Aufsatz
Artikel (Publikationen im Open Access gefördert durch die UB)
A simple retroelement based knock-down system in Dictyostelium
(Further insights into RNA interference mechanisms)
Zusammenfassung
Characteristics of DIRS-1 Mediated Knock-Downs __ We have previously shown that the most abundant Dictyostelium discoideum retroelement DIRS-1 is suppressed by RNAi mechanisms. Here we provide evidence that both inverted terminal repeats have strong promoter activity and that bidirectional expression apparently generates a substrate for Dicer. A cassette containing the inverted terminal repeats and a fragment of a gene of interest was sufficient to activate the RNAi response, resulting in the generation of ~21 nt siRNAs, a reduction of mRNA and protein expression of the respective endogene. Surprisingly, no transitivity was observed on the endogene. This was in contrast to previous observations, where endogenous siRNAs caused spreading on an artificial transgene. Knock-down was successful on seven target genes that we examined. In three cases a phenotypic analysis proved the efficiency of the approach. One of the target genes was apparently essential because no knock-out could be obtained; the RNAi mediated knock-down, however, resulted in a very slow growing culture indicating a still viable reduction of gene expression.
Advantages of the DIRS-1–RNAi System __ The knock-down system required a short DNA fragment (~400 bp) of the target gene as an initial trigger. Further siRNAs were generated by RdRPs since we have shown some siRNAs with a 5’-triphosphate group. Extrachromosomal vectors facilitate the procedure and allowed for molecular and phenotypic analysis within one week. The system provides an efficient and rapid method to reduce protein levels including those of essential genes.
Zitierform
In: PLoS one. - Lawrence, Kan. : PLoS, 2015, 10 (6), e0131271, 1-17Förderhinweis
Gefördert durch den Publikationsfonds der Universität KasselSammlung(en)
Publikationen (Fachgebiet Entwicklungsgenetik)Artikel (Publikationen im Open Access gefördert durch die UB)
Zitieren
@article{urn:nbn:de:hebis:34-2016011849634,
author={Friedrich, Michael and Meier, Doreen and Schuster, Isabelle and Nellen, Wolfgang},
title={A simple retroelement based knock-down system in Dictyostelium},
journal={PLoS one},
year={2015}
}
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2016-01-18T13:34:53Z 2016-01-18T13:34:53Z 2015 1932-6203 urn:nbn:de:hebis:34-2016011849634 http://hdl.handle.net/123456789/2016011849634 Gefördert durch den Publikationsfonds der Universität Kassel eng PLoS Urheberrechtlich geschützt https://rightsstatements.org/page/InC/1.0/ 570 A simple retroelement based knock-down system in Dictyostelium Aufsatz Characteristics of DIRS-1 Mediated Knock-Downs __ We have previously shown that the most abundant Dictyostelium discoideum retroelement DIRS-1 is suppressed by RNAi mechanisms. Here we provide evidence that both inverted terminal repeats have strong promoter activity and that bidirectional expression apparently generates a substrate for Dicer. A cassette containing the inverted terminal repeats and a fragment of a gene of interest was sufficient to activate the RNAi response, resulting in the generation of ~21 nt siRNAs, a reduction of mRNA and protein expression of the respective endogene. Surprisingly, no transitivity was observed on the endogene. This was in contrast to previous observations, where endogenous siRNAs caused spreading on an artificial transgene. Knock-down was successful on seven target genes that we examined. In three cases a phenotypic analysis proved the efficiency of the approach. One of the target genes was apparently essential because no knock-out could be obtained; the RNAi mediated knock-down, however, resulted in a very slow growing culture indicating a still viable reduction of gene expression. Advantages of the DIRS-1–RNAi System __ The knock-down system required a short DNA fragment (~400 bp) of the target gene as an initial trigger. Further siRNAs were generated by RdRPs since we have shown some siRNAs with a 5’-triphosphate group. Extrachromosomal vectors facilitate the procedure and allowed for molecular and phenotypic analysis within one week. The system provides an efficient and rapid method to reduce protein levels including those of essential genes. open access Further insights into RNA interference mechanisms In: PLoS one. - Lawrence, Kan. : PLoS, 2015, 10 (6), e0131271, 1-17 Friedrich, Michael Meier, Doreen Schuster, Isabelle Nellen, Wolfgang Lawrence, Kan. doi:10.1371/journal.pone.0131271 6 PLoS one S. 1-17 10
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