Simultaneous cloning of open reading frames into several different expression vectors

Genomic sequencing has enabled the prediction of thousands of genes, most of which either cannot be assigned a function or can be only broadly categorized on the basis of sequence alone. High-throughput strategies for elucidating protein function are of high priority, and numerous approaches are being developed. Many of these approaches require the cloning of open reading frames (ORFs) into expression vectors that enable the encoded proteins to be tested for biological and biochemical activities. Typically, more than one type of vector must be employed, as different experiments require different conditions of protein production. Here we show that it is possible to simultaneously transfer a single ORF from a source vector to four target vectors using a commercially available in vitro recombination system. To test the approach, we constructed new vectors for expression of fusion proteins in yeast, including vectors for the LexA two-hybrid system. We show that individual ORFs can be efficiently transferred to four different vectors in a single in vitro reaction. The resulting expression plasmids can be separated using prototrophic markers specific to each vector. Using this system to produce multiple expression constructs simultaneously could greatly facilitate high-throughput subcloning and proteomic studies.     

T载体介导的基于attL核心区LR反应的简化快速Gateway克隆系统

Gateway克隆技术已得到广泛的应用。该技术先通过BP反应将目标片段连到带有完整attL特异识别位点的入门载体,然后与终载体通过LR反应得到表达载体。Gateway克隆方法与传统的酶切连接方法相比有快速简单等优点。但是,BP和LR酶都非常昂贵。本研究首先对3个常用Gateway载体的atts特异位点序列比对发现,attL序列核心交换位点“core attL”的21~22 bp长的碱基是保守和必要的。由此,设计含有core-attL序列的引物,通过PCR克隆得到DNA片段并连入pMD18-T载体,然后进行LR反应,可成功得到目标表达载体,并在保守的位点上正确重组。本研究还对其中一个带有绿色荧光蛋白基因的表达载体转化至烟草,能够正常表达该蛋白质。结果表明,通过将含有attL核心位点基因片段连接到pMD18-T载体上,可以省略BP反应而将目标片段连接到终载体上,节约了反应时间和成本。     

Exploration of Human ORFeome: High-Throughput Preparation of ORF Clones and Efficient Characterization of Their Protein Products.

In this study, we established new systematic protocols for the preparation of cDNA clones, conventionally termed open reading frame (ORF) clones, suitable for characterization of their gene products by adopting a restriction-enzyme-assisted cloning method using the Flexi((R)) cloning system. The system has following advantages: (1) preparation of ORF clones and their transfer into other vectors can be achieved efficiently and at lower cost; (2) the system provides a seamless connection to the versatile HaloTag((R)) labeling system, in which a single fusion tag can be used for various proteomic analyses; and (3) the resultant ORF clones show higher expression levels both in vitro and in vivo. With this system, we prepared ORF clones encoding 1929 human genes and characterized the HaloTag-fusion proteins of its subset that are expressed in vitro or in mammalian cells. Results thus obtained have demonstrated that our Flexi((R)) ORF clones are efficient for the production of HaloTag-fusion proteins that can provide a new versatile set for a variety of functional analyses of human genes.     

A versatile and efficient high-throughput cloning tool for structural biology

Methods for the cloning of large numbers of open reading frames into expression vectors are of critical importance for challenging structural biology projects. Here we describe a system termed fragment exchange (FX) cloning that facilitates the high-throughput generation of expression constructs. The method is based on a class IIS restriction enzyme and negative selection markers. FX cloning combines attractive features of established recombination- and ligation-independent cloning methods: It allows the straightforward transfer of an open reading frame into a variety of expression vectors and is highly efficient and very economic in its use. In addition, FX cloning avoids the common but undesirable feature of significantly extending target open reading frames with cloning related sequences, as it leaves a minimal seam of only a single extra amino acid to either side of the protein. The method has proven to be very robust and suitable for all common pro- and eukaryotic expression systems. It considerably speeds up the generation of expression constructs compared to traditional methods and thus facilitates a broader expression screening.     

几种新型植物基因表达载体的构建方法

利用基因工程技术手段研究基因功能过程中,构建基因表达载体处于转基因植物的主导地位,采用合适的构建方法会使实验效果事半功倍。植物基因表达载体的构建方法除了传统构建法、Gateway技术、三段T-DNA法、一步克隆法等,还有近年来出现的几种新型的载体构建方法:基于竞争性连接原理快速构建小片段基因表达载体;MicroRNA前体PCR置换法适用于构建小分子RNA表达载体;重组融合PCR法特别适用于插入片段中含有较多限制性酶切位点的载体构建;利用In-Fusion试剂盒可以将任何目的片段插入一个线性化载体的某个区域;构建多片段复杂载体可采用不依赖序列和连接的克隆方法(Sequence and ligation-independent cloning,SLIC)法;Gibson等温拼接法;Golden Gate拼接法。本文将在总结分析前人工作的基础上,结合自己工作的体会和经验分析这7种新方法的特点,期望通过这几种新的方法给植物基因工程表达载体的构建提供新的思路。     

A Modified MultiSite Gateway Cloning Strategy for Consolidation of Genes in Plants

The genome information is offering opportunities to manipulate genes, polygenic characters and multiple traits in plants. Although a number of approaches have been developed to manipulate traits in plants, technical hurdles make the process difficult. Gene cloning vectors that facilitate the fusion, overexpression or down regulation of genes in plant cells are being used with various degree of success. In this study, we modified gateway MultiSite cloning vectors and developed a hybrid cloning strategy which combines advantages of both traditional cloning and gateway recombination cloning. We developed Gateway entry (pGATE) vectors containing attL sites flanking multiple cloning sites and plant expression vector (pKM12GW) with specific recombination sites carrying different plant and bacterial selection markers. We constructed a plant expression vector carrying a reporter gene (GUS), two Bt cry genes in a predetermined pattern by a single round of LR recombination reaction after restriction endonuclease-mediated cloning of target genes into pGATE vectors. All the three transgenes were co-expressed in Arabidopsis as evidenced by gene expression, histochemical assay and insect bioassay. The pGATE vectors can be used as simple cloning vectors as there are rare restriction endonuclease sites inserted in the vector. The modified multisite vector system developed is ideal for stacking genes and pathway engineering in plants.     

Generation of expression vectors for high-throughput functional analysis of target genes in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

An immediate challenge in the post-genomic era is to assign a biological functions to proteins unraveled by genome analysis. This report is based on studies conducted using Schizosaccharomyces pombe, a simple model organism, and presents various vector systems as tools for high-throughput functional analysis of human genes. We constructed S. pombe expression vectors for efficient cloning of genes via the Gateway system. We modified the pREP and pSLF series vectors, which are widely used for gene expression in S. pombe. The vectors constructed have a uniform backbone of S. pombe autonomously replicating sequence (ARS) elements with different selective markers, namely, urw4 ⁺ and Saccharomyces cerevisiae LEU2 complementing leul. These vectors contain 3 different strengths of the inducible promoter nmtl, which affect the expression levels of the cloned open reading frames (ORFs). Further, target proteins can be fused with an N-terminal or C-terminal tag such as triple hemagglutinin (3× HA), enhanced green fluorescent protein (EGFP), or Discosoma red fluorescent protein (DsRed). We tested the feasibility of the constructed vectors by using 3 human genes, namely, RAB18, SCC-112, and PTEN. Proper expression of tagged RAB18 was confirmed by western blot analysis. Further, localization of RAB18, SCC112, and PTEN was demonstrated. The constructed vectors can be utilized for high-throughput functional analysis of heterologous genes.     

用于通路(Gateway)克隆技术的植物表达载体研究进展

通路(Gateway)克隆技术是根据λ噬菌体基因组和大肠杆菌基因组之间的位点专一性重组分子机制开发的一套分子克隆新技术.利用该技术LR反应构建目的基因的表达载体时不需要经过酶切和连接等繁琐而又费时的过程,因此,可以节省很多时间.为了扩大Gateway技术在植物基因工程领域的应用,最近有很多研究机构和研究小组开发了能用于组成型或诱导型表达目的基因、基因沉默、启动子分析、蛋白质亚细胞定位、蛋白质/蛋白质相互作用、多个DNA片段的模块化组装和DNA组片段功能验证等研究用的植物表达载体.该文对这些技术的研究进展进行了综述.     

Gateway-compatible transposon vector to genetically modify human embryonic kidney and adipose-derived stromal cells

The Gateway technology cloning system and transposon technology represent state-of-the-art laboratory techniques. Combination of these molecular tools allows rapid cloning of target genes into expression vectors. Here, we describe a novel Gateway technology-compatible transposon plasmid that combines the advantages of Gateway recombination cloning with the Sleeping Beauty (SB) transposon-mediated transgene integrations. In our system the transposition is catalyzed by the novel hyperactive SB100x transposase, and provides highly efficient and precise transgene integrations into the host genome. A Gateway-compatible transposon plasmid was generated in which the potential target gene can be fused with a yellow fluorescent protein (YFP) tag at the N-terminal. The vector utilizes the CAGGS promoter to control fusion protein expression. The transposon expression vector encoding the YFP-interferon-β protein (IFNB1) fusion protein together with the hyperactive SB100x transposase was used to generate stable cell lines in human embryonic kidney (HEK293) and rat adipose-derived stromal cells (ASC). ASCs and HEK293 cells stably expressed and secreted the human IFNB1 for up to 4 weeks after transfection. The generated Gateway-compatible transposon plasmid can be utilized for numerous experimental approaches, such as gene therapy or high-throughput screening methods in primary cells, representing a valuable molecular tool for laboratory applications.     

使用与Gateway技术兼容的T载体获得入门克隆

与Gateway技术兼容的农杆菌双元载体系统已开始应用于植物功能基因组的研究,但应用这些载体系统的一个瓶颈问题,是如何简单、经济和高效地将PCR产物或其他来源的目的DNA片段构建到入门载体上获得入门克隆.为此,将传统的TA克隆技术与Gateway重组克隆技术进行整合,构建了与Gateway技术兼容的两种TA克隆载体,用于在克隆PCR产物或其他来源的目的DNA片段的同时获得入门克隆.利用兼容Gateway技术的TA克隆载体有效地解决了上述瓶颈问题.     

Rapid cloning and purification of proteins: gateway vectors for protein purification by self-cleaving tags

We have combined Invitrogen's Gateway cloning technology with self-cleaving purification tags to generate a new system for rapid production of recombinant protein products. To accomplish this, we engineered our previously reported DeltaI-CM cleaving intein to include a Gateway cloning recognition sequence, and demonstrated that the resulting Gateway-competent intein is unaffected. This intein can therefore be used in several previously reported purification methods, while at the same time being compatible with Gateway cloning. We have incorporated this intein into a set of Gateway vectors, which include self-cleaving elastin-like polypeptide (ELP), chitin binding domain (CBD), phasin (polyhydroxybutyrate-binding), or maltose binding domain (MBD) tags. These vectors were verified by Gateway cloning of TEM-1 beta-lactamase and Escherichia coli catalase genes, and the expressed target proteins were purified using the four methods encoded on the vectors. The purification methods were unaffected by replacing the DeltaI-CM intein with the Gateway intein. It was observed that some purification methods were more appropriate for each target than others, suggesting utility of this technology for rapid process identification and optimization. The modular design of the Gateway system and intein purification method suggests that any tag and promoter can be trivially added to this system for the development of additional expression vectors. This technology could greatly facilitate process optimization, allowing several targets and methods to be tested in a high-throughput manner.     

A high-throughput platform for lentiviral overexpression screening of the human ORFeome

In response to the growing need for functional analysis of the human genome, we have developed a platform for high-throughput functional screening of genes overexpressed from lentiviral vectors. Protein-coding human open reading frames (ORFs) from the Mammalian Gene Collection were transferred into lentiviral expression vector using the highly efficient Gateway recombination cloning. Target ORFs were inserted into the vector downstream of a constitutive promoter and upstream of an IRES controlled GFP reporter, so that their transfection, transduction and expression could be monitored by fluorescence. The expression plasmids and viral packaging plasmids were combined and transfected into 293T cells to produce virus, which was then used to transduce the screening cell line. We have optimised the transfection and transduction procedures so that they can be performed using robotic liquid handling systems in arrayed 96-well microplate, one-gene-per-well format, without the need to concentrate the viral supernatant. Since lentiviruses can infect both dividing and non-dividing cells, this system can be used to overexpress human ORFs in a broad spectrum of experimental contexts. We tested the platform in a 1990 gene pilot screen for genes that can increase proliferation of the non-tumorigenic mammary epithelial cell line MCF-10A after removal of growth factors. Transduced cells were labelled with the nucleoside analogue 5-ethynyl-2'-deoxyuridine (EdU) to detect cells progressing through S phase. Hits were identified using high-content imaging and statistical analysis and confirmed with vectors using two different promoters (CMV and EF1α). The screen demonstrates the reliability, versatility and utility of our screening platform, and identifies novel cell cycle/proliferative activities for a number of genes.     

转录因子DREB1A基因的克隆与Gateway克隆技术构建植物表达载体

目的:研究转录因子DREB1A在植物抗渗透胁迫反应中的作用,并探讨利用Gateway克隆技术构建植物表达载体的方法。方法:根据GenBank中登录的DREB1A基因的全长mRNA序列设计引物,克隆了拟南芥的转录因子DREBIA基因。根据Gateway克隆技术的要求,设计含有attB接头的引物,利用高保真的PlatinumpfxDNA聚合酶,通过PCR方法在克隆基因的两端加上B序列。通过BP反应将包含有attB接头的PCR产物克隆到含有attP的donor载体上以产生Entry克隆,通过LR反应将已经重组入Entry载体的DREB1A基因再克隆到pH2GW7双元载体。结果:对重组载体pH2GW7-DREB1A的鉴定结果表明成功构建了DREB1A基因的植物表达载体。结论:利用Gateway克隆技术构建植物表达载体简便易行,该结果为遗传转化研究奠定了基础。     

利用TA克隆的方法简便构建入门克隆

Gateway技术是一种通用型克隆方法,其基于λ噬菌体位点特异性重组,将目的DNA快速克隆到各种与Gateway技术兼容的目的载体上,不需要进行酶切和连接反应。但存在获得入门克隆过程中相关反应酶制剂价格昂贵,且药品订购时间较长等问题。通过对入门载体pDONR207的改造,使之产生3’端具有单个T 末端的线性化的入门载体,采用TA克隆的方法替代BP反应,从而简便、经济和快速地获得入门克隆。利用改造后的Gateway技术构建拟南芥SOS2基因的原核表达载体和真核表达载体,通过原核表达和原生质体瞬时表达证明通过此方法构建的表达载体在原核细胞和真核细胞中都得到了很好的表达。     

Improved solubility of TEV protease by directed evolution

The efficiency and high specificity of tobacco etch virus (TEV) protease has made it widely used for cleavage of recombinant fusion proteins. However, the production of TEV protease in E. coli is hampered by low solubility. We have subjected the gene encoding TEV protease to directed evolution to improve the yield of soluble protein. Libraries of mutated genes obtained by error-prone PCR and gene shuffling were introduced into the Gateway cloning system for facilitated transfer between vectors for screening, purification, or other applications. Fluorescence based in vivo solubility screening was carried out by cloning the libraries into a plasmid encoding a C-terminal GFP fusion. Mutant genes giving rise to high GFP fluorescence intensity indicating high levels of soluble TEV-GFP were subsequently transferred to a vector providing a C-terminal histidine tag for expression, purification, and activity tests of mutated TEV. We identified a mutant, TEV(SH), in which three amino acid substitutions result in a five-fold increase in the yield of purified protease with retained activity.     

成簇pri-miRNAs表达载体的构建及表达验证

目的:旨在建立一个多重表达任意miRNA的方案,实现多个pri-miRNA表达阅读框的串联,用于miRNA簇或者miRNA家族的功能研究。方法:通过改造CRISPR/Cas9系统共表达载体42230,插入EGFP编码序列以及单个pri-miRNA序列,实现单个pri-miRNA表达载体可视化的构建。在单个pri-miRNA表达载体的基础上,通过同源重组的方式,插入下一个pri-miRNA序列,实现多个pri-miRNA表达阅读框的串联表达。结果:以miR29家族的三个miRNA为例,验证了在pri-miRNA表达阅读框串联方面的可行性;在多重的pri-miRNA表达载体中,miRNA成熟体验证结果表明,pri-miR29a和pri-miR29b能够在哺乳动物细胞中被加工成熟,其成熟体明显地过表达。在转染的293A细胞中,miR29家族的靶基因PTEN的表达水平显著降低。结论:所构建的pri-miRNA多重表达方案,能够实现多个外源miRNA在同一细胞中的共表达,对于探索miRNA家族和miRNA簇的拮抗或协同调控功能起到重要的推进作用。