基因工程外文翻译.doc

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。然而,许多基因的功能仍是未知尽管在生物信息学上以及转基因和基因敲除小鼠做出了大量的努力工作。逆转录病毒介导的基因转移是一个功能强大的工具,可以用来了解基因的功能。我们已经开发出多种逆转录病毒载体和高效的包装细胞系,有利于发展高效功能表达克隆的方法。本文中,我们描述了逆转录病毒介导的方法,用于基因功能和功能为基础的筛选策略的研究。2003 International Society for Experimental Hematology. Published by Elsevier Inc.Function-based gene cloningIt was only 30 years ago that recombinant DNA technology was initiated 1. The globin gene was cloned as the first mammalian gene in 1976 that was reverse transcribed from the purified mRNA for globin 2. For less abundant mRNAs, the cDNAs were frequently isolated based on the amino acid sequences of purified proteins. In late 1970s, a method called hybrid selection was developed. The principle of this method was to detect proteins translated from the mRNA hybridized to a particular pool of subdivided cDNA library fixed on nitrocellulose membranes, thereby identifying a pool that contains a cDNA of interest. Xenopus oocyte was used for production of proteins. This type of experiment is called “expression cloning,” which means “cloning of cDNA by detection of proteins expressed from cDNA libraries.” This strategy is suitable for isolation of rare cDNAs, such as cDNAs for cytokines and cytokine receptors. Levels of the protein expression are low, but a small amount of protein is enough to exert biologic functions.基于功能基因的克隆在仅仅30 年前,DNA重组技术才刚刚出现1。1976年珠蛋白基因被成功克隆,作为首次被克隆的哺乳动物基因,是从珠蛋白的纯化的mRNA反转录而来的。对于含量不丰富的mRNA来说,其cDNA经常以纯化的蛋白质的氨基酸序列为基础来纯化。在20世纪70年代末,一种称为混合选择的方法发展起来。这种方法的原理是检测与固定于硝酸纤维素膜上的特定细分的cDNA文库池杂交的mRNA转录而来的蛋白质,从而确定一个cDNA文库池,其中包含特定的基因。爪蟾卵母细胞用于蛋白质的生物合成。这种类型的实验称作表达克隆,意思是通过检测cDNA文库中表达而来的蛋白质而进行的cDNA克隆。这种方法适用于稀有cDNA的纯化,比如细胞因子和细胞因子受体的cDNA。虽然蛋白质表达水平低,但是少量的蛋白质足够发挥生物学功能。 A variety of expression cloning strategies has been established and utilized for cloning of cDNAs based on the biologic functions of their protein products. One of the early expression cloning methods used the Escherichia coli expression system for expression of cDNAs followed by detection by antibodies. In the early 1980s, the hybrid selection method was used for identification of cDNAs for cytokines using growth stimulation as a screening method. This method was later modified to directly transcribe cDNAs in Xenopus oocyte using the SP6 promoter. Alternatively, genomic DNAs were used to functionally clone cDNAs. The famous oncogene screening method, the focus-forming assay, using NIH3T3 cells falls into this category. It was notable that the invention of COS cells, in which plasmids can be amplified for the first time in mammalian cells, enabled expression cloning using mammalian cells 3. A variety of cDNAs were isolated by the COS cells-based functional cloning method. However, this strategy depended on specific cells such as COS7 cells where the SV40 large T antigens are expressed to enable amplification of SV40 origin-bearing plasmids 4. Therefore, only transient assays can be used for the screening多种表达克隆的方法已经建立,并可以其蛋白质产物的生物学功能来克隆cDNA。早期的表达克隆的方法之一是用大肠杆菌表达系统表达cDNAs随后进行抗体检测。在20世纪80年代初,混合选择法用于鉴定细胞因子的cDNAs,以生长刺激作为一种筛选方法。这种方法后来被修改,直接在爪蟾卵母细胞转录cDNA,使用SP6启动子。此外,基因组DNA用来功能性的克隆cDNAs。著名的基因筛选方法,聚焦实验,使用NIH3T3细胞属于这一范畴。值得注意的是COS细胞的出现,是质粒在哺乳动物中的首次扩增,是其在哺乳动物中克隆表达成为可能。通过COS细胞为基础的功能性克隆方法,许多cDNAs已被纯化。然而,这一方法依赖于特定的细胞,如COS7细胞中SV40大T抗体表达是SV40起源的质粒的扩增成为可能。因此只有为时较短的方法可以用来筛选。 To overcome the limitations of the conventional expression cloning system using COS cells, we and others turned to the idea of harnessing the power of retrovirus gene transfer to develop function-based screening of cDNAs为了克服使用COS细胞的传统的克隆表达系统的限制,我们和其他研究者转向另一种方法,即利用逆转录病毒基因转移来进行cDNAs的功能性筛选。Retrovirus-mediated expression screening; rationale and application逆转录病毒介导的表达筛选,基本原理和应用 Retrovirus-mediated expression cloning was developed in mid 1990s5?8. Construction of a cDNA library in a retrovirus vector is not different from that in a plasmid vector9. One can generate either unidirectional or bi-directional cDNA libraries depending on the intended application. Complimentary DNAs are generated using either oligo-dT primers or random hexamer primers. The library is kept as DNA solution, and is converted to retroviruses by using packaging cell lines. To generate retroviruses that represent and cover a high complexity of cDNA libraries, it is recommended to use 293-based packaging cell lines that are efficient in transient packaging 10,11The virus stock containing high-titer retroviruses is used to infect target cells, and the infected cells are selected for the phenotype of interest. The integrated cDNA then is recovered by genomic polymerase chain reaction PCR or reverse transcriptase RT-PCR to deter-mine which cDNA is responsible for the phenotype and is subjected to the sequence.逆转录病毒介导的表达克隆在20世纪90年代中期发展起来5-8。借助逆转录病毒载体的cDNA基因库建设和用质粒载体没有区别9。根据这个方法可以生成单向或双向的cDNA文库。互补的DNA用oligo-dT uo随即六聚体核苷酸作引物来生产。文库一DNA溶液的形式保存,并且使用包装细胞系统转换成逆转录病毒。要生成代表并包含cDNA文库的高复杂性的逆转录病毒,建议使用293包装细胞系,瞬时包装效率跟更高10,11。病毒的遗传特性包括高效价的逆转录病毒用来感染靶细胞,被感染的细胞可具有特定的性状而被选择出来。然后完整的cDNA可以用聚合酶链式反应(PCR)或逆转录PCR来恢复,老确定哪些cDNA与表现型有关哪些与基因序列有关。 The retrovirus-mediated expression cloning method is efficient because the number of the provirus integrations in each cell is limited. Therefore, it is not necessary to recover and reintroduce the plasmid from, and into, the cells repeatedly, as in the conventional method using COS7 cells. In retrovirus-mediated expression cloning, the infection efficiencies should be controlled between 10% and 30% to avoid multiple integration in a cell as much as possible. Alternatively, one can recover the integrated retroviruses by transfecting a helper construct harboring gag-pol and env genes into the isolated clone that has acquired a phenotype of interest after transduction of the cDNA library. In this case, the recovered retroviruses are infected to the target cells to determine which integration was responsible for the phenotype.逆转录病毒介导的表达克隆的方法是有效的,因为在每个细胞的前病毒整合的数量是有限的。因此,它没有必要像在COS7细胞的常规方法一样恢复和重新引入质粒。逆转录病毒介导的表达克隆,感染效率应控制在10%和30%之间,以尽可能避免多个整合到一个细胞内。另外,我们可以通过转染一个辅助子构建包含gag-pol和env基因到一个独立的克隆来回收整合的逆转录病毒,在cDNA文库转导后已经获得了特定表型。在这种情况下,回收的逆转录病毒感染靶细胞从而确定其中哪个整合是与表现型有关的。The most important advantage over the conventional method is that any functional assay can be applied to identify cDNAs by their functions because, once integrated, the expression of the retrovirally transduced cDNA usually is stable相对于传统方法最重要的优点是可应用于任何功能性的鉴定即通过功能识别DNAs,因为一旦整合,逆转录病毒转移的cDNA表达一般是稳定的。Retrovirus-mediated expression cloning: some examples逆转录病毒介导的表达克隆:一些实例 A variety of functional assays can be utilized in retrovirus- mediated expression cloning. For instance, cellular receptors for various viruses were identified based on infectability of the viruses. Infection-resistant cells transduced with the library derived from infectible cells are screened by infection of the virus vector harboring a reporter gene such as GFP. The cDNA recovered from reporter gene-positive cell i.e., infectible cell should encode a receptor for a virus of interest. Co-receptors for human immunodeficiency virus HIV and simian immunodeficiency virus SIV were identified in this way from cDNA libraries derived from human T cells 12,13A receptor for polytropic and xenotropic retro- virus, which had been searched for, was identified using the same approach14 各种功能检测都可以利用逆转录病毒介导的表达克隆。例如,许多病毒的细胞受体被确定是基于病毒感染。转染了从被感染细胞产生的文库的抗感染细胞用包含识别基因如GFP基因的感染病毒来筛选。从识别基因阳性细胞(即,被感染的细胞)恢复的cDNA编码一种他定的病毒的受体。人类免疫缺陷病毒(HIV)的联合受体和猿免疫缺陷病毒(SIV)通过这种方法识别,及人类T细胞的cDNA文库12,13。一个多变的和亲异的逆转录病毒的受体,它已被查处出被认定使用同样的方法14。Tumor necrosis factor TNF and Fas induce apoptosis through activation of downstream signaling pathways. After introduction of cDNA library into the cells, some cells may become resistant to TNF and Fas stimulation by expressing a retrovirally introduced cDNA. Using this strategy, a novel transcription factor BSAC and an adhesion molecule ICAM- 2 have been identified as inhibitors of TNF-induced and TNF- and Fas-induced apoptosis, respectively 15,16Intriguingly, ICAM-2 was found to activate PI3K, PDK-1, and Akt, leading to inhibition of apoptosis16Thus, identification of proteins based on their functions sometimes leads to unexpected and important findings. Another example of the unexpected result was encountered in our study. Mouse leukemic M1 cells differentiate into macrophages and undergo apoptosis upon interleukin-6 IL-6 stimulation. Starr et al. 17 identified an inhibitor of the cytokine signal, SOCS-1, by isolating an IL-6?resistant M1 clone after transducing a cDNA library to M1 cells via retrovirus infection. By the same strategy, we identified A1, which is an anti- apoptotic protein of the bcl-2 family and protected M1 cells from IL-6?induced apoptosis 18We also discovered a novel GAP MgcRacGAP in the anti-sense orientation from an IL-6?resistant clone; expression of anti-sense MgcRac- GAP protected M1 cells from IL-6?induced differentiation and apoptosis. On the other hand, overexpression of Mgc- RacGAP induced differentiation into macrophages in HL60 cells. An unexpected finding brought by the subsequent study was that MgcRac-GAP is required for cell division, especially for cytokinesis 19,20It would be interesting to investigate the link between cytokinesis and cell differentiation. Thus, functional identification of protein is a powerful tool in cell biology.肿瘤坏死因子(TNF)和Fas通过激活下游信号通路诱导凋亡。在细胞内引入cDNA文库方法后,一些细胞有可能通过表达逆转录引入的基因对肿瘤坏死因子和Fas的刺激有抗性。通过这种方法,一种新的转录因子BSAC和粘附分子ICAM-2已被分别确认为TNF诱导的和TNF和Fas一起诱导的细胞凋亡抑制剂15,16。有趣的是, ICAM-2被发现激活的PDK-1,PI3K和Akt,从而抑制细胞凋亡16。然而,根据功能对蛋白质的鉴定,有时会导致意想不到的重要发现。在另一个例子我们的研究中遇到了意想不到的结果。小鼠白血病M1细胞分化成巨噬细胞,并在白细胞介素-6的刺激下经历了细胞凋亡。Starr 等人17 在通过逆转录病毒感染转导M1细胞cDNA文库后通过隔离IL-6抗性的M1克隆发现了一种细胞因子信号抑制剂,SOCS-1。同样的策略,我们确定了A1,一种bcl-2家族的抗凋亡蛋白,保护M1细胞免受IL-6诱导的细胞凋亡18。我们还从IL-6抗性克隆发现了一种新型反义方向的GAP MgcRacGAP; 反义MgcRac-GAP M1的表达保护细胞免受IL-6诱导分化和凋亡。另一方面,在HL60细胞过度的MGC-RacGAP诱导其分化为巨噬细胞。后续研究带来了一个意外的发现是,MgcRac-gap对细胞分裂是必需的,尤其是胞质19,20。调查细胞分裂和细胞分化之间的联系将会很有前景。因此,鉴定蛋白质的功能是在细胞生物学的有力工具。Structure/function analysis using retrovirus-mediated screening combined with PCR-driven random mutagenesis采用逆转录病毒介导的利用PCR的随机突变筛选结合的结构/功能分析In addition to applications for a variety of expression cloning strategies, the retrovirus-mediated expression system can be used to identify a mutant molecule with altered functions. For instance, we identified constitutively active forms of a cytokine receptor MPL21 and a transcription factor STAT5 22In brief, we introduced random mutations into MPL and STAT5 by PCR. The PCR products then were ligated into the retrovirus vector pMX 9, and the ligated DNA was amplified in E. coliThus, the resulting plasmid DNA represented a mutation library of the protein of interest. This library was transiently transfected into a 293-based packaging cell to generate the retrovirus stock representing the mutation library of a particular molecule. It then was infected to target cells, and the infected cells were selected for a phenotype of interest, followed by retrieving and sequencing the integrated retrovirus of the selected clones. In our experiments, we used mouse IL-3?dependent Ba/F3 cells as targets, and selected the library-transduced Ba/F3 cells in the absence of IL-3 to isolate factor-independent clones. In that manner, we were able to identify constitutively active mutants of MPL 21 and STAT5A 22 that induced factor- independent growth in Ba/F3 cells as well as other IL-3? dependent cell lines. The constitutively active mutants of various signaling molecules will be useful for analyzing signaling pathways. In addition to isolation of active mutants of various molecules, this strategy will be applicable in generating various mutants with acquired functions. For instance, one may want to generate restriction enzymes that recognize altered restriction sites, or cytokines with stronger activities or with less side effect in vivo.除了应用多种表达克隆策略,逆转录病毒介导的表达系统可用于识别改变功能的突变分子。比如,我们确定了一个组成型细胞因子受体MPL21 和一个转录因子STAT5 22 的活跃形式。总之,我们通过PCR引入MPL和STAT5随机突变。PCR产物连接到逆转录病毒载体PMX9,连接好的DNA在大肠杆菌中扩增。所以,由此产生的质粒DNA代表了特定蛋白质的基因突变库。这个库瞬时转染到293包装细胞产生逆转录病毒的互补代表一个特定的分子突变库。然后,用它感染靶细胞,被感染的细胞通过特定表型而筛选,其次是对选择的整合的克隆逆转录进行检索和测序。在我们的实验中,我们用小鼠IL-3依赖的Ba/F3细胞作为靶细胞,并选择文库转染的没有IL-3的Ba/F3细胞来纯化不依赖细胞因子的细胞克隆。以这种方式,我们能够识别组成型的MPL突变21和STAT5A22 ,即诱导因子非依赖性的Ba/F3细胞以及其他IL-3依赖性细胞株的生长。各种组成型的信号分子的有益突变将有益于信号通路的分析。除了纯化各种分子的有益突变,这一方法将适用于产生各种功能的获得性突变。例如,产生的限制性内切酶可识别改变的酶切位点,或者具有更高活性或在体内副作用更少的细胞因子。SST-REX, an efficient signal sequence trap based on retrovirus-mediated gene transferSST - REX,基于逆转录病毒介导的基因转移的一个有效的信号序列陷阱Sorting of the protein between the cellular components is regulated by various sorting signals in the proteins, such as the nuclear localization signal and the mitochondrial targeting sequence. Signal sequence is one such sorting signal found in type I transmembrane proteins and secreted proteins. Tashiro et al. 23 developed an elegant method called signal sequence trap SST by which signal sequence-harboring cDNAs are specifically isolated. The rationale of the method is to search for a cDNA fragment that contains a signal sequence and directs a signal sequence-defective CD25 to the cell surface by the fusion. This method, how- ever, is time consuming and leads to frequent isolation of false-positive clones. Klein et al. 24 invented a modified SST method using growth of a yeast mutant YT455 as a screening method. We applied retrovirus-mediated gene transfer and developed an efficient and accurate method SST- REX signal sequence trap? retrovirus-mediated expression screening using mammalian cells 25We used the constitutively active MPL 21 in developing SST-REX, as illustrated in Figure 1. In brief, we construct a cDNA library in a retrovirus vector in which cDNA fragments are fused to an extracellular deletion mutant of the constitutively active MPL. The fusion library then is transduced into IL-3?dependent Ba/F3 cells via retrovirus infection. When the inserted cDNA fragment contains a signal sequence, it directs the mutant MPL on the cell surface and confers Ba/F3 cells factor independence. We then recover the integrated cDNA from the factor-independent Ba/F3 clones. Ten milliliters of the virus supernatant gives rise to isolation of about 1000 to 2000 signal sequences with 95 to 100% accuracy. Whereas previous methods collected shorter cDNA fragments for SST, we use cDNA fragments in the range from 0.5 to 6 kbp. In the results, the average length of cDNA fragments isolated in SST-REX is about 1 kbp. In SST, generally speaking, shorter cDNA fragments shorter than 100?200 bp will give false-positive results more frequently unpublished results, and we believe this is a reason why SST- REX achieves higher accuracy than other methods.细胞成分之间的蛋白质分选受各种蛋白质上分选信号的调节,如核定位信号和线粒体定位序列。信号序列是发现在I型跨膜蛋白和分泌蛋白的一个序列信号。Tashiro 等人 23发现了一种温和的方法被称为信号序列陷阱(SST),包含信号序列的cDNAs被专门纯化。该方法的基本原理是搜索一个包含信号序列并且指信号序列缺陷的CD25表达通过细胞融合到细胞表面的cDNA片段。这种方法费时,且导致频繁出现假阳性克隆。Klein 等人 24 提出了一种改进的SST方法,用酵母突变YT455的生长作为一种筛选途径。我们使用哺乳动物细胞采用逆转录病毒介导的基因转移和生产一个高效,准确的方法即SST-REX(信号序列陷阱-逆转录病毒介导的表达筛选) 25。我们在研究SST-REX中使用组成活跃的MPL21,如图1所示。总之,我们在逆转录病毒载体构建cDNA文库,在此cDNA片段融合到细胞外缺失突变体的一个组成型活跃的MPL。然后通过逆转录病毒感染融合库转成IL-3依赖性的Ba