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In Vivo RNAi-Based Screens: Studies in Model Organisms.

Yamamoto-Hino M, Goto S - Genes (Basel) (2013)

Bottom Line: RNA interference (RNAi) is a technique widely used for gene silencing in organisms and cultured cells, and depends on sequence homology between double-stranded RNA (dsRNA) and target mRNA molecules.Drosophila and Caenorhabditis elegans are two model organisms whose whole bodies and individual body parts have been subjected to RNAi-based genome-wide screening.Moreover, Drosophila RNAi allows the manipulation of gene function in a spatiotemporal manner when it is implemented using the Gal4/UAS system.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Rikkyo University, 3-34-1 Nishi-Ikebukuro, Toshima-ku, Tokyo 171-8501, Japan. hinomiki@rikkyo.ac.jp.

ABSTRACT
RNA interference (RNAi) is a technique widely used for gene silencing in organisms and cultured cells, and depends on sequence homology between double-stranded RNA (dsRNA) and target mRNA molecules. Numerous cell-based genome-wide screens have successfully identified novel genes involved in various biological processes, including signal transduction, cell viability/death, and cell morphology. However, cell-based screens cannot address cellular processes such as development, behavior, and immunity. Drosophila and Caenorhabditis elegans are two model organisms whose whole bodies and individual body parts have been subjected to RNAi-based genome-wide screening. Moreover, Drosophila RNAi allows the manipulation of gene function in a spatiotemporal manner when it is implemented using the Gal4/UAS system. Using this inducible RNAi technique, various large-scale screens have been performed in Drosophila, demonstrating that the method is straightforward and valuable. However, accumulated results reveal that the results of RNAi-based screens have relatively high levels of error, such as false positives and negatives. Here, we review in vivo RNAi screens in Drosophila and the methods that could be used to remove ambiguity from screening results.

No MeSH data available.


Inductive expression of double-stranded RNA (dsRNA) by the Gal4/UAS system. Fly strains expressing Gal4 proteins in a spatiotemporally regulated manner under the control of enhancers are genetically crossed with fly strains bearing a gene expressing hairpin RNA downstream of an UAS. In the progeny, dsRNA is expressed in a regulated pattern and induces its target gene silencing.
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genes-04-00646-f001: Inductive expression of double-stranded RNA (dsRNA) by the Gal4/UAS system. Fly strains expressing Gal4 proteins in a spatiotemporally regulated manner under the control of enhancers are genetically crossed with fly strains bearing a gene expressing hairpin RNA downstream of an UAS. In the progeny, dsRNA is expressed in a regulated pattern and induces its target gene silencing.

Mentions: In contrast to C. elegans, Drosophila does not incorporate dsRNAs by feeding. In Drosophila, therefore, dsRNA is expressed in target cells as hairpin RNA (Section 3) using the Gal4/UAS system (Figure 1). The yeast Gal4 transcription factor binds to the UAS and activates expression of the downstream gene; theoretically, the gene downstream of the UAS is not expressed in the absence of Gal4. Consequently, a genetic cross between UAS- and Gal4-fly strains will induce expression of the gene downstream of the UAS. By placing genes expressing hairpin RNAs downstream of an UAS, RNAi is readily induced by genetic crossing. In addition, there are a large number of Gal4 strains in which the Gal4 gene is conditionally expressed, for example, in a specific tissue, or developmental stage, or under specific temperature conditions. Therefore, spatiotemporal patterns and levels of expression of hairpin RNAs are dependent on the Gal4 strain used.


In Vivo RNAi-Based Screens: Studies in Model Organisms.

Yamamoto-Hino M, Goto S - Genes (Basel) (2013)

Inductive expression of double-stranded RNA (dsRNA) by the Gal4/UAS system. Fly strains expressing Gal4 proteins in a spatiotemporally regulated manner under the control of enhancers are genetically crossed with fly strains bearing a gene expressing hairpin RNA downstream of an UAS. In the progeny, dsRNA is expressed in a regulated pattern and induces its target gene silencing.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3927573&req=5

genes-04-00646-f001: Inductive expression of double-stranded RNA (dsRNA) by the Gal4/UAS system. Fly strains expressing Gal4 proteins in a spatiotemporally regulated manner under the control of enhancers are genetically crossed with fly strains bearing a gene expressing hairpin RNA downstream of an UAS. In the progeny, dsRNA is expressed in a regulated pattern and induces its target gene silencing.
Mentions: In contrast to C. elegans, Drosophila does not incorporate dsRNAs by feeding. In Drosophila, therefore, dsRNA is expressed in target cells as hairpin RNA (Section 3) using the Gal4/UAS system (Figure 1). The yeast Gal4 transcription factor binds to the UAS and activates expression of the downstream gene; theoretically, the gene downstream of the UAS is not expressed in the absence of Gal4. Consequently, a genetic cross between UAS- and Gal4-fly strains will induce expression of the gene downstream of the UAS. By placing genes expressing hairpin RNAs downstream of an UAS, RNAi is readily induced by genetic crossing. In addition, there are a large number of Gal4 strains in which the Gal4 gene is conditionally expressed, for example, in a specific tissue, or developmental stage, or under specific temperature conditions. Therefore, spatiotemporal patterns and levels of expression of hairpin RNAs are dependent on the Gal4 strain used.

Bottom Line: RNA interference (RNAi) is a technique widely used for gene silencing in organisms and cultured cells, and depends on sequence homology between double-stranded RNA (dsRNA) and target mRNA molecules.Drosophila and Caenorhabditis elegans are two model organisms whose whole bodies and individual body parts have been subjected to RNAi-based genome-wide screening.Moreover, Drosophila RNAi allows the manipulation of gene function in a spatiotemporal manner when it is implemented using the Gal4/UAS system.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Rikkyo University, 3-34-1 Nishi-Ikebukuro, Toshima-ku, Tokyo 171-8501, Japan. hinomiki@rikkyo.ac.jp.

ABSTRACT
RNA interference (RNAi) is a technique widely used for gene silencing in organisms and cultured cells, and depends on sequence homology between double-stranded RNA (dsRNA) and target mRNA molecules. Numerous cell-based genome-wide screens have successfully identified novel genes involved in various biological processes, including signal transduction, cell viability/death, and cell morphology. However, cell-based screens cannot address cellular processes such as development, behavior, and immunity. Drosophila and Caenorhabditis elegans are two model organisms whose whole bodies and individual body parts have been subjected to RNAi-based genome-wide screening. Moreover, Drosophila RNAi allows the manipulation of gene function in a spatiotemporal manner when it is implemented using the Gal4/UAS system. Using this inducible RNAi technique, various large-scale screens have been performed in Drosophila, demonstrating that the method is straightforward and valuable. However, accumulated results reveal that the results of RNAi-based screens have relatively high levels of error, such as false positives and negatives. Here, we review in vivo RNAi screens in Drosophila and the methods that could be used to remove ambiguity from screening results.

No MeSH data available.