哺乳动物细胞小干扰RNA库的建立和应用

双链小干扰RNA（siRNA）在多种类型细胞中介导特异性的基因沉默,这一现象的发现为深入研究单个基因的功能提供了重要的方法学基础,从而得到了广泛的应用.最近的文献报道了全基因组siRNA库的建立,为高通量基因功能分析和研究提供了新的方法,成为新的研究热点.小干扰RNA库可以用来筛选和研究介导细胞复杂表型和生物学过程的关键基因,通过建立一系列具有目的表型的细胞系,有可能对特定细胞信号调节通路进行更为全面的解析.本文综述了目前在siRNA建库方法方面的进展,并探讨了建立小干扰RNA库中的关键问题.     

A primer on using pooled shRNA libraries for functional genomic screens

The discovery of RNA interference (RNAi) has revolutionized genetic analysis in mammalian cells. Loss-of-function RNAi screens enable rapid, functional annotation of the genome. Of the various RNAi approaches, pooled shRNA libraries have received considerable attention because of their versatility. A number of genome-wide shRNA libraries have been constructed against the human and mouse genomes, and these libraries can be readily applied to a variety of screens to interrogate the function of human and mouse genes in an unbiased fashion. We provide an introduction to the technical aspects of using pooled shRNA libraries for genetic screens.     

High-throughput RNAi screening to dissect cellular pathways: A how-to guide

RNA interference (RNAi) has become a powerful tool to dissect cellular pathways and characterize gene functions. The availability of genome-wide RNAi libraries for various model organisms and mammalian cells has enabled high-throughput RNAi screenings. These RNAi screens successfully identified key components that had previously been missed in classical forward genetic screening approaches and allowed the assessment of combined loss-of-function phenotypes. Crucially, the quality of RNAi screening results depends on quantitative assays and the choice of the right biological context. In this review, we provide an overview on the design and application of high-throughput RNAi screens as well as data analysis and candidate validation strategies.     

Cellular phenotyping by RNAi.

A systematic characterization of genes with unknown function is a key challenge after the sequencing of the human genome and the genomes of many model organisms. High-throughput RNA-interference (RNAi) screenings have become a widely used approach in invertebrate model organisms and also promise to revolutionize cell biology in mammals. Genome-wide RNAi screens in Caenorhabditis elegans and Drosophila, and in a smaller scale in mammalian cells have proven to be a valuable and successful method for the dissection of diverse biological processes. A number of RNAi libraries have become available that rely on different technologies, such as long double-stranded (ds) RNAs, in vitro diced short-interfering (si) RNAs, synthetic siRNAs and short-hairpin (sh) RNAs, which all have specific advantages and disadvantages. In addition, progress in screening technologies and data analysis allows the adaptation of screening methods to analyse more complex cellular processes. This review will summarize strategies in combining genome-scale RNAi libraries, high-throughput screening technologies, integrated high-content data analysis and will discuss future challenges.     

Technique review: how to use RNA interference.

RNA interference (RNAi) has been rapidly adopted as a general method for inhibiting gene expression in most laboratory organisms. This paper discusses how libraries of RNAi reagents are being used to perform genome-wide reverse genetic screens in both model organisms and mammalian cells. Guidelines for designing effective small interfering RNAs and appropriate controls for mammalian RNAi experiments will also be discussed.     

A Genome-Wide CRISPR Library for High-Throughput Genetic Screening in Drosophila Cells

The simplicity of the CRISPR/Cas9 system of genome engineering has opened up the possibility of performing genome-wide targeted mutagenesis in cell lines,enabling screening for cellular phenotypes resulting from genetic aberrations.Drosophila cells have proven to be highly effective in identifying genes involved in cellular processes through similar screens using partial knockdown by RNAi.This is in part due to the lower degree of redundancy between genes in this organism,whilst still maintaining highly conserved gene networks and orthologs of many human disease-causing genes.The ability of CRISPR to generate genetic loss of function mutations not only increases the magnitude of any effect over currently employed RNAi techniques,but allows analysis over longer periods of time which can be critical for certain phenotypes.In this study,we have designed and built a genome-wide CRISPR library covering 13,501 genes,among which 8989 genes are targeted by three or more independent single guide RNAs(sg RNAs).Moreover,we describe strategies to monitor the population of guide RNAs by high throughput sequencing(HTS).We hope that this library will provide an invaluable resource for the community to screen loss of function mutations for cellular phenotypes,and as a source of guide RNA designs for future studies.     

High-throughput RNA interference screening using pooled shRNA libraries and next generation sequencing

RNA interference (RNAi) screening is a state-of-the-art technology that enables the dissection of biological processes and disease-related phenotypes. The commercial availability of genome-wide, short hairpin RNA (shRNA) libraries has fueled interest in this area but the generation and analysis of these complex data remain a challenge. Here, we describe complete experimental protocols and novel open source computational methodologies, shALIGN and shRNAseq, that allow RNAi screens to be rapidly deconvoluted using next generation sequencing. Our computational pipeline offers efficient screen analysis and the flexibility and scalability to quickly incorporate future developments in shRNA library technology.     

RNA干扰文库及其在功能基因组学研究中的应用

随着人类基因组大规模测序的完成,下一步的挑战是了解每一个基因的功能 . RNA 干扰文库为大规模基因功能筛选提供了可能 . 虽然用于线虫等模式生物的 RNAi 文库,已经证明是大规模基因功能筛选的有效方法,但这些文库不能用于高等动物的细胞 . 自 2003 年以来,用于人的细胞和哺乳动物细胞的 RNAi 文库取得了突破,相继出现构建已知基因 RNAi 文库和构建随机 RNAi 文库的报道,并成功地应用于大规模基因功能的筛选 . RNAi 文库作为一种简单、高效、大规模、高通量的功能基因组学研究的工具,将在基因功能研究、发现新的药物靶基因、发现疾病相关基因等方面有广阔的应用前景 .     

Genome-wide RNAi of C. elegans using the hypersensitive rrf-3 strain reveals novel gene functions

RNA-mediated interference (RNAi) is a method to inhibit gene function by introduction of double-stranded RNA (dsRNA). Recently, an RNAi library was constructed that consists of bacterial clones expressing dsRNA, corresponding to nearly 90% of the 19,427 predicted genes of C. elegans. Feeding of this RNAi library to the standard wild-type laboratory strain Bristol N2 detected phenotypes for approximately 10% of the corresponding genes. To increase the number of genes for which a loss-of-function phenotype can be detected, we undertook a genome-wide RNAi screen using the rrf-3 mutant strain, which we found to be hypersensitive to RNAi. Feeding of the RNAi library to rrf-3 mutants resulted in additional loss-of-function phenotypes for 393 genes, increasing the number of genes with a phenotype by 23%. These additional phenotypes are distributed over different phenotypic classes. We also studied interexperimental variability in RNAi results and found persistent levels of false negatives. In addition, we used the RNAi phenotypes obtained with the genome-wide screens to systematically clone seven existing genetic mutants with visible phenotypes. The genome-wide RNAi screen using rrf-3 significantly increased the functional data on the C. elegans genome. The resulting dataset will be valuable in conjunction with other functional genomics approaches, as well as in other model organisms.     

Genome-wide high-throughput screens in functional genomics

The availability of complete genome sequences from many organisms has yielded the ability to perform high-throughput, genome-wide screens of gene function. Within the past year, rapid advances have been made towards this goal in many major model systems, including yeast, worms, flies, and mammals. Yeast genome-wide screens have taken advantage of libraries of deletion strains, but RNA-interference has been used in other organisms to knockdown gene function. Examples of recent large-scale functional genetic screens include drug-target identification in yeast, regulators of fat accumulation in worms, growth and viability in flies, and proteasome-mediated degradation in mammalian cells. Within the next five years, such screens are likely to lead to annotation of function of most genes across multiple organisms. Integration of such data with other genomic approaches will extend our understanding of cellular networks.     

High-throughput approaches to dissecting MAPK signaling pathways

With the development of genome-wide RNAi libraries, it is now possible to screen for novel components of mitogen-activated protein kinase (MAPK) pathways in cell culture. Although genetic dissection in model organisms and biochemical approaches in mammalian cells have been successful in identifying the core signaling cassettes of these pathways, high-throughput assays can yield unbiased, functional genomic insight into pathway regulation. We describe general high-throughput approaches to assaying MAPK signaling and the receptor tyrosine kinase (RTK)/extracellular signal-regulated kinase (ERK) pathway in particular using a phospho-specific antibody-based readout of pathway activity. We also provide examples of secondary validation screens and methods for managing large datasets for future in vivo functional characterization.     

A novel class of temperature-sensitive mutants generated by RNAi-mediated knockdown

Temperature-sensitive (ts) mutants are powerful tools with which to investigate gene function, but it has been difficult to generate ts mutants in mammalian cells. Recently, RNA interference (RNAi) has been widely used for loss of function analyses. In addition, in various organisms, hypothermic-temperature-sensitive RNAi has been reported. By using this characteristic of RNAi, we attempted to generate ts mutants in mammalian cells and were able to successfully generate ts mutants of cell cycle regulator cdc2 and ubiquitin-activating enzyme E1. We compared ts mutants previously isolated by mutagenesis with those generated by RNAi knockdown, and observed similar phenotypes. This method enabled us to generate ts mutants (KDts, knockdown temperature-sensitive mutants) of the genes of interest and will be utilized to facilitate understanding of the biological processes regulated by an essential gene in mammalian cells.     

Dissecting mitosis by RNAi in<Emphasis Type="Italic">Drosophila</Emphasis> tissue culture cells

Here we describe a detailed methodology to study the function of genes whose products function during mitosis by dsRNA-mediated interference (RNAi) in cultured cells ofDrosophila melanogaster. This procedure is particularly useful for the analysis of genes for which genetic mutations are not available or for the dissection of complicated phenotypes derived from the analysis of such mutants. With the advent of whole genome sequencing it is expected that RNAi-based screenings will be one method of choice for the identification and study of novel genes involved in particular cellular processes. In this paper we focused particularly on the procedures for the proper phenotypic analysis of cells after RNAi-mediated depletion of proteins required for mitosis, the process by which the genetic information is segregated equally between daughter cells. We use RNAi of the microtubule-associated protein MAST/Orbit as an example for the usefulness of the technique. Published: June 15, 2003     

Using Functional Genetics to Understand Breast Cancer Biology

Genetic screens were for long the prerogative of those that studied model organisms. The discovery in 2001 that gene silencing through RNA interference (RNAi) can also be brought about in mammalian cells paved the way for large scale loss-of-function genetic screens in higher organisms. In this article, we describe how functional genetic studies can help us understand the biology of breast cancer, how it can be used to identify novel targets for breast cancer therapy, and how it can help in the identification of those patients that are most likely to respond to a given therapy.Much remains to be learned regarding the function of mammalian genes. Only some quarter of all human genes have well-described functions. It is likely that quite a few of these currently unannotated genes will turn out to play key parts in cancer biology. For example, a 70-gene gene signature that can discriminate breast tumors of good and poor prognosis contained some 20 genes of currently unknown function (van ‘t Veer et al. 2002). The fact that these genes of unknown function foretell breast cancer prognosis hints at a role for at least some of these genes in breast cancer biology. The unbiased search for genes that contribute to breast cancer development is therefore likely to yield a rich harvest of new insights. RNA interference allows us to suppress genes systematically on a large scale and study the effects of gene suppression on specific cellular processes or signaling pathways. Consequently, RNA interference-based genetic screens have the potential to deepen our understanding of the molecular events that cause breast cancer, to find novel targets for therapy and to find biomarkers of drug responsiveness. In this article, we will describe the technologies available to perform both gain-of-function and loss-of-function genetic screens and will illustrate how such functional genetic screens have been used in the recent past to study a variety of outstanding questions in the biology of breast cancer.     

Automated genome-wide visual profiling of cellular proteins involved in HIV infection

Recent genome-wide RNAi screens have identified >842 human genes that affect the human immunodeficiency virus (HIV) cycle. The list of genes implicated in infection differs between screens, and there is minimal overlap. A reason for this variance is the interdependence of HIV infection and host cell function, producing a multitude of indirect or pleiotropic cellular effects affecting the viral infection during RNAi screening. To overcome this, the authors devised a 15-dimensional phenotypic profile to define the viral infection block induced by CD4 silencing in HeLa cells. They demonstrate that this phenotypic profile excludes nonspecific, RNAi-based side effects and viral replication defects mediated by silencing of housekeeping genes. To achieve statistical robustness, the authors used automatically annotated RNAi arrays for seven independent genome-wide RNAi screens. This identified 56 host genes, which reliably reproduced CD4-like phenotypes upon HIV infection. The factors include 11 known HIV interactors and 45 factors previously not associated with HIV infection. As proof of concept, the authors confirmed that silencing of PAK1, Ku70, and RNAseH2A impaired HIV replication in Jurkat cells. In summary, multidimensional, visual profiling can identify genes required for HIV infection.     

CDC-42 regulates PAR protein localization and function to control cellular and embryonic polarity in C. elegans.

BACKGROUND: The polarization of the anterior-posterior axis (A-P) of the Caenorhabditis elegans zygote depends on the activity of the par genes and the presence of intact microfilaments. Functional links between the PAR proteins and the cytoskeleton, however, have not been fully explored. It has recently been shown that in mammalian cells, some PAR homologs form a complex with activated Cdc42, a Rho GTPase that is implicated in the control of actin organization and cellular polarity. A role for Cdc42 in the establishment of embryonic polarity in C. elegans has not been described. RESULTS: To investigate the function of Cdc42 in the control of cellular and embryonic polarity in C. elegans, we used RNA-mediated interference (RNAi) to inhibit cdc-42 activity in the early embryo. Here, we demonstrate that RNAi of cdc-42 disrupts manifestations of polarity in the early embryo, that these phenotypes depend on par-2 and par-3 gene function, and that cdc-42 is required for the localization of the PAR proteins. CONCLUSIONS: Our genetic analysis of the regulatory relationships between cdc-42 and the par genes demonstrates that Cdc42 organizes embryonic polarity by controlling the localization and activity of the PAR proteins. Combined with the recent biochemical analysis of their mammalian homologs, these results simultaneously identify both a regulator of the PAR proteins, activated Cdc42, and effectors for Cdc42, the PAR complex.     

High-throughput RNAi screening in mammalian cells with esiRNAs

The development of advanced functional genomic tools has paved the way for systematic investigations of biological processes in health and disease. In particular, the implementation of RNA interference (RNAi) as a genome-wide, loss-of-function screening tool has enabled scientists to probe the role for every gene in cellular assays and many new factors for various processes have been discovered employing RNAi screens in recent years. However, the results also demonstrate the complexity of biological systems and indicate that we are still a long way from understanding functional networks in depth. Nevertheless, RNAi screens present a powerful method to interrogate gene function in high-throughput and different methods to elicit RNAi in mammalian cells have been developed. Here, we describe steps that should be considered when planning an RNAi screen employing endoribonuclease prepared (e)siRNAs. We provide useful information on how to implement the screen and analyze the results. Furthermore, we discuss strategies for hit validation and present an outline on how to follow-up on verified hits to gain a molecular understanding of the underlying phenotypes.     

Novel genes exhibit distinct patterns of function acquisition and network integration

Background  

Genes are created by a variety of evolutionary processes, some of which generate duplicate copies of an entire gene, while others rearrange pre-existing genetic elements or co-opt previously non-coding sequence to create genes with 'novel' sequences. These novel genes are thought to contribute to distinct phenotypes that distinguish organisms. The creation, evolution, and function of duplicated genes are well-studied; however, the genesis and early evolution of novel genes are not well-characterized. We developed a computational approach to investigate these issues by integrating genome-wide comparative phylogenetic analysis with functional and interaction data derived from small-scale and high-throughput experiments.     

Coexisting/Coexpressing Genomic Libraries (CoGeL) identify interactions among distantly located genetic loci for developing complex microbial phenotypes

In engineering novel microbial strains for biotechnological applications, beyond a priori identifiable pathways to be engineered, it is becoming increasingly important to develop complex, ill-defined cellular phenotypes. One approach is to screen genomic or metagenomic libraries to identify genes imparting desirable phenotypes, such as tolerance to stressors or novel catabolic programs. Such libraries are limited by their inability to identify interactions among distant genetic loci. To solve this problem, we constructed plasmid- and fosmid-based Escherichia coli Coexisting/Coexpressing Genomic Libraries (CoGeLs). As a proof of principle, four sets of two genes of the l-lysine biosynthesis pathway distantly located on the E. coli chromosome were knocked out. Upon transformation of these auxotrophs with CoGeLs, cells growing without supplementation were found to harbor library inserts containing the knocked-out genes demonstrating the interaction between the two libraries. CoGeLs were also screened to identify genetic loci that work synergistically to create the considerably more complex acid-tolerance phenotype. CoGeL screening identified combination of genes known to enhance acid tolerance (gadBC operon and adiC), but also identified the novel combination of arcZ and recA that greatly enhanced acid tolerance by 9000-fold. arcZ is a small RNA that we show increases pH tolerance alone and together with recA.