http://www.springerprotocols.com/cdp/access/showWebFeedListing This feed provides the latest 25 protocols in the given category. http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-191-8_1 An efficient RNAi largely depends on optimal design of the siRNA. In recent studies, Dicer substrates were found to be more potent than classical synthetic 21-mer siRNAs, suggesting a coupling of the Dicer-mediated processing step to the efficient assembly of the silencing complex, RISC. We describe the fundamental principles and experimental results leading to optimal Dicer substrates. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-191-8_10 RNA-mediated interference (RNAi) has been a valuable tool for the analysis of gene function in Caenorhabditis elegans (C. elegans). In C. elegans, the injection of double-stranded RNA (dsRNA) or plasmid DNA expressing dsRNA under the control of a C. elegans promoter results in gene inactivation through the specific degradation of the targeted endogeneous mRNA. It is also possible to initiate RNAi either by soaking worms in a solution of dsRNA or by feeding worms with E. coli expressing the dsRNA. Using studies of the DNA repair in C. elegans as an example, we describe the use of RNAi against the C. elegans POLH gene (Ce-POLH), which encodes DNA polymerase η (pol η). 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-191-8_11 Containment of the SARS coronavirus (SCV) outbreak was accompanied by the rapid characterization of this new pathogen's genome sequence in 2003, encouraging the development of anti-SCV therapeutics using short interfering RNA (siRNA) inhibitors. A pair of siRNA duplexes identified as potent SCV inhibitors in vitro was evaluated for in vivo efficacy and safety in a rhesus macaque SARS model using intranasal administration with clinical viable delivery carrier in three dosing regimens. Observations of SCV-induced SARS-like symptoms, measurements of SCV RNA presence in the respiratory tract, microscopic inspections of lung histopathology, and immunohistochemistry sections from 21 tested macaques consistently demonstrated siRNA-mediated anti-SCV activity. The prophylactic and therapeutic efficacies resulted in relief of animals from SCV infection-induced fever, diminished SCV in upper airway and lung alveoli, and milder acute diffuse alveoli damage (DAD). The dosages of siRNA used, 10 to 40 mg/kg, did not show any sign of siRNA-induced toxicity. These results support that a clinical investigation of this anti-SARS siRNA therapeutic agent is warranted. The study also illustrates the capability of siRNA to enable a massive reduction in development time for novel targeted therapeutic agents. We detail a representative example of large-mammal siRNA use. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-191-8_12 We describe the protocols of using siRNAs, or shRNAs delivered by a lentiviral vector, as a means to silence cancer-causing genes. We use cervical cancer as a model to demonstrate the inhibition of the human papillomavirus (HPV) oncogenes E6 and E7 in cervical cancer cells by RNAi and inhibition of the cell growth in vitro and tumor growth in mouse models. The protocols include methods on siRNA and shRNA design, production of lentiviral-vectored shRNA, transfection or transduction of cervical cancer cells with siRNA or shRNA, and detection of the inhibitory effects of siRNA or shRNA both in vitro and in vitro. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-191-8_13 RNA interference (RNAi) is a sequence-specific gene regulatory mechanism in which the specificity is determined by small RNAs. Three major classes of endogenous small RNAs, namely microRNAs (miRNAs), small interfering RNAs (siRNAs), and piwi-interacting RNAs (piRNAs/gsRNAs), have been characterized in vertebrates. The miRNAs are mainly involved in development and differentiation and alter gene expression through translational repression or mRNA cleavage. The siRNAs, in contrast, mainly defend against molecular parasites including viruses, transposons, and transgenes. We reported on the expression profile of miRNAs during Xenopus development using a combination of cloning and Northern blot analysis of stage-specific small RNAs. The expression of most miRNAs appeared to be regulated, and some were only expressed at specific stages of development. We also reported on small RNAs specifically expressed during gametogenesis in the mouse. The study revealed the existence of retrotransposon-derived siRNAs in oocytes and a novel class of small RNA (piRNAs/gsRNAs) in testes. In this chapter, we describe methods of low molecular weight RNA preparation, small RNA cloning, annotation of small RNAs, and analysis of expression during development. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-191-8_14 RNA silencing, including RNA interference, is a novel method of gene regulation and one of the potent host-defense mechanisms against the viruses. In the course of evolution, the viruses have encoded proteins with the potential to suppress the host RNA silencing mechanism as a counterdefense strategy. The virus-encoded RNA silencing suppressors (RSSs) can serve as important biological tools to dissect the detailed RNA silencing pathways and also to evolve the antiviral strategies. Screening and identification of the RSSs are indeed of utmost significance in the field of plant biotechnology. We describe two Green Fluorescent Protein (GFP) reporter-based plant assay systems that rely on two different principles, namely reversal of silencing and enhancement of rolling circle replication (RCR) of geminiviral replicon. These proof-of-concept examples and assay systems could be used to screen various plant, animal, and insect viral ORFs for identification of the RSS activities. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-191-8_15 Social insects represent a group of organisms that have dual importance from perspectives relating to both basic and applied science. From a basic perspective, social insects serve as excellent model systems for studying social organization, behavioral ecology, neurobiology, and phenotypic plasticity. From applied perspectives, social insects play important roles in the pollination of agricultural crops, in the damage of human structures and commodities, and in cellulose processing in natural ecosystems. With the advent of insect sociogenomics research (and the ability to identify dozens or hundreds of relevant candidate genes from a single experiment) has come a great demand for functional genomics tools for application in gene characterization. To date, RNAi is one of the most powerful tools to have become available for such functional characterizations, and it has broad relevance across a range of insect sociobiology research topics. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-191-8_16 The analysis of gene function during embryonic development asks for tight temporal control of gene expression. Classic genetic tools do not allow for this, as the absence of a gene during the early stages of development will preclude its functional analysis during later stages. In contrast, RNAi technology allows one to achieve temporal control of gene silencing especially when used with oviparous animal models. In contrast to mammals, reptiles and birds are easily accessible during embryonic development. We have developed approaches to use RNAi for the analysis of gene function during nervous system development in the chicken embryo. Although the protocol given here describes a method for gene silencing in the developing spinal cord, it can easily be adapted to other parts of the developing nervous system. The combination of the easy accessibility of the chicken embryo and RNAi provides a unique opportunity for temporal and spatial control of gene silencing during development. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-191-8_17 Flower color is mainly determined by the structure of flavonoids, a group of secondary metabolites of plants. The biosynthetic pathway and the genes involved in the pathway are well characterized such that it is possible to change flower color by engineering the pathway by overexpression of heterologous genes and/or suppression of endogenous genes in transgenic plants. Trimming an unnecessary pathway by suppression of endogenous genes is often essential to achieve successful engineering of the pathway and the resultant accumulation of desirable compounds. RNAi by transcription of double-stranded RNA (dsRNA) is a powerful and efficient method to command such suppression and is widely used for artificial gene suppression in transgenic plants. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-191-8_18 RNA interference (RNAi) is a powerful tool in deciphering gene function. It has been used extensively, especially in cultured mammalian cells. We have shown that RNAi-induced gene silencing can be generated in mice. With conventional transgenic techniques, shRNA-expressing constructs can be introduced into one-cell mouse embryos. The transgenic animals so obtained exhibit reduced expression of the targeted genes. Furthermore, the knockdown effect can be transmitted through the germline in these animals. We describe a method of generating a transgenic RNAi mouse line. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-191-8_2 RNA interference (RNAi) is a form of posttranscriptional gene silencing mediated by microRNA (miRNA) and small interfering RNA (siRNA). In Drosophila melanogaster, the RNase III enzymes Dicer-1 and Dicer-2 generate miRNA and siRNA, respectively. We describe the methods for the expression, purification, and analysis of recombinant Dicer-1 and Dicer-2 enzymes. Our studies demonstrate that Dicer-1 and Dicer-2 display different substrate specificities and ATP requirements. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-191-8_3 Recent studies have revealed that Argonaute proteins are crucial components of the RNA-induced silencing complexes (RISCs) that direct both small interfering RNA (siRNA)- and microRNA (miRNA)-mediated gene silencing. Full complementarity between the small RNA and its target messenger RNA (mRNA) results in RISC-mediated cleavage (“Slicing”) of the target mRNA. A subset of Argonaute proteins directly contributes to the target cleavage (“Slicer”) activity of the RISC. We describe (in vitro) Slicer assays using endogenous Argonaute protein immunopurified from animal cells and recombinant Argonaute protein produced in and purified from Escherichia coli. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-191-8_4 RNA interference (RNAi) is an endogenous gene regulatory pathway that the research community has adopted to facilitate the creation of a functional map of the human genome. To achieve this, small interfering RNAs (siRNAs), short synthetic duplexes having complete homology to the intended target, are introduced into cells to silence gene expression via a posttranscriptional cleavage mechanism. While siRNAs can be designed to effectively knock down any target gene, recent studies have shown that these small molecules frequently trigger off-target effects. These unintended events can have a significant impact on experimental outcomes and subsequent data interpretation. As RNAi is envisioned to play a central role in developing a functional map of the human genome, the development of reliable protocols for identifying off-targeted genes is essential. This chapter focuses on the underlying features of siRNA-mediated off-targeting and the state-of-the-art methodology used to identify off-targeted genes via microarray-based gene expression analysis. Future adoption of standards in this field will allow a clean distinction between sequence-specific off-target gene regulation and other forms of gene modulation resulting from delivery effects and other events unrelated to the RNAi pathway. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-191-8_5 The use of siRNA in vivo as well as in animal models has become more widespread in recent years, leading to further questions as to the best mode of delivery that will achieve optimal knockdown. While the exact mechanism of siRNA uptake at a cellular level has yet to be fully elucidated, various delivery techniques are being researched, including the use of viral vectors of shRNA, liposome encapsulations, and hydrodynamic delivery of naked siRNA. We describe the use of hydrodynamic administration as a technique to deliver, in vivo, naked siRNA constructs into experimental animals as a method of transient gene knockdown. This method may prove useful in situations where knockout animals do not exist, or to determine the effect of gene knockdown at specific time points during an experiment. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-191-8_6 The intranasal administration of siRNA has opened new vistas in drug delivery and respiratory therapy. In this strategy, synthetic siRNA with or without chemical modifications can be applied intranasally. Various delivery vehicles have been tested and optimized. With a few exceptions, all promote significant uptake of siRNA into the lung tissue and offer protection against respiratory viruses such as respiratory syncytial virus (RSV), parainfluenza virus (PIV), and influenza virus. No major adverse immune reaction has been encountered. Nasally applied siRNA remains within the lung and does not have systemic access, as judged by its absence in other major organs such as the lung, liver, heart, and kidney. We provide techniques for using the nose as a specific route for siRNA delivery into the lung of laboratory animals, which has enormous potential for clinical applications. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-191-8_7 RNA interference (RNAi) is a cellular mechanism that is often exploited as a technique for quelling the expression of a specific gene. RNAi studies are carried out in vivo, making this a powerful means for the study of protein function in situ Several trypanosomatids, including those organisms responsible for human and animal diseases, naturally possess the machinery necessary for RNAi manipulations. This allows for the use of RNAi in unraveling many of the pressing questions regarding the parasite's unique biology. The completion of the Trypanosoma brucei genome sequence, coupled with several powerful genetic tools, has resulted in widespread utilization of RNAi in this organism. The key steps for RNAi-based reduction of gene expression, including parasite cell culture, DNA transfection, RNAi expression, and experimental execution, are discussed with a focus on procyclic forms of Trypanosoma brucei. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-191-8_8 The ability to manipulate RNAi in cultured mammalian cells has provided scientists with a very powerful tool to influence gene expression. Neurons represent a cell type that initially displayed resistance to transduction by siRNAs or shRNA, when attempting to silence expression of endogenous genes. However, the development of lentiviral systems with that goal has facilitated the exogenous manipulation of RNAi in these postmitotic cells. Lentiviral-mediated RNAi experiments in cultured mammalian neurons can be designed to address a wide variety of biological questions or to test potential therapeutic hairpins before moving to treatment trials in vivo. We provide a practical approach to accomplish siRNA-mediated silencing of the disease-linked protein torsinA in primary neuronal cultures through the generation of lentiviral vectors expressing shRNAs. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-191-8_9 Experimental procedures using the RNA interference (RNAi) approach have recently emerged as a powerful tool for gene silencing in eukaryotic microbes for which gene replacement techniques have not yet been developed. Our group has recently explored RNAi to knock down gene-specific expression in the protozoan parasite Entamoeba histolytica, through delivery of small interfering RNA (siRNA) oligonucleotides by the soaking approach. 2008-04-01T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-248-3_1 Retroviral vectors have been widely used for research and clinical trials in gene therapy because of their high transduction efficiency. Retroviruses interact with target cells through their surface molecules (i.e., envelope proteins) and cellular receptors, which limit the susceptibility of target cells to retroviral vectors. Murine leukemia retrovirus (MuLV) pseudotyped with vesicular stomatitis virus G glycoprotein (VSV-G) overcomes the species barrier and is more resistant to mechanical and biochemical inactivation. A cell line producing VSV-G pseudotyped MuLV vector can be established by transfecting 293T cells expressing Gag, Pol, and VSV-G (293 GPG cell line) with a retroviral vector plasmid. Transduction potency of the resulting VSV-G pseudotyped MuLV retroviral supernatant can be quantified by titration, electron microscopy (EM), and the reverse transcriptase (RT) assay. These protocols provide methods to prepare and quantify a pseudotyped retroviral vector with high transduction rates for most types of target cells. 2008-03-07T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-248-3_10 A large amount of research activity worldwide is currently directed towards developing lipid- or polymer-based, non-viral gene vectors for therapeutic applications. This strong interest is motivated by their low toxicity, ease of production, ability to transfer large pieces of DNA into cells, and lack of immunogenic protein components. Cationic liposomes (CLs) are one of the most powerful non-viral vectors. In fact, CL-based vectors are among the prevalent synthetic carriers of nucleic acids currently used in human clinical gene therapy trials as well as in cell transfection applications for biological research. Our understanding of the mechanisms of action of CL–DNA complexes is still in its infancy. However, the relevance of a few crucial parameters, such as the lipid/DNA charge ratio ( $\rho_{\rm chg}$ ) and the membrane charge density of lamellar complexes ( $\sigma_{\rm M}$ ), is well established. To arrive at true comparisons of lipid performance, one must optimize both these parameters using a reproducible, reliable transfection assay. In this chapter, we aim to provide the reader with detailed procedures for liposome formation and transfection. It is our hope that the use of such optimized protocols will improve the comparability of transfection data obtained with novel lipids. 2008-03-07T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-248-3_11 Noninvasive imaging of molecular-genetic and cellular processes is an effective way to determine the location(s), magnitude, and time variation of action of gene products used for many therapeutic strategies. Lentiviral vectors provide effective means for the delivery, integration, and expression of transgenes in cultured mammalian cells as well as in vivo. Therefore, the combination of lentiviral vector-mediated therapeutic and imaging-targeted reporter gene delivery to various target organs holds promise for the future treatment of diseases. In this chapter, we provide protocols for developing lentiviral vectors that can be utilized for noninvasive monitoring/imaging of reporter gene expression. We have described the procedures to perform cellular assays and animal imaging based on positron emission tomography (PET), optical bioluminescence, and fluorescence reporter genes. The protocols described here are standardized for mouse models, which can also be adapted for other small animal models (e.g., rats). 2008-03-07T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-248-3_12 Mesenchymal stem cells (MSCs) are a promising target for the delivery of secreted proteins due to their ease of isolation, expansion, and genetic modification. The bleeding disorder hemophilia A results from the deficiency of a secreted blood clotting factor termed factor VIII (fVIII). Hemophilia A could be cured by gene-transfer-based procedures targeting virtually any cell type, including MSCs. Here, we describe methods for retroviral modification of MSCs incorporating a high-expression porcine (HEP)-fVIII transgene and a murine model of hemophilia A. MSCs were isolated from bone marrow of hemophilia A mice, expanded, and transduced ex vivo. Genetically modified MSCs secreted high levels of HEP-fVIII into the conditioned medium. HEP-fVIII was purified from the conditioned medium and demonstrated to have a specific activity, relative electrophoretic mobility, and proteolytic activation pattern similar to HEP-fVIII produced by other commercial cell lines. Collectively, these data support the concept that MSCs can be utilized as a cellular vehicle for successful gene-transfer-based therapy of hemophilia A and other disorders resulting from the deficiency of a secreted protein. 2008-03-07T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-248-3_13 Hematopoietic stem cells (HSCs) were among the first targets of genetic manipulation for the purpose of treating human diseases. The translational aspects of the first human clinical trials were based on results obtained using the mouse as an experimental model. Murine studies have shown that the major limitations of HSC gene therapy are similar to those encountered when using non-hematopoietic cells as targets and include (1) an inability to genetically modify sufficient numbers of target cells, (2) the loss of transgene function over time, and (3) potential complications due to vector integration. With continued improvements in transduction protocols, murine HSC transduction and transplantation are now routine with transduction efficiencies >50% easily achievable and even >90% feasible. However, attaining high-level engraftment of gene-modified cells after transplantation is still problematic. Basic transduction conditions entail cytokine stimulation of HSC populations, such as stem cell antigen-1 positive (Sca-1 $^{+}$ ) cells isolated from bone marrow, in serum-free media followed by multiple additions of recombinant retrovirus. Analysis of peripheral blood 12 weeks post transplantation of transduced cells into lethally irradiated recipients shows genetic marking in all hematopoietic lineages. Transduction of HSCs is then confirmed by transplanting bone marrow cells harvested from primary transplant recipients into lethally irradiated secondary recipients. Analysis of these mice shows that recombinant retroviruses transduce murine HSCs efficiently and stably and that the genetically modified cells are capable of completely repopulating the hematopoietic system. 2008-03-07T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-248-3_14 Cystic fibrosis (CF) a monogenic lethal disease and, therefore, ideally suited for the development of gene therapy. The first clinical trials were carried out shortly after cloning the CF gene in 1989. Since then, 25 trials have been carried out. Proof of principle for low-level airway gene transfer was established in most, but not all, trials. It is currently unclear whether current gene transfer efficiency will lead to improvements in clinically relevant endpoints such as inflammation or infection. In addition to addressing this important question, we and others are further improving airway gene transfer, by modifying existing and developing new gene transfer agents. Here, we describe pre-clinical methods related to assessing correction of the CF chloride transport defect. 2008-03-07T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-248-3_15 The use of replication-competent oncolytic viruses has largely advanced cancer gene therapy. Oncolytic virus not only possesses unique mechanisms of action that are distinct from other treatment modalities, its self-perpetuating nature provides an ideal platform for therapeutic transgene insertion. Tumor selectivity can be achieved by deleting viral genes that are critical for growth in normal cells but dispensable in tumor cells, transcriptional control under tumor-specific promoters, fiber modification targeting tumor-specific cellular receptors, or the use of inherent tumor-specific viruses. Transgene products can be amplified along with viral replication, thus maximizing therapeutic effect. Using adenovirus as a template, this chapter describes common assays used for the study of oncolytic viruses, with special emphasis on in vitro and in vivo viral replication determination. 2008-03-07T05:00:00Z