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A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila.

Nagarkar-Jaiswal S, Lee PT, Campbell ME, Chen K, Anguiano-Zarate S, Gutierrez MC, Busby T, Lin WW, He Y, Schulze KL, Booth BW, Evans-Holm M, Venken KJ, Levis RW, Spradling AC, Hoskins RA, Bellen HJ - Elife (2015)

Bottom Line: The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or mutant phenotypes.Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies.This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States.

ABSTRACT
Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.

No MeSH data available.


Dnc expression pattern in mushroom bodies in the adult head.(A) Dnc-EGFP-Dnc expression in adult mushroom body: α/β and α′/β′ lobe (a–c), γ lobe (d–f). Adult heads are immuostained with ani GFP (a and d) and anti Dnc (b and e) antibodies. Scale bar, 50 μm. (B) Expression pattern of MB driver 117y-GAL4. Scale bar, 50 μm.DOI:http://dx.doi.org/10.7554/eLife.05338.021
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fig9s1: Dnc expression pattern in mushroom bodies in the adult head.(A) Dnc-EGFP-Dnc expression in adult mushroom body: α/β and α′/β′ lobe (a–c), γ lobe (d–f). Adult heads are immuostained with ani GFP (a and d) and anti Dnc (b and e) antibodies. Scale bar, 50 μm. (B) Expression pattern of MB driver 117y-GAL4. Scale bar, 50 μm.DOI:http://dx.doi.org/10.7554/eLife.05338.021

Mentions: To correlate the removal of a protein with a behavioral assay, we tagged the Dunce protein via RMCE using MI03415 (Figure 9A). dunce (dnc) encodes a cAMP phosphodiesterase (PDE) that is required for learning and memory (Davis and Kiger, 1981; Qiu et al., 1991). The EGFP cassette exon is positioned to tag all but one protein isoform (Figure 9A). The Dnc-EGFP-Dnc protein is expressed in the adult brain and is mostly restricted to mushroom bodies (MB), the center for learning and memory in Drosophila (Figure 9B). A more detailed analysis reveals that the fusion protein is expressed in all MB lobes (α, α′, β, β′ and γ) in the adult brain (Figure 9B), and the GFP staining reveals a much crisper expression pattern than the anti Dunce antibody (Figure 9—figure supplement 1A) (Nighorn et al., 1991). To knock down Dnc we used the 117y-GAL4 driver (Armstrong et al., 1998) which is expressed in MBs (Figure 9—figure supplement 1). The dnc-EGFP-dnc;117y-GAL4/+;UAS-deGradFP/+ flies were raised at 18°C. 1 day after eclosion, the flies were transferred to 28°C for 3 days, and then returned to 18°C for 2 days. We observe a progressive loss of Dnc-EGFP-Dnc, and after 48 hr at 28°C we could not or barely detect the tagged protein (Figure 9C). Protein expression is restored 48 hr after a shift back to 18°C (Figure 9C). To determine whether the decrease in Dnc-EGFP-Dnc expression levels correlates with a known behavioral phenotype associated with loss of dnc, we performed the aversive olfactory learning assay (Tully and Quinn, 1985). As expected, the dnc mutant flies, dnc1/dnc1, dnc1/dncML and dnc1/dncM14 (all partial loss of function combinations of dnc alleles) exhibit a 50% reduction in performance index when compared to dnc1/+, y w, Canton-S, and dnc-EGFP-dnc flies (Figure 9D–E). In contrast, dnc-EGFP-dnc flies expressing deGradFP under the control of the MB specific driver and maintained at 28°C for 3 days exhibit a performance index that is decreased by 70%, similar to the most severe dunce alleles (Davis and Kiger, 1981). The same flies were then returned to 18°C, Dnc-EGFP-Dnc expression in MB was re-established within 24 hr, and the learning phenotype was recovered fully within 48 hr. Hence, deGradFP is able to reversibly alter protein expression levels. In addition to reestablishing Dnc-EGFP-Dnc expression, the learning score is reestablished after 2 days at 18°C (Figure 9F). These data indicate that combining MiMIC-based EGFP tagging, UAS-deGradFP and GAL4 drivers allows us to achieve not only spatial and temporal control of protein expression, but to do so in a reversible manner, in order to reduce and then restore gene function in live flies.10.7554/eLife.05338.020Figure 9.MiMIC mediated intronic tagging with EGFP permits a reversible spatial and temporal removal of proteins in flies.


A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila.

Nagarkar-Jaiswal S, Lee PT, Campbell ME, Chen K, Anguiano-Zarate S, Gutierrez MC, Busby T, Lin WW, He Y, Schulze KL, Booth BW, Evans-Holm M, Venken KJ, Levis RW, Spradling AC, Hoskins RA, Bellen HJ - Elife (2015)

Dnc expression pattern in mushroom bodies in the adult head.(A) Dnc-EGFP-Dnc expression in adult mushroom body: α/β and α′/β′ lobe (a–c), γ lobe (d–f). Adult heads are immuostained with ani GFP (a and d) and anti Dnc (b and e) antibodies. Scale bar, 50 μm. (B) Expression pattern of MB driver 117y-GAL4. Scale bar, 50 μm.DOI:http://dx.doi.org/10.7554/eLife.05338.021
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4379497&req=5

fig9s1: Dnc expression pattern in mushroom bodies in the adult head.(A) Dnc-EGFP-Dnc expression in adult mushroom body: α/β and α′/β′ lobe (a–c), γ lobe (d–f). Adult heads are immuostained with ani GFP (a and d) and anti Dnc (b and e) antibodies. Scale bar, 50 μm. (B) Expression pattern of MB driver 117y-GAL4. Scale bar, 50 μm.DOI:http://dx.doi.org/10.7554/eLife.05338.021
Mentions: To correlate the removal of a protein with a behavioral assay, we tagged the Dunce protein via RMCE using MI03415 (Figure 9A). dunce (dnc) encodes a cAMP phosphodiesterase (PDE) that is required for learning and memory (Davis and Kiger, 1981; Qiu et al., 1991). The EGFP cassette exon is positioned to tag all but one protein isoform (Figure 9A). The Dnc-EGFP-Dnc protein is expressed in the adult brain and is mostly restricted to mushroom bodies (MB), the center for learning and memory in Drosophila (Figure 9B). A more detailed analysis reveals that the fusion protein is expressed in all MB lobes (α, α′, β, β′ and γ) in the adult brain (Figure 9B), and the GFP staining reveals a much crisper expression pattern than the anti Dunce antibody (Figure 9—figure supplement 1A) (Nighorn et al., 1991). To knock down Dnc we used the 117y-GAL4 driver (Armstrong et al., 1998) which is expressed in MBs (Figure 9—figure supplement 1). The dnc-EGFP-dnc;117y-GAL4/+;UAS-deGradFP/+ flies were raised at 18°C. 1 day after eclosion, the flies were transferred to 28°C for 3 days, and then returned to 18°C for 2 days. We observe a progressive loss of Dnc-EGFP-Dnc, and after 48 hr at 28°C we could not or barely detect the tagged protein (Figure 9C). Protein expression is restored 48 hr after a shift back to 18°C (Figure 9C). To determine whether the decrease in Dnc-EGFP-Dnc expression levels correlates with a known behavioral phenotype associated with loss of dnc, we performed the aversive olfactory learning assay (Tully and Quinn, 1985). As expected, the dnc mutant flies, dnc1/dnc1, dnc1/dncML and dnc1/dncM14 (all partial loss of function combinations of dnc alleles) exhibit a 50% reduction in performance index when compared to dnc1/+, y w, Canton-S, and dnc-EGFP-dnc flies (Figure 9D–E). In contrast, dnc-EGFP-dnc flies expressing deGradFP under the control of the MB specific driver and maintained at 28°C for 3 days exhibit a performance index that is decreased by 70%, similar to the most severe dunce alleles (Davis and Kiger, 1981). The same flies were then returned to 18°C, Dnc-EGFP-Dnc expression in MB was re-established within 24 hr, and the learning phenotype was recovered fully within 48 hr. Hence, deGradFP is able to reversibly alter protein expression levels. In addition to reestablishing Dnc-EGFP-Dnc expression, the learning score is reestablished after 2 days at 18°C (Figure 9F). These data indicate that combining MiMIC-based EGFP tagging, UAS-deGradFP and GAL4 drivers allows us to achieve not only spatial and temporal control of protein expression, but to do so in a reversible manner, in order to reduce and then restore gene function in live flies.10.7554/eLife.05338.020Figure 9.MiMIC mediated intronic tagging with EGFP permits a reversible spatial and temporal removal of proteins in flies.

Bottom Line: The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or mutant phenotypes.Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies.This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, United States.

ABSTRACT
Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.

No MeSH data available.