑皮层的传递过程。

下面将重点介绍几种嗅觉通路中具有重要功能的蛋白质。

1. 神经元特异性磷酸化酪氨酸酶（NCS-1）神经元特异性磷酸化酪氨酸酶（NCS-1）是一种小分子蛋白质，主要表达于嗅觉神经元的钙信号传导通路中。

NCS-1可与钙离子结合，从而对细胞内钙离子水平的调节起到重要作用。

此外，NCS-1还能与磷脂酰肌醇结合，参与细胞膜的信号转导。

在清鼻液中，NCS-1的表达水平与嗅觉衰退相关。

2. 鼠鼻状结构转运体（MOR28）鼠鼻状结构转运体（MOR28）是嗅觉神经元细胞膜上的一个特定通道，它能够转运感知气味的分子，从而触发钙离子的内流和一系列下游信号的传递。

MOR28的表达受嗅觉体系的调控，只在特定类型的嗅觉感知神经元中表达。

研究表明，MOR28与嗅觉通路中其他重要蛋白质如受体和转导蛋白有协同作用，共同构建了嗅觉信号的转导通路。

3. 嗅觉G蛋白（Golf）嗅觉G蛋白（Golf），与视网膜G蛋白相似，是嗅觉膜上的一种重要蛋白质。

Golf可在气味调节器上呈现表达，连接气味受体和腺苷酸环化酶的信号转导通路。

Golf与其他嗅觉信号转导通路蛋白质的结合，可使其自身活性得到调节，发挥出更为重要的功能作用。

4. 嗅觉受体蛋白（ORs）嗅觉受体蛋白是嗅觉通路中起着主要作用的蛋白质。

它们能够识别气味分子，并触发钙离子的内流和一系列下游信号的传递，实现气味的感知。

嗅觉受体蛋白质属于G蛋白偶联受体家族，可与前文提到的Golf相互作用，共同构建嗅觉信号转导通路。

目前，已发现将近400种嗅觉受体蛋白，各有不同的结构和功能，它们的出现和调节与人类的嗅觉神经系统相关。

结论：这些嗅觉通路中的蛋白质利用其特定的结构和功能，实现了信息的传递和信号的转导，为嗅觉感知和神经系统正常的运转提供了重要支持，也为人类对元素感知和判断提供了巨大帮助。

Central dogma （中心法则):DNA 的遗传信息经RNA 一旦进入蛋白质就不能再输出了。

Reductionism （还原论)：把问题分解为各个部分，然后再按逻辑顺序进行安排的研究方法. Genome （基因组）:单倍体细胞的全部基因.transcriptome（转录组)：一个细胞、组织或有机体在特定条件下的一组完整基因。

roteome （蛋白质组）：在大规模水平上研究蛋白质特征,获得蛋白质水平上的关于疾病的发生、细胞代谢等过程的整体而全面的认识。

Metabolome （代谢组)：对生物体内所有代谢物进行定量分析并寻找代谢物与生病理变化的相关关系的研究方法。

Gene （基因）：具有遗传效应的DNA 片段。

Epigenetics （表观遗传学现象）：DNA 结构上完全相同的基因，由于处于不同染色体状态下具有不同的表达方式，进而表现出不同的表型。

Cistron (顺反子）：即结构基因,决定一条多肽链合成的功能单位.Muton（突变子)：顺反子中又若干个突变单位，最小的突变单位被称为突变子。

recon（交换子）：意同突变子。

Z DNA(Z型DNA）:DNA 的一种二级结构，由两条核苷酸链反相平行左手螺旋形成. Denaturation （变性)：物质的自然或非自然改变。

Renaturation (复性）：变形的生物大分子恢复成具有生物活性的天然构想的现象。

egative superhelix （负超螺旋)：B—DNA 分子被施加左旋外力,使双螺旋体局部趋向松弛，ＤＮＡ分子会出现向右旋转的力的超螺旋结构。

C value paradox （Ｃ值矛盾）：生物overlapping gene（重叠基因）：不同的基因公用一段相同的ＤＮＡ序列。

体的大Ｃ值与小ｃ值不相等且相差非常大。

interrupted gene （断裂基因）：由若干编码区和非编码区连续镶嵌而成的基因。

splitting gene(间隔基因)：意思与断裂基因相同。

基因转换的生物学意义及分子机制 来源： 2008年11月25日09:40基因转换(gene conversion)是指遗传信息从一个分子向其同源分子单向传递的过程，使受体序列部分或者全部被供体序列所替代，而供体本身的序列不变。

这种现象不仅在真菌中普遍存在，在线虫和哺乳动物中也存在。

迄今已知该现象在原核生物和真核生物中均普遍存在。

深入的研究表明基因转换在基因的致同进化(concerted evolution)、降低突变率等方面均有重要作用。

本文则主要对在其他不同生物类群上的研究情况以及基因转换的分子机制等方面取得的新进展做一概述。

1. 不同生物类群中基因转换及其生物学意义从细菌到植物乃至高等的哺乳动物，大多数非编码重复基因(研究最多的是多拷贝rRNA 基因)和多基因家族都是通过该机制保持其多拷贝基因序列的一致性，它是致同进化现象的背后机制。

1.1 原核生物中的基因转换现象在多种原核生物上，研究表明基因转换导致了它们的多拷贝rRNA 操纵子的基因致同进化现象。

如：在大肠杆菌中有七个rRNA 操纵子，rrnA、B、C、D、E、G 和H，每个操纵子上rRNA 基因的排列顺序为16S-23S-5S，编码rRNA 的基因rrn 往往是多拷贝的。

Ammons 等在研究敲除5S rRNA 基因对细胞的影响时，发现rrnB 操纵子上敲除了其中一个5S rRNA 基因后它可以通过基因转换的方式从别的操纵子上重新获得。

在副溶血弧菌的一个菌株的基因组中有11 个拷贝的rRNA 操纵子，其中10 个位于1 号染色体上，另一个位于2 号染色体上，其16S rRNA 基因序列是完全相同的；而在另一菌株中则含有两类操纵子，其中7 个为一类型，另外4 个为另一类型，它们的差异是在编码16S rRNA可变的主干环的25 bp中有10 bp的差异，Gonzalez-Escalona等认为这种操纵子的差异是基因转换的结果。

在分析比较肠球菌属16S RNA 序列时，发现在三个亲缘关系很近的种属菌株基因中的相同位置都含有一段相同的可变区域，这种情况被认为是因为在不同种属16S RNA 操纵子之间发生了基因转换而实现了遗传信息的传递的结果。

基因水平转移—搜狗百科水平基因转移（horizontal gene transfer, HGT），又称侧向基因转移（lateral gene transfer, LGT），是指在差异生物个体之间，或单个细胞内部细胞器之间所进行的遗传物质的交流。

差异生物个体可以是同种但含有不同的遗传信息的生物个体，也可以是远缘的，甚至没有亲缘关系的生物个体。

单个细胞内部细胞器主要指的是叶绿体、线粒体及细胞核。

水平基因转移是相对于垂直基因转移（亲代传递给子代）而提出的，它打破了亲缘关系的界限，使基因流动的可能变得更为复杂。

1959年，一系列的文章报道了大肠杆菌（Escherichia coli）的高频转导（Hfr）菌株可以将遗传信息传递给特定的鼠伤寒沙门氏菌（Salmonella typhimurium）突变菌株。

同年，Tomochiro Akiba和Kunitaro Ochiai发现病原菌中的抗性质粒，而这一发现直接导致了携带抗性的质粒可以在不同菌种间转移现象的发现，这实际上就宣告了野生型菌株间存在着水平基因转移。

然而，水平基因转移作为一种概念，并不是一开始就伴随着其现象的发现而出现的。

直到20世纪90年代，由于下列原因，人们才逐步使用水平基因转移的概念来解释所遇到的水平基因转移现象，并形成研究热点。

基因工程生物，特别是基因工程微生物（gene engineered organisms, GEOs, or gene engineered microorganisms, GEMs）的应用，及被释放到环境中后的安全性问题。

抗药性病原菌的大量出现，许多药物，特别是抗生素已经不能抑制或杀死原来敏感的病原菌，这已不仅仅是基因突变可解释的，可能与抗药性基因的水平转移有关。

已发现基因的转移不仅仅是发生在细菌之间，而且也发生在细菌与高等生物之间，甚至是高等生物之间。

1 由质粒或病毒等介导的水平基因转移质粒和病毒是在各生物间进行遗传物质传递的重要媒介。

基因工程外文翻译(中英对照)Retrovirus-mediated gene transfer and expression cloning: Powerful tools in functional genomics Most of the human genome has now been sequenced and about 30,000 potential open reading frames have been identified, indicating that we use these 30,000 genes to functionally organize our biologic activities. However, functions of many genes are still unknown despite intensive efforts using bioinformatics as well as transgenic and knockout mice. Retrovirus-mediated gene transfer is a powerful tool that can be used to understand gene functions. We have developed a variety of retrovirus vectors and efficient packaging cell lines that have facilitated the development of efficient functional expression cloning methods. In this review, we describe retrovirus-mediated strategies used for investigation of gene functions and function-based screening strategies 2003 International Society for Experimental Hematology. Published by Elsevier Inc.摘要:人类基因组的大部分现在已经测序完成,大约30,000潜在的开放阅读框已经确定,表明我们使用这30,000个基因管理我们的生物学活和功能性。

鱼腥草离体再生及农杆菌介导的遗传转化1. 背景介绍鱼腥草（Houttuynia cordata Thunb.）是一种具有药用和食用价值的多年生草本植物，广泛分布于中国南方及周边地区。

由于其发育迅速、适应性强、营养价值高等特点，近年来受到了越来越多的关注。

然而，鱼腥草在种质资源的保护、栽培与利用中仍面临着重要的问题，如不良基因型选择、缺乏鉴定表征等，制约了其进一步发展。

离体组织培养技术是一种有效的手段，可用于解决植物繁殖及基因转化等重要问题。

而农杆菌介导的遗传转化技术又是进行植物基因转化的重要方法之一，广泛应用于农作物和观赏植物的改良，是提高作物品质和增加产量的重要途径。

因此，研究鱼腥草离体再生及农杆菌介导的遗传转化技术，对于推进鱼腥草种质资源的保护和利用具有重要的意义。

2. 鱼腥草离体再生技术离体组织培养技术是在无菌条件下，将植物组织分离培养于适当的培养基上，以获得新的诱导植株或组织器官的技术。

鱼腥草离体再生技术是指在离体培养环境中，从鱼腥草的愈伤组织或植株的各个部分（如叶片、茎等）中诱导产生新的植株或组织。

目前，鱼腥草离体再生技术已经有了较多的研究报道。

一些研究表明，适当的培养基配方和培养条件对鱼腥草的离体再生具有很大的影响。

例如，张芳等人研究发现，在以Murashige和Skoog培养基为基础配方，添加不同的生长调节剂的条件下，可以诱导鱼腥草叶片愈伤组织的快速分化和增殖；杜东芹等人研究发现，加入腐植酸对鱼腥草离体芽发生的影响较大。

这些结果表明，调节培养基配方和培养条件可以有效地优化鱼腥草离体再生过程。

3. 农杆菌介导的遗传转化技术农杆菌介导的遗传转化技术是通过农杆菌（Agrobacterium tumefaciens）把携带外源基因的T-DNA区域导入到宿主植物细胞的过程，以达到植物基因转化的目的。

农杆菌介导的遗传转化技术可以用于鱼腥草的基因转化研究。

通过构建合适的载体和选择合适的农杆菌菌株，可实现鱼腥草的遗传转化。

科研Nature：免疫逃避型人胰岛类器官改善糖尿病编译：怀瑾，编辑：景行、江舜尧。

原创微文，欢迎转发转载。

导读干细胞来源的胰岛有望成为治疗胰岛素依赖型糖尿病的有效策略，但仍面临挑战。

本研究诱导多能干细胞产生人胰岛类器官（HILO），驱动非经典的WNT4信号促进其代谢成熟。

这些功能成熟的HILO包含内分泌类细胞，且移植后可在糖尿病NOD / SCID小鼠中快速重建葡萄糖稳态。

PD-L1的过表达保护HILO异种移植，使它们能够在具有免疫活性的糖尿病小鼠中复原葡萄糖稳态达50天。

此外，IFN-γ离体刺激可诱导内源性PD-L1表达，并抑制T细胞活化和移植排斥。

这类能够逃避免疫检测的葡萄糖反应性胰岛类器官的产生，为尸体和设备依赖的糖尿病治疗提供了极具前景的替代方案。

论文ID原名：Immune-evasive human islet-like organoids ameliorate diabetes译名：免疫逃避型人胰岛类器官改善糖尿病期刊：NatureIF：43.07发表时间：2020年8月通讯作者：Ronald M Evans通讯作者单位：索尔克生物研究所，霍华德·休斯医学院DOI号：10.1038/s41586-020-2631-z结果1 WNT4诱导HILOs的功能成熟胰岛移植可为1型和2型晚期糖尿病患者提供长期有效的血糖，但尸体来源的胰岛因质量和可利用性而限制了其实用价值。

尽管诱导性多能干细胞（iPS细胞）向胰岛β样细胞的分化取得重大进展，但产生适合于人类治疗的功能性β样细胞仍待探究。

研究者先前证明核雌激素相关受体γ（ERRγ）能够驱动葡萄糖刺激下β细胞胰岛素分泌（GSIS）所必需的产后代谢成熟。

ERRγ在iPS来源的β样细胞中的过表达足以实现体内/外功能。

为了产生适合移植的功能性细胞，研究者探索能够复制细胞结构以及胰岛细胞类型多样性的培养条件。

最初利用人类脂肪干细胞（hADSCs）和人脐静脉内皮细胞（HUVECs）（分别模拟胰腺成纤维细胞和胰腺内皮细胞）在三维（3D）培养和多糖悬浮凝胶培养中形成器官和血管结构的固有能力。

软骨细胞代谢基因-概述说明以及解释1.引言1.1 概述软骨细胞代谢基因是指参与软骨细胞代谢调控的基因，它们在维持软骨组织结构和功能中发挥关键作用。

软骨细胞代谢基因的表达水平和功能异常与多种软骨相关疾病的发生和发展密切相关，如骨关节炎、软骨缺损等。

软骨细胞代谢基因受到复杂的调控机制影响，包括转录因子、信号通路和非编码RNA等多种调控因子的参与。

这些调控机制对于维持软骨细胞的稳态以及其在软骨组织修复和再生过程中的功能发挥起着重要的作用。

研究表明，软骨细胞代谢基因的异常表达和功能紊乱与多种软骨相关疾病的发生和发展密切相关。

例如，软骨细胞代谢基因的过度表达可能导致软骨细胞的异常增殖和分化，进而引发疾病的发生。

相反，软骨细胞代谢基因的缺失或功能丧失可能导致软骨组织的退化和功能减退。

因此，深入了解软骨细胞代谢基因的定义、特点和调控机制，以及其与疾病的关系，对于揭示软骨相关疾病的发生机制和寻找新的治疗靶点具有重要的意义。

此外，对软骨细胞代谢基因的研究还有助于进一步认识软骨组织的生物学特性，推动软骨组织工程和再生医学领域的发展。

在未来的研究中，应进一步深入探究软骨细胞代谢基因的功能和调控网络，以及其在不同疾病中的具体作用和机制。

此外，应结合基因工程技术和生物信息学方法，全面分析软骨细胞代谢基因的表达谱、调控网络和相互作用，以期为软骨相关疾病的预防和治疗提供新的策略和方法。

总而言之，软骨细胞代谢基因是软骨组织功能维持和疾病发生发展的关键因素。

加深对其定义、特点和调控机制的认识，以及研究其与疾病的关系，对于揭示软骨相关疾病发生的机制和寻找新的治疗策略具有重要意义。

未来的研究应继续深入探索软骨细胞代谢基因的功能和调控网络，并结合基因工程技术和生物信息学方法进行全面分析，以期推动软骨组织工程和再生医学领域的发展。

1.2 文章结构文章结构部分的内容可以包括以下内容：本文主要分为引言、正文和结论三个部分。

引言部分介绍了软骨细胞代谢基因的主题，并概述了该主题的重要性和研究的背景。

易位子名词解释细胞生物学
在细胞生物学中，"易位子"（Transposon）是指一类能够在基因组内移动位置的DNA序列。

它们也被称为跳跃基因（Jumping Genes）。

易位子是一种遗传元件，具有自主复制和插入到新位置的能力。

易位子通常由两个重要的DNA序列构成：转座酶基因（transposase gene）和转座序列（transposon sequence）。

转座酶基因编码转座酶（transposase），是易位子移动的关键酶。

转座序列则是易位子的DNA片段，包含了转座酶基因以及其他可能的功能基因。

易位子通过转座酶的作用，在细胞的基因组内进行移动和复制。

它们可以插入到基因组中的新位置，也可以从一个位置剪切出来再插入到另一个位置。

易位子的移动可以导致基因组结构的改变，进而影响基因的表达和调控。

它们在生物进化、基因多样性和基因组重排等方面发挥着重要的作用。

尽管易位子在细胞生物学中扮演着重要角色，但过多或异常的易位子活动可能会对细胞功能和基因组稳定性造成不利影响。

因此，细胞通常通过一系列机制来控制易位子的活动，以维持基因组的稳定性。

原核生物基因转移的机制原核生物基因转移是指原核生物（如细菌和古菌）之间传递基因的过程。

这种转移可以通过三种主要机制实现：转化（Transformation）、转导（Transduction）和共轭（Conjugation）。

转化是指从外界环境中获取游离的DNA片段并集成到接收细胞的基因组中。

转化过程主要分为三个步骤：捕获、外化和吸收。

在捕获阶段，接收细胞通过一种称为自然转化的过程来吸附来自供体细胞的DNA片段。

在外化阶段，DNA片段进入了细胞的外部环境，这个过程通常由外膜的发生局部解离来实现。

在吸附阶段，接收细胞利用其表面的受体和附着因子，将外化的DNA片段捕获并吸收到细胞内。

转导是指利用噬菌体（即细菌病毒）作为载体，将DNA片段从供体细胞传递到接收细胞中。

这种机制发生在细菌感染噬菌体的过程中。

病毒将其遗传物质注入宿主细菌中后，可以寄生在细菌的染色体上，形成纵横交错的线粒体。

这样，当噬菌体复制时，也会复制附着的细菌DNA片段。

在噬菌体释放后，它可能会感染另一个细菌，将带有小片段DNA的包壳噬菌体注入接收细胞，再将其整合到接收细胞的染色体中。

共轭是一种直接的细胞间基因传递方式，需要供体和接收细胞之间的物质交换。

这种机制发生在两个细菌细胞之间，其中一种细菌含有称为共轭质粒（Conjugative plasmid）的DNA分子。

共轭质粒是一种环状DNA分子，它可以复制自身并传递给另一细菌细胞。

共轭过程包括以下几个步骤：首先，供体细胞通过一种称为丙氨酸炎细胞机制（Auxotrophic mechanism）诱导自身形成细胞连接管（pilus）。

然后，细胞连接管通过细胞壁的孔道将共轭质粒从供体细胞转移到接收细胞中。

共轭质粒DNA进入接收细胞后，会通过螺旋自由担体和重组酶等机制集成到细胞染色体中。

总的来说，原核生物基因转移的机制多种多样，这些机制可以在不同的条件下发生。

这种基因转移可以为原核生物提供新的遗传物质，并帮助细菌适应不同的环境压力，提高其生存能力。

转录中介体复合物的研究进展转录中介体复合物是一种多分子复合物，它在基因转录中起重要作用。

它由多个蛋白质组成，包括RNA聚合酶、转录因子和调节蛋白质等。

转录中介体复合物负责在基因转录的过程中协调转录因子和RNA聚合酶之间的作用，从而向DNA序列转录RNA。

由此，它对基因表达的调节起到了至关重要的作用。

在过去的几十年中，人们对转录中介体复合物的研究越来越深入，逐渐揭示了其作用机制和组成成分。

最早发现的复合物是具有转录激活功能的Mediator，它由多个亚组分组成，并在基因转录中发挥着极为关键的作用。

近年来，随着技术的进步，人们对转录中介体复合物的研究也越来越深入。

例如，研究人员通过将RNA聚合酶结合到DNA上，并利用电镜技术的图像分析，揭示出Mediator和RNA聚合酶的相互作用方式。

该研究揭示了Mediator和RNA聚合酶之间的4个接触点，这些接触点是通过近距离相互作用而形成的。

此外，还有研究人员使用大规模质谱分析技术，鉴定出了许多新的转录中介体复合物的成分。

例如，蛋白质TFIID是一种由14个亚单位组成的复合物，它在基因转录中起重要作用。

同时，还有许多新的蛋白质因子被发现，它们是Mediator与其他复合物之间联系的关键因子。

此外，还有研究人员在转录中介体复合物中发现了一些非蛋白质成分，如小RNA和某些小分子。

通过这些非蛋白质成分的存在，转录中介体复合物的功能变得更加复杂，与基因表达的调控相互作用更紧密。

总的来说，转录中介体复合物是一个复杂而关键的系统，在维持正常的基因表达方面具有重要的作用。

目前，越来越多的研究人员致力于深入研究这个领域，以期更好地理解转录中介体复合物的组成，作用机制以及与人类疾病的相关性，为后续的治疗提供更好的帮助。

Gene Therapy (2000)7,1063–1066©2000Macmillan Publishers Ltd All rights reserved 0969-7128/00$/gtVIRAL TRANSFER TECHNOLOGY BRIEF COMMUNICATIONPlat-E:an efficient and stable system for transient packaging of retrovirusesS Morita,T Kojima and T KitamuraDepartment of Hematopoietic Factors,Institute of Medical Science,University of Tokyo,4-6-1Shirokanedai Minato-ku,Tokyo 108-8639,JapanA potent retrovirus packaging cell line named Platinum-E (Plat-E)was generated based on the 293T cell line.Plat-E is superior to existing packaging cell lines regarding efficiency,stability and safety.The novel packaging constructs utilized in establishment of Plat-E ensure high and stable expression of viral structural proteins.Conventional packaging con-structs made use of the promoter of MuLV-LTR for expression of viral structural genes gag-pol and env,while our packaging constructs utilized the EF1␣promoter,which is 100-fold more potent than the MuLV-LTR in 293T cells in combination with the Kozak’s consensus sequence upstream of the initiation codon resulting in high expression Keywords:packaging cell;retroviruses;ecotropic;EF1␣promoterRetroviral vectors and packaging cells are important tools for gene transfer applications.Introduction of retroviral vectors containing the gene of interest into suitable pack-aging cells enables production of infectious retroviruses,and these particles can infect target cells and stably trans-mit the gene of interest into chromosomes.In conven-tional strategies,stable high producers of a retrovirus vector harboring a gene of interest were established by transducing the retrovirus construct into NIH3T3-based packaging cells such as PA317,1and 2–3months were usually needed to acquire high producers.Pear et al 2developed a unique packaging system by which high titer retroviruses can be obtained in 3days by transient transfection.The expression of viral structural genes was driven by the MuLV LTR in Bosc23cells.For transient transfection,the combination of Bosc23cells and the pMX-neo vector 3produced 1–3×106/ml viruses,assessed based on the number of neomycin resistant col-onies of the infected NIH3T3cells (data not shown).Since Bosc23cells carry the large T antigen,we attempted to increase titers of the retroviruses by introducing the SV40origin to the pMX vector for amplification of the vector.However,this proved unfeasible (data not shown),sug-gesting that the limiting factor was the expression level of the viral structural proteins in the packaging cells.Bosc23was obtained by cotransfection of the plasmid encoding gag-pol together with the plasmid encoding the hygromycin-resistant gene and the plasmid encoding envCorrespondence:T KitamuraReceived 3November 1999;accepted 3March 2000of virus structural proteins in Plat-E cells.To maintain the high titers of retroviruses under drug selection pressure,we inserted the IRES (internal ribosome entry site)sequence between the gene encoding gag-pol or env,and the gene encoding a selectable marker in the packaging constructs.Plat-E cells can stably produce retroviruses with an average titer of 1×107/ml for at least 4months.In addition,as we used only the coding sequences of viral structural genes to avoid inclusion of unnecessary retrovirus sequences in the packaging constructs,the probability of generating the repli-cation competent retroviruses (RCR)by recombination can virtually be ruled out.Gene Therapy (2000)7,1063–1066.together with the plasmid encoding another selection gene GPT (guanine phosphoribosyl transferase),one after the other.Therefore,expression of selectable markers did not guarantee the expression of gag-pol or env genes,which may account for the instability of the cells in producing high-titer retroviruses.A similar packaging cell line Phoenix-E 4has also been developed.In Phoenix-E cells,the plasmids encoding the gag-pol and env genes were cotransfected with selection markers,which did not warrant the stable expression of the gag-pol and env genes in the selection drug,hygromy-cin and diphtheria toxin,respectively.There were several improvements in the Phoenix-E cells when compared with Bosc23cells.First,the RSV and CMV promoters,which are much stronger than MuLV-LTR in 293T cells,were used to express the gag-pol and env genes,respect-ively.Second,the internal ribosome entry site (IRES 5)sequence was used to express gag-pol and a cell surface marker CD8simultaneously which enables sorting of high expressers of the gag-pol gene.However,one needs to sort cells from time to time to maintain the expression levels of the gag-pol gene.To design a packaging cell line which can stably pro-duce retroviruses with high titer,we searched for a strong promoter to drive expression of viral structural proteins in 293T cells using the FACS-GAL assay.6Among seven promoters tested,the EF1␣and CMV pro-moters induced high expression of lacZ (Figure 1).The activities driven by these promoters were 100-fold higher than those by LTR utilized in Bosc23cells,and even exceeded those by SV40and SR ␣promoters,which enable amplification of vectors in 293T cells expressingPlat-E:a system for transient packaging of retrovirusesS Morita et al1064 GeneTherapyFigure1Activities of various promoters in293T cells.The activities ofthe seven promoters,SV40,SR␣,EF1␣,RSV,TK,MuLV LTR and CMVwere evaluated by expression of lacZ under the control of each promoter in293T7cells by the FACS-GAL assay as described.6Briefly,cells(1×106)transfected with each promoter construct were suspended in50␮l of phos-phate buffered saline(PBS),then incubated for5min at37°C.FDG(fluorescein di-␤-d-galactopyranoside;Molecular Probes,Eugene,OR,USA)was dissolved in distilled water,warmed at37°C and50␮l of2m m FDG solution was added to50␮l of cell suspension.After1min ofincubation at37°C,1ml of PBS was added followed by incubation on icefor2h.To stop the reaction,20␮l of50m m PETG(phenylethyl-␤-d-thiogalactoside;Sigma,St Louis,MO,USA)was added,and the prep-aration was placed on ice until being subjected to FACS analysis.the SV40large T antigen.Because we thought that thepromoters of housekeeping genes were more suitablethan the viral promoters for driving stable geneexpression in mammalian cells,we used the EF1␣pro-moter to express the viral structural proteins in293T cells(Figure2).In addition,IRES was inserted between thegag-pol or env gene and the selection marker in the pack-aging constructs described here.Therefore,expression ofthe selection marker is a direct reflection of gag-pol or envexpression in the same cells.Packaging constructs pEnv-IRES-puro r and pGag-pol-IRES-bs r,which were constructed as described above,were sequentially transfected into293T cells and50sub-clones resistant to both puromycin and blasticidin wereisolated.Among50subclones,clone1named Platinum-E(Plat-E)produced the retrovirus which had the highestinfection efficiency and was used for further analysis.Thetiter of the retroviruses was about1×107/ml when testedon NIH3T3cells,using serially diluted virus super-natants of Plat-E cells transfected with pMX-lacZ(datanot shown).We next compared early passages of Plat-Ecells with those of Bosc23cells and Phoenix-E cells withregards to long-term stability to produce high-titer retro-viruses by transient transfection(Figure3).Culture con-ditions of the three packaging cell lines were as follows:Bosc23cells were grown in DMEM with10%fetal bovineserum containing the GPT selection reagents as indicatedby the manufacturer(Specialty Media,Lavallette,NJ,USA).Phoenix-E cells were sorted by FACS forexpression of CD8and maintained in DMEM with10%Figure2Schematic diagrams of packaging constructs.The packagingconstructs used for development of Plat-E are shown.The fragment carry-ing the selectable marker,the blasticidin resistant gene(bs r)or the puro-mycin resistant gene(puro r),was obtained by PCR using a pair of oligo-nucleotides(for bs r:5Ј-AAAACATTTAACATTTCTCAACAAG-3Ј,5Ј-ACGCGTCGACTTAATTTCGGGTATATTTGAGTG-3Ј,for puro r:5Ј-ACCGAGTACAAGCCCACG-3Ј,5Ј-ACGCAGATCTTCAGGCACCGGGCTTG-3Ј),and were inserted in the NcoI and SalI site(for bs r),or inthe NcoI and BglII site(for puro r)of pMX-IRES-EGFP.8The fragmentscontaining the IRES sequence and either of bs r and puro r were excisedfrom the vector by NotI and SalI for bs r,or NotI and BglII for puro r,respectively.The viral structural genes,gag-pol and env were amplifiedby PCR,using the MoMuLV genome as a template,and the oligonucleo-tide primers were used as follows.Each primer contains either the EcoRIsite or the NotI site(underlined)and the5Јprimers also contain a Kozak’sconsensus sequence GCCGCCACC located upstream of the initiationcodon.gag-pol:5Ј-CGAATTCGCCGCCACCATGGGCCAGACTGTTACCACTCCCTTAA-3Ј;5Ј-TACGCGGCCGCTCTGAGCATCAGAAGAA-3Ј;env:5Ј-cGAATTCGCCGCCACCATGGCGCGTTCAACGCTCTCAAAA-3Ј;5Ј-TACGCGGCCGCTATGGCTCGTACTCTAT-3Ј.Theresulting PCR fragments were digested with the EcoRI and the NotI frag-ment.Finally,the fragment containing the viral structural genes,and thefragment containing the IRES sequence and the selection marker wereinserted downstream of the EF1␣promoter in the pCHO vector,a deriva-tive of pEF-BOS.9For construction of the pGag-pol-IRES-bs r,pCHO wasdigested with BamHI,and converted to a blunt end by Klenow reaction,and then ligated with SalI linker(Stratagene,La Jolla,CA,USA).TheEcoRI–NotI fragment of gag-pol,and the NotI–SalI fragment of IRES-bs rwere inserted into the EcoRI and the SalI site of pCHO by triple ligation.To construct pEnv-IRES-puro r,pCHO was digested with EcoRI andBamHI,and the EcoRI–NotI fragment of env,and the NotI–SalI fragmentof IRES-puro r were inserted into the EcoRI and the BamHI sites of pCHO.Packaging constructs pEnv-IRES-puro r and pGag-pol-IRES-bs r weresequentially transfected into293T cells using Fugene(BoehringerMannheim,Germany)according to the manufactuer’s recommendations.One day after transfection with pEnv-IRES-puro r,293T cells were selectedin DMEM containing1␮g/ml puromycin.The selected cells were thentransfected with the pGag-pol-IRES-bs r vector,and subcloned in the pres-ence of puromycin and blasticidin(10␮g/ml).The selected clones weretested for their potential to produce retroviruses.EF1␣,EF1␣promoter;IRES,internal ribosome entry site;bs r,blasticidin resistant gene;puro r,puromycin resistant gene.fetal bovine serum containing hygromycin(300␮g/ml)and diphtheria toxin(1␮g/ml)for1week,then cellswere transferred to DMEM with10%fetal bovine serumwithout hygromycin and diphtheria toxin.Plat-E cellswere always maintained in DMEM with10%fetal bovineserum containing blasticidin(10␮g/ml)and puromycin(1␮g/ml).On one hand,infection efficiency of retro-viruses produced from Bosc23was decreased within3months,and that of retroviruses produced from the Pho-enix-E cells also decreased in time(Figure3).On theother hand,Plat-E produced retroviruses with an infec-tion efficiency greater than75%with a titer of about1×107/ml for at least4months under drug selectionpressure.To compare the expression level of gag-pol and envmRNA in Plat-E,Bosc23and Phoenix-E packaging cellPlat-E:a system for transient packaging of retroviruses S Morita et al1065Figure3Long-term stability of Plat-E in producing high titer retro-viruses.The infection efficiencies of Ba/F3cells using retroviruses derivedfrom pMX-GFP produced by Plat-E,Bosc23and Phoenix E were exam-ined at the indicated times.pMX-GFP was constructed as follows.TheGFP fragment was excised from the pEGFP-N1vector(Clontech,PaloAlto,CA,USA)by EcoRI and NotI,and was inserted into the EcoRI–NotI site of the pMX vector.3Transfection and infection were performedas described10except that we used Fugene(Boehringer Mannheim)insteadof LipofectAmine(Gibco-BRL,Rockville,MD,USA).lines,Northern blot analysis was done using the cells cul-tured for3weeks.The expression levels of gag-pol andenv mRNA was four-fold and10-fold higher,respect-ively,in Plat-E cells than in the other packaging cell lines(Figure4a).The RT activity in the cell lysate was alsoanalyzed.Plat-E produced at least twice more RT activitywhen compared with Bosc23and Phoenix-E cells(Figure4b).In addition,the expression level of env protein wasmuch greater than that of Bosc23and Phoenix-E(Figure4c)when evaluated by antibody staining raised againstthe env gene product.As the retroviral structural genes were encoded on thetwo different plasmids,three recombination events arenecessary to generate the replication competent retro-viruses(RCR).In addition,the probability of recombi-nation was minimized by using only the coding sequenceof gag-pol and env genes isolated by PCR from MuLV gen-ome in the packaging constructs.In fact,production ofRCR was tested by the XC plaque assay,13and no RCRwas detected from Plat-E cells after transfection of pMX-GFP.As for a positive control,a supernatant ofMoMuLV-infected C3H2K cells(a gift from Dr Hoshino)was used after serial dilutions,and the viral titer of thewild-type MoMuLV produced from C3H2K cells wasestimated as1×104/ml.In conclusion,we report here a stable retrovirus pack-aging cell line Plat-E which has several advantages overthe existing packaging cell lines.First,the EF1␣promoterin the packaging constructs,in combination with theKozak’s consensus sequence,allows production of retro-viruses with a titer of1×107/ml.Second,a bicistronicvector carrying the IRES sequence was used in the pack-aging constructs to ensure stable expression of the viralstructural genes under the drug selection pressure,whichGeneTherapyFigure4Comparison of gag-pol and env expression in Bosc23,Phoenix-E and Plat-E.(a)Northern hybridization of gag-pol and env.Expressionof gag-pol and env in Plat-E(3)was compared with that in Bosc23(1)and Phoenix-E(2)by Northern hybridization.The probes used were theEcoRI–NotI fragment of pGag-pol-IRES-bs r and pEnv-IRES-puro r.(b)RTassay of cell lysate of Bosc23,Phoenix-E and Plat-E.Cell lysate of293Tcells was used as a negative control(1),1ng of HIV RT was mixed withthe cell lysate of293T cells(2),and was used as a positive control.RTactivities derived from Bosc23(3),Phoenix-E(4),Plat-E(5)were meas-ured as described.11(c)Expression of env in Bosc23,Phoenix E and Plat-E cells.The expression of env was determined by cell surfacefluorescenceof Bosc23,Phoenix-E and Plat-E using the rat monoclonal antibody raisedagainst the env proteins termed83A25.12Staining procedure was perfor-med as described12and then subjected to FACS analysis.As a control,these cells were stained only with the second antibody(FITC-conjugatedgoat anti-rat IgG as second antibody).Plat-E:a system for transient packaging of retrovirusesS Morita et al1066Gene Therapymakes it possible to maintain the titer of retroviruses derived from the Plat-E cells by simply culturing the cells in the presence of selection drugs.Finally,to lessen the possibility of generation of RCR,the minimum virus sequences were used in the packaging constructs.Thus,Plat-E cells can stably produce helper-free retroviruses at high titers for a long time.Using retroviruses produced by Plat-E cells,we can efficiently transfer genes to many different cells including cells in primary culture such as T cells and mast cells (data not shown).Recently,it has been reported that by introducing the coding region of the polyomavirus early gene into the packaging cell lines,the titers of recombi-nant retrovirus produced by these cell lines were 10–100times higher than those produced by the parent cell line.14Introduction of the polyomavirus early region into Plat-E may lead to more efficient production of retro-viruses with high titer.Plat-E is an ecotropic packaging cell line and generation of its amphotropic counterpart,the Plat-A cell line should prove useful in human gene therapy.AcknowledgementsWe would like to thank Dr Hiroo Hoshino (Department of Hygiene and Virology,Gunma University School of Medicine)for his kind gift of MoMuLV-infected C3H2K cells,and Dr Leonard H Evans (Laboratory of Persistent Viral Disease,National Institute of Allergy and Infectious Disease)for anti-Env antibody,and Dr Kunitada Shimo-tohno (Department of Viral Oncology,Institute for Virus Research,Kyoto University)for his valuable discussions.We also thank Ms Mariko Ohara for her providing langu-age assistance.This work was supported in part by grant-in-aid from the Ministry of Education,Science,Sports,and Culture and the Ministry of Health and Welfare ofJapan.The Department of Hematopoietic Factors is supported by Chugai Pharmaceutical Company Ltd.References1Miller AD,Buttimore C.Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production.Mol Cell Biol 1986;6:2983–2902.2Pear WS,Nolan GP,Scott ML,Baltimore D.Production of high-titer helper-free retroviruses by transient transfection.Proc Natl Acad Sci USA 1993;90:8392–8396.3Onishi M et al .Application of retrovirus-mediated expression cloning.Exp Hematol 1996;24:324–329.4/group/nolan/.5Gattas IR,Sanesm JR,Major JE.The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryo.Mol Cell Biol 1991;11:5848–5859.6Fiering SN et al .Improved FACS-Gal:flow cytometric analysis and sorting of viable eukaryotic cells expressing reporter gene constructs.Cytometry 1991;12:291–301.7Dubridge RB,Tang P,Hsia HC.Analysis of mutation in human cells by using an Epstein–Barr virus shuttle system.Mol Cell Biol 1987;7:379–387.8Nosaka T et al .STAT5as a molecular regulator of proliferation,differentiation,and apoptosis in hematopoietic cells.EMBO J 1999;18:4754–4765.9Mizushima S,Nagata S.pEF-BOS,a powerful mammalian expression vector.Nucleic Acids Res 1990;18:5332.10Kitamura T et al .Efficient screening of retroviral cDNAexpression libraries.Proc Natl Acad Sci USA 1995;92:9146–9150.11Mathias S et al .Reverse transcriptase encoded by a human trans-posable element.Science 1991;254:1808–1810.12Evans LH et al .A neutralizable epitope common to the envelopeglycoproteins of ecotropic,polytropic,xenotropic,and ampho-tropic murine leukeia viruses.J Virol 1990;64:6176–6183.13Rowe WP,Pugh WE,Hartly JW.Plaque assay techniques formurine leukemia viruses.Virology 1970;42:1136–1139.14Yoshimatsu T et al .Improvement of retroviral packaging celllines by introducing the polyomavirus early region.Hum Gene Ther 1998;9:161–172.。

diversity-generating retroelementsRetroelements are genetic elements that can replicate and transfer between organisms. Among them, diversity-generating retroelements are a special type that can generate genetic diversity in organisms through mutation and recombination. This article will introduce the characteristics, classification, and functions of diversity-generating retroelements.一、特点Diversity-generating retroelements are a special type of retroelements that can generate genetic diversity in organisms through mutation and recombination. They are characterized by their high mutation rate, high recombinability, and the ability to integrate into new sequences. This allows diversity-generating retroelements to adapt to changes in the environment and provide genetic variation for organisms to adapt to new challenges.二、分类Diversity-generating retroelements can be classified into different types according to their structure and function. Some common types include retrotransposons, long interspersed nuclear elements (LINEs), and short interspersed nuclear elements (SINEs). Retrotransposons are a large group of retroelements that can replicate and transfer through an RNAintermediate. LINEs and SINEs are another two types of diversity-generating retroelements that are commonly found in higher eukaryotes.三、功能Diversity-generating retroelements play an important role in maintaining genome diversity and adaptation. Through mutation and recombination, they generate new genes, sequences, and even whole chromosomes that provide organisms with adaptive potential. Diversity-generating retroelements can also play a role in epigenetic modification, regulating gene expression, and affecting organismal development and function.四、应用Diversity-generating retroelements have important applications in genetic engineering and biotechnology. Through genetic manipulation, researchers can introduce diversity-generating retroelements into organisms to generate genetic variation and improve their adaptability to new environments. This can be used in plant breeding, animal breeding, and medical research to enhance the performance and quality of crops, livestock, and human beings.此外，diversity-generating retroelements还在物种演化和生态系统稳定中起到重要作用。

逆转录酶名词解释逆转录酶（Reverse Transcriptase）是一种酶类，具有将RNA模板反向转录为DNA的能力。

这种酶最早发现于1965年，由美国生物化学家霍华德·泰曼和广岛立博士等人发现，是研究逆转录病毒的重要工具。

逆转录酶是逆转录病毒（如HIV 等）和一些植物和细菌的核糖体元件中的重要成分，它在病毒的复制逆过程中发挥着关键作用。

逆转录酶主要有两个重要的功能：1. RNA依赖DNA聚合酶（RNA-dependent DNA polymerase）: 逆转录酶可以使用RNA作为模板，在硫酸核糖酸核苷下合成DNA链。

它能够使RNA的核酸序列转录为相应的DNA链，该过程称为反转录。

逆转录酶具有较高的酶活性，可以将RNA链转录为DNA链，该能力使得科学家们可以通过逆转录酶来研究RNA的结构、功能以及RNA参与的生物学过程，如基因表达、蛋白质合成等。

2. RNA酶H（Ribonuclease H）活性: 逆转录酶还具有RNA酶H活性，可以水解RNA- DNA杂合体。

在反转录的过程中，逆转录酶合成的DNA链和RNA链形成杂合物，杂合物的RNA部分可以通过RNA酶H活性被逆转录酶水解，从而释放出单链的DNA。

RNA酶H的活性可以在病毒的逆转录复制过程中发挥重要作用，尤其是在合成和修复DNA链的过程中。

逆转录酶在科学研究中的应用广泛。

科学家们可以利用逆转录酶将RNA转录为DNA，并进一步进行PCR扩增、测序、克隆等实验操作。

逆转录酶也被广泛应用于分子诊断、基因表达调控、病毒学研究等领域。

此外，在药物研发上，逆转录酶被用作目标酶的抑制剂，从而阻止逆转录病毒的复制和传播。

总之，逆转录酶是一种能够将RNA转录为DNA的酶类，具有RNA依赖DNA聚合酶和RNA酶H活性。

它在逆转录病毒复制、基因表达调控、分子诊断等方面具有重要的应用价值。

逆转录酶的发现和研究对于理解逆转录病毒的生物学特性、疾病治疗等方面都起到了重要的推动作用。