Limits...
The iBeetle large-scale RNAi screen reveals gene functions for insect development and physiology.

Schmitt-Engel C, Schultheis D, Schwirz J, Ströhlein N, Troelenberg N, Majumdar U, Dao VA, Grossmann D, Richter T, Tech M, Dönitz J, Gerischer L, Theis M, Schild I, Trauner J, Koniszewski ND, Küster E, Kittelmann S, Hu Y, Lehmann S, Siemanowski J, Ulrich J, Panfilio KA, Schröder R, Morgenstern B, Stanke M, Buchhholz F, Frasch M, Roth S, Wimmer EA, Schoppmeier M, Klingler M, Bucher G - Nat Commun (2015)

Bottom Line: Therefore, although deep sequencing is revealing the genes of ever more insect species, the functional studies predominantly focus on candidate genes previously identified in Drosophila, which is biasing research towards conserved gene functions.RNAi screens in other organisms promise to reduce this bias.This work transcends the restrictions of the candidate gene approach and opens fields of research not accessible in Drosophila.

View Article: PubMed Central - PubMed

Affiliation: 1] Johann-Friedrich-Blumenbach-Institut, GZMB, Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany [2] Department Biologie, Entwicklungsbiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstraße 5, 91058 Erlangen, Germany.

ABSTRACT
Genetic screens are powerful tools to identify the genes required for a given biological process. However, for technical reasons, comprehensive screens have been restricted to very few model organisms. Therefore, although deep sequencing is revealing the genes of ever more insect species, the functional studies predominantly focus on candidate genes previously identified in Drosophila, which is biasing research towards conserved gene functions. RNAi screens in other organisms promise to reduce this bias. Here we present the results of the iBeetle screen, a large-scale, unbiased RNAi screen in the red flour beetle, Tribolium castaneum, which identifies gene functions in embryonic and postembryonic development, physiology and cell biology. The utility of Tribolium as a screening platform is demonstrated by the identification of genes involved in insect epithelial adhesion. This work transcends the restrictions of the candidate gene approach and opens fields of research not accessible in Drosophila.

No MeSH data available.


Related in: MedlinePlus

Sensitivity and reproducibility.(a) Recognition rates of 41 different positive controls shown separately for the larval and pupal injection screens (left and middle bars) and for both together (right bar). About 80% of the positive controls were fully recognized while another 10% were ‘partially recognized' (that is, not all phenotypic aspects were annotated). Only 4% of the positive controls were missed. ‘technical lethality': Expected phenotype not recognized owing to lethality of the animals for example, by injection. (b) Recognition rates for dsRNAs targeting 48 genes with published phenotypes, which had by chance been included in the screen. Of all, 78% were recognized with the published phenotype while 17% were annotated with a ‘reproducibly different phenotype'; that is, the differing phenotype was reproduced in independent experiments under iBeetle conditions. Hence, these different phenotypes are biologically meaningful and reflect that the timing and the degree of gene knockdown influences the phenotype. See Supplementary Note 1 for discussion of these cases. (c) Only 2% of all buffer injections led to false positive annotations. (d) A total of 158 dsRNAs were tested in independent injections with non-overlapping fragments. When the phenotype differed from the screening result, we analysed whether it was a false positive (‘not reproduced'), or whether the genetic background was the reason for the difference (strain specific). Finally, we tested whether the outcome depended on the dsRNA fragment used (fragment specific), which indicated off-target effects or splice variant specific knockdown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4525174&req=5

f1: Sensitivity and reproducibility.(a) Recognition rates of 41 different positive controls shown separately for the larval and pupal injection screens (left and middle bars) and for both together (right bar). About 80% of the positive controls were fully recognized while another 10% were ‘partially recognized' (that is, not all phenotypic aspects were annotated). Only 4% of the positive controls were missed. ‘technical lethality': Expected phenotype not recognized owing to lethality of the animals for example, by injection. (b) Recognition rates for dsRNAs targeting 48 genes with published phenotypes, which had by chance been included in the screen. Of all, 78% were recognized with the published phenotype while 17% were annotated with a ‘reproducibly different phenotype'; that is, the differing phenotype was reproduced in independent experiments under iBeetle conditions. Hence, these different phenotypes are biologically meaningful and reflect that the timing and the degree of gene knockdown influences the phenotype. See Supplementary Note 1 for discussion of these cases. (c) Only 2% of all buffer injections led to false positive annotations. (d) A total of 158 dsRNAs were tested in independent injections with non-overlapping fragments. When the phenotype differed from the screening result, we analysed whether it was a false positive (‘not reproduced'), or whether the genetic background was the reason for the difference (strain specific). Finally, we tested whether the outcome depended on the dsRNA fragment used (fragment specific), which indicated off-target effects or splice variant specific knockdown.

Mentions: To assess the sensitivity of our screen, roughly 5% of screened genes were positive controls from a set of 41 different genes (Supplementary Fig. 1). In addition, 48 previously published genes that happened to be within our gene set served as additional blind positive controls. Ninety-three per cent of the selected controls and 95% of the previously published genes were identified during the screen (Fig. 1a,b; Supplementary Table 2), which is similar to the findings in a genome-wide RNAi screen in C. elegans11. Interestingly, we found reproducibly different or additional phenotypes for 17% of the published genes, which likely reflects the dependence of the RNAi effect on injection time and dsRNA concentration (see Supplementary Note 1). As expected, negative control injections usually produced no phenotype (Fig. 1c; Supplementary Note 1; Supplementary Fig. 2).


The iBeetle large-scale RNAi screen reveals gene functions for insect development and physiology.

Schmitt-Engel C, Schultheis D, Schwirz J, Ströhlein N, Troelenberg N, Majumdar U, Dao VA, Grossmann D, Richter T, Tech M, Dönitz J, Gerischer L, Theis M, Schild I, Trauner J, Koniszewski ND, Küster E, Kittelmann S, Hu Y, Lehmann S, Siemanowski J, Ulrich J, Panfilio KA, Schröder R, Morgenstern B, Stanke M, Buchhholz F, Frasch M, Roth S, Wimmer EA, Schoppmeier M, Klingler M, Bucher G - Nat Commun (2015)

Sensitivity and reproducibility.(a) Recognition rates of 41 different positive controls shown separately for the larval and pupal injection screens (left and middle bars) and for both together (right bar). About 80% of the positive controls were fully recognized while another 10% were ‘partially recognized' (that is, not all phenotypic aspects were annotated). Only 4% of the positive controls were missed. ‘technical lethality': Expected phenotype not recognized owing to lethality of the animals for example, by injection. (b) Recognition rates for dsRNAs targeting 48 genes with published phenotypes, which had by chance been included in the screen. Of all, 78% were recognized with the published phenotype while 17% were annotated with a ‘reproducibly different phenotype'; that is, the differing phenotype was reproduced in independent experiments under iBeetle conditions. Hence, these different phenotypes are biologically meaningful and reflect that the timing and the degree of gene knockdown influences the phenotype. See Supplementary Note 1 for discussion of these cases. (c) Only 2% of all buffer injections led to false positive annotations. (d) A total of 158 dsRNAs were tested in independent injections with non-overlapping fragments. When the phenotype differed from the screening result, we analysed whether it was a false positive (‘not reproduced'), or whether the genetic background was the reason for the difference (strain specific). Finally, we tested whether the outcome depended on the dsRNA fragment used (fragment specific), which indicated off-target effects or splice variant specific knockdown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525174&req=5

f1: Sensitivity and reproducibility.(a) Recognition rates of 41 different positive controls shown separately for the larval and pupal injection screens (left and middle bars) and for both together (right bar). About 80% of the positive controls were fully recognized while another 10% were ‘partially recognized' (that is, not all phenotypic aspects were annotated). Only 4% of the positive controls were missed. ‘technical lethality': Expected phenotype not recognized owing to lethality of the animals for example, by injection. (b) Recognition rates for dsRNAs targeting 48 genes with published phenotypes, which had by chance been included in the screen. Of all, 78% were recognized with the published phenotype while 17% were annotated with a ‘reproducibly different phenotype'; that is, the differing phenotype was reproduced in independent experiments under iBeetle conditions. Hence, these different phenotypes are biologically meaningful and reflect that the timing and the degree of gene knockdown influences the phenotype. See Supplementary Note 1 for discussion of these cases. (c) Only 2% of all buffer injections led to false positive annotations. (d) A total of 158 dsRNAs were tested in independent injections with non-overlapping fragments. When the phenotype differed from the screening result, we analysed whether it was a false positive (‘not reproduced'), or whether the genetic background was the reason for the difference (strain specific). Finally, we tested whether the outcome depended on the dsRNA fragment used (fragment specific), which indicated off-target effects or splice variant specific knockdown.
Mentions: To assess the sensitivity of our screen, roughly 5% of screened genes were positive controls from a set of 41 different genes (Supplementary Fig. 1). In addition, 48 previously published genes that happened to be within our gene set served as additional blind positive controls. Ninety-three per cent of the selected controls and 95% of the previously published genes were identified during the screen (Fig. 1a,b; Supplementary Table 2), which is similar to the findings in a genome-wide RNAi screen in C. elegans11. Interestingly, we found reproducibly different or additional phenotypes for 17% of the published genes, which likely reflects the dependence of the RNAi effect on injection time and dsRNA concentration (see Supplementary Note 1). As expected, negative control injections usually produced no phenotype (Fig. 1c; Supplementary Note 1; Supplementary Fig. 2).

Bottom Line: Therefore, although deep sequencing is revealing the genes of ever more insect species, the functional studies predominantly focus on candidate genes previously identified in Drosophila, which is biasing research towards conserved gene functions.RNAi screens in other organisms promise to reduce this bias.This work transcends the restrictions of the candidate gene approach and opens fields of research not accessible in Drosophila.

View Article: PubMed Central - PubMed

Affiliation: 1] Johann-Friedrich-Blumenbach-Institut, GZMB, Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany [2] Department Biologie, Entwicklungsbiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstraße 5, 91058 Erlangen, Germany.

ABSTRACT
Genetic screens are powerful tools to identify the genes required for a given biological process. However, for technical reasons, comprehensive screens have been restricted to very few model organisms. Therefore, although deep sequencing is revealing the genes of ever more insect species, the functional studies predominantly focus on candidate genes previously identified in Drosophila, which is biasing research towards conserved gene functions. RNAi screens in other organisms promise to reduce this bias. Here we present the results of the iBeetle screen, a large-scale, unbiased RNAi screen in the red flour beetle, Tribolium castaneum, which identifies gene functions in embryonic and postembryonic development, physiology and cell biology. The utility of Tribolium as a screening platform is demonstrated by the identification of genes involved in insect epithelial adhesion. This work transcends the restrictions of the candidate gene approach and opens fields of research not accessible in Drosophila.

No MeSH data available.


Related in: MedlinePlus