RNAi In Reverse Genetic Studies

Traditionally, the function of a gene was determined by forward genetic experiments that start with a mutant phenotype and the analysis of the protein defect and end with the conversion of the protein sequence into genetic information. Mutant phenotypes were found in patients with inherited diseases or knockout animals (64). Today, most gene functions are determined by reverse genetics, which work in the opposite direction. This approach was made possible by huge progress in recombinant DNA technology and the sequencing of a variety of genomes (65,66). Reverse genetic studies, sometimes also referred to as functional genomics, meet the challenge of deciphering the steadily accumulating genetic data into functional information. Meanwhile, the genetic information of several organisms has been deciphered, including C. elegans, D. melanogaster, and humans (65,66). So far, many methods have been developed to manipulate the expression of genetic information at different levels.

Gene silencing at the genomic level mainly proceeds by subjecting the isolated wildtype gene to in vitro mutagenesis by nucleotide substitution, deletion, or insertion. The mutated gene is then placed back into cultured cells or into the organism of interest, where it replaces the functional gene after homologous recombination. This method has been especially exploited to silence genes in animals such as mice and flies. Many practical approaches have been published, including inducible systems that enable the spatial, tissue-specific, and temporal inhibition ofgene expression (67-69). Many ofthese methods are very time-consuming and laborious.

Other common methods act at the posttranslational level, such as the depletion of proteins by antibodies masking the protein, inhibitors that block protein function by imitating substrates, or docking sites for other interaction partners, as well as RNA-based aptamers and intramers (70,71). With the discovery of RNAi, posttranscriptional silencing is receiving more and more interest.

In antisense technology the target mRNA is bound by homologous strands of antisense DNA, modified DNA, or PNA (peptide nucleic acids) to prevent the binding of the ribo-some and, thus, translation. Many modifications have been introduced to stabilize the antisense binding partner and a few systems have made their way to clinical trials. Yet, antisense technology has never quite met its high expectations, whereas RNAi with its catalytic nature seems to create a real hype.

In the generation of loss-of-function phenotypes, RNAi procedures are much faster and straightforward than traditional genetic approaches (64), certainly displaying the method for initial and high-throughput experiments.

In addition, it offers many advantages over other methods comprising specificity and efficiency. Only mRNA sharing perfect homology with catalytic amounts of exogenously applied dsRNA is cleaved, whereas other mRNAs, even those with point mutations, remain unaffected (1,72,73). It further offers a very simple handling. In C. elegans, RNAi can be induced by simply injecting adult worms with dsRNA, by soaking the animals in the dsRNA, by electroporation of dsRNA, or by engineering Escherichia coli to produce the appropriate dsRNA and feeding the bacteria to the worms (3,9,74-76). Soaking also functions in Drosophila S2 cell culture (77). Moreover, in plants and worms, the RNAi effect can diffuse across tissue boundaries and can be transmitted to the progeny (78,79). Moreover, RNAi can be used to simultaneously silence several genes.

Many techniques have already been established by a variety of laboratories, as described earlier. In C. elegans, RNAi has already yielded impressive results in investigating the functions ofthe whole genome (80), including genes implicated in cell division (81,82)— fat regulatory genes (83).

Genomewide RNAi screens became feasible with the generation of a library ofbacterial strains that each produce dsRNA for an individual nematode gene. The current library contains 16,757 bacterial strains targeting approx 86% of the 19,427 currently predicted genes of the C. elegans genome. The loss-of-function phenotype when performing systemic RNAi on a genomewide scale is estimated to be approx 65% (75,84) (For review, see refs. 83 and 85-87). These investigations revealed not only a detailed knowledge about the function of the targeted genes, but also allowed to estimate its relationship to conserved homologs in other species.

Like C. elegans, Drosophila is a prominent organism for genomewide functional RNAi studies. A huge number of Drosophila genes have been silenced since the early days of RNAi research, starting with frizzled-2 and wingless, which are involved in wing development (88). Recently, a genomewide RNAi screen in Drosophila Schneider cells (S2 cells) has been reported for the study of phenotypes affecting cell morphology (89,90). Moreover, RNAi has been used to successfully dissect mitosis and cytokinesis and to unravel cell signaling pathways in Drosophila tissue culture and cell lines (91,92). All Drosophila kinesins and cytoplasmic dynein have been targeted for mitotic phenotypes in S2 cells. For the analysis of functional redundancy and coordinated activity, RNAi was subsequently performed to simultaneously target multiple kinesin genes, an approach that was made feasible only by the RNAi technique (93).

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