Mammalian RNAi: a practical guide (original) (raw)

Gene silencing in mammals by small interfering RNAs

Nature Reviews Genetics, 2002

In the early days of molecular biology, genes were first defined through the description of their mutant phenotype. FORWARD GENETICS has the advantage that the phenotype of the mutant gives a clue to the function of the gene. But with the advent of large-scale genome sequencing the situation is different -literally thousands of genes have been identified, but we know nothing of their function. REVERSE GENETICS is now the most effective way to assess the function of a gene, but so far there has been no general method for reverse genetics other than gene targeting by homologous recombination, which is slow and costly. Antisense approaches, such as antisense oligonucleotide and RIBOZYME technologies, have been useful in reverse genetics, but only to a limited degree. By contrast, the promise of small interfering RNA (siRNA) technology to 'knock down' the expression of any gene in vertebrate cells is set to revolutionize reverse genetic approaches.

Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells

Genes & Development, 2002

RNA interference (RNAi) was first recognized in Caenorhabditis elegans as a biological response to exogenous double-stranded RNA (dsRNA), which induces sequence-specific gene silencing. RNAi represents a conserved regulatory motif, which is present in a wide range of eukaryotic organisms. Recently, we and others have shown that endogenously encoded triggers of gene silencing act through elements of the RNAi machinery to regulate the expression of protein-coding genes. These small temporal RNAs (stRNAs) are transcribed as short hairpin precursors (approximately 70 nt), processed into active, 21-nt RNAs by Dicer, and recognize target mRNAs via base-pairing interactions. Here, we show that short hairpin RNAs (shRNAs) can be engineered to suppress the expression of desired genes in cultured Drosophila and mammalian cells. shRNAs can be synthesized exogenously or can be transcribed from RNA polymerase III promoters in vivo, thus permitting the construction of continuous cell lines or transgenic animals in which RNAi enforces stable and heritable gene silencing.

Effective RNAi-mediated gene silencing without interruption of the endogenous microRNA pathway

Nature, 2007

Systemic administration of synthetic small interfering RNAs (siRNAs) effectively silences hepatocyte gene expression in rodents and primates 1-3. Whether or not in vivo gene silencing by synthetic siRNA can disrupt the endogenous microRNA (miRNA) pathway remains to be addressed. Here we show that effective target-gene silencing in the mouse and hamster liver can be achieved by systemic administration of synthetic siRNA without any demonstrable effect on miRNA levels or activity. Indeed, siRNA targeting two hepatocyte-specific genes (apolipo-protein B and factor VII) that achieved efficient (~80%) silencing of messenger RNA transcripts and a third irrelevant siRNA control were administered to mice without significant changes in the levels of three hepatocyte-expressed miRNAs (miR-122, miR-16 and let-7a) or an effect on miRNA activity. Moreover, multiple administrations of an siRNA targeting the hepatocyte-expressed gene Scap in hamsters achieved long-term mRNA silencing without significant changes in miR-122 levels. This study advances the use of siRNAs as safe and effective tools to silence gene transcripts in animal studies, and supports the continued advancement of RNA interference therapeutics using synthetic siRNA. Recently, it was reported that adeno-associated viral (AAV)-expressed short hairpin RNA (shRNA), when administered to mice, can result in profound toxicity, presumably by saturation of the cellular miRNA pathway 4. AAV expression of shRNA seemed to elicit toxicological effects by interference of cellular pathways for miRNA biogenesis, including Correspondence and requests for materials should be addressed to D.B.

RNAi Codex: a portal/database for short-hairpin RNA (shRNA) gene-silencing constructs

Nucleic Acids Research, 2006

Use of RNA interference (RNAi) in forward genetic screens is proliferating. Currently, short-interfering RNAs (siRNAs) and short-hairpin RNAs (shRNAs) are being used to silence genes to tease out functional information. It is becoming easier to harness RNAi to silence specific genes, owing to the development of libraries of readymade shRNA and siRNA genesilencing constructs by using a variety of sources. RNAi Codex, which consists of a database of shRNA related information and an associated website, has been developed as a portal for publicly available shRNA resources and is accessible at http:// codex.cshl.org. RNAi Codex currently holds data from the Hannon-Elledge shRNA library and allows the use of biologist-friendly gene names to access information on shRNA constructs that can silence the gene of interest. It is designed to hold usercontributed annotations and publications for each construct, as and when such data become available. We will describe features of RNAi Codex and explain the use of the tool.

A status report on RNAi therapeutics

Silence, 2010

Fire and Mello initiated the current explosion of interest in RNA interference (RNAi) biology with their seminal work in Caenorhabditis elegans. These observations were closely followed by the demonstration of RNAi in Drosophila melanogaster. However, the full potential of these new discoveries only became clear when Tuschl and colleagues showed that 21-22 bp RNA duplexes with 3" overhangs, termed small interfering (si)RNAs, could reliably execute RNAi in a range of mammalian cells. Soon afterwards, it became clear that many different human cell types had endogenous machinery, the RNA-induced silencing complex (RISC), which could be harnessed to silence any gene in the genome. Beyond the availability of a novel way to dissect biology, an important target validation tool was now available. More importantly, two key properties of the RNAi pathway -sequence-mediated specificity and potency -suggested that RNAi might be the most important pharmacological advance since the advent of protein therapeutics. The implications were profound. One could now envisage selecting disease-associated targets at will and expect to suppress proteins that had remained intractable to inhibition by conventional methods, such as small molecules. This review attempts to summarize the current understanding on siRNA lead discovery, the delivery of RNAi therapeutics, typical in vivo pharmacological profiles, preclinical safety evaluation and an overview of the 14 programs that have already entered clinical practice.

Lentivirus-delivered stable gene silencing by RNAi in primary cells

RNA, 2003

Genome-wide genetic approaches have proven useful for examining pathways of biological significance in model organisms such as Saccharomyces cerevisiae, Drosophila melanogastor, and Caenorhabditis elegans, but similar techniques have proven difficult to apply to mammalian systems. Although manipulation of the murine genome has led to identification of genes and their function, this approach is laborious, expensive, and often leads to lethal phenotypes. RNA interference (RNAi) is an evolutionarily conserved process of gene silencing that has become a powerful tool for investigating gene function by reverse genetics. Here we describe the delivery of cassettes expressing hairpin RNA targeting green fluorescent protein (GFP) using Moloney leukemia virus-based and lentivirus-based retroviral vectors. Both transformed cell lines and primary dendritic cells, normally refractory to transfection-based gene transfer, demonstrated stable silencing of targeted genes, including the tumor suppressor gene TP53 in normal human fibroblasts. This report demonstrates that both Moloney leukemia virus and lentivirus vector-mediated expression of RNAi can achieve effective, stable gene silencing in diverse biological systems and will assist in elucidating gene functions in numerous cell types including primary cells.