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A genetic toolkit for tagging intronic MiMIC containing genes.

Nagarkar-Jaiswal S, DeLuca SZ, Lee PT, Lin WW, Pan H, Zuo Z, Lv J, Spradling AC, Bellen HJ - Elife (2015)

Bottom Line: These modified genes permit numerous applications including assessment of protein expression pattern, identification of protein interaction partners by immunoprecipitation followed by mass spec, and reversible removal of the tagged protein in any tissue.At present, these conversions remain time and labor-intensive as they require embryos to be injected with plasmid DNA containing the exon tag.We document the efficiency and tag 60 mostly uncharacterized genes.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Baylor College of Medicine, Houston, United States.

ABSTRACT
Previously, we described a large collection of Minos-Mediated Integration Cassettes (MiMICs) that contain two phiC31 recombinase target sites and allow the generation of a new exon that encodes a protein tag when the MiMIC is inserted in a codon intron (Nagarkar-Jaiswal et al., 2015). These modified genes permit numerous applications including assessment of protein expression pattern, identification of protein interaction partners by immunoprecipitation followed by mass spec, and reversible removal of the tagged protein in any tissue. At present, these conversions remain time and labor-intensive as they require embryos to be injected with plasmid DNA containing the exon tag. In this study, we describe a simple and reliable genetic strategy to tag genes/proteins that contain MiMIC insertions using an integrated exon encoding GFP flanked by FRT sequences. We document the efficiency and tag 60 mostly uncharacterized genes.

No MeSH data available.


Expression of EGFP tagged protein in various tissues.(A) Colocalization of GFP tagged proteins with gene specific antibodies: anti-GFP signals (green) show colocalization with anti-Eys (red) in inter-rhabdomere space in adult eye (top panel) and anti-Delta (blue) in L3 eye imaginal disc in bottom panel. Scale bars, 2 μm and 5 μm. (B) Examples of EGFP expression pattern in different tissues from third instar larvae: brain; l(2)gl (A), Dl (B), twins (tws) (C), Sap-r (E), Rgk3 (F), and Hrb98DE (G), wing imaginal disc: kay (D), hindgut: CG10086 (H), and cells of the cuticle: CG5656 (I). Scale bars 100 μm. (A–I, except G), 25 μm (G). Eys, eyes shut.DOI:http://dx.doi.org/10.7554/eLife.08469.006
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fig3: Expression of EGFP tagged protein in various tissues.(A) Colocalization of GFP tagged proteins with gene specific antibodies: anti-GFP signals (green) show colocalization with anti-Eys (red) in inter-rhabdomere space in adult eye (top panel) and anti-Delta (blue) in L3 eye imaginal disc in bottom panel. Scale bars, 2 μm and 5 μm. (B) Examples of EGFP expression pattern in different tissues from third instar larvae: brain; l(2)gl (A), Dl (B), twins (tws) (C), Sap-r (E), Rgk3 (F), and Hrb98DE (G), wing imaginal disc: kay (D), hindgut: CG10086 (H), and cells of the cuticle: CG5656 (I). Scale bars 100 μm. (A–I, except G), 25 μm (G). Eys, eyes shut.DOI:http://dx.doi.org/10.7554/eLife.08469.006

Mentions: To ensure that the expression pattern and protein distribution correspond to the endogenous protein, we costained two tagged lines with GFP for which specific monoclonal antibodies are available: Eyes shut (Eys) (mAb 21A6,) and Delta (Dl) (mAb C594.9B) (Figure 3A). In both cases, the protein recognized by the mAb colocalizes with the GFP and match the described expression patterns (Das et al., 2013; Haltom et al., 2014). Note, however, that the GFP tagged Eys protein is present in the cytoplasm of the photoreceptors and the inter-rhabdomere spaces (IRS) of the photoreceptors, whereas the mAb against Eys mostly localizes to the IRS (Figure 3A). These data are in agreement with what we previously observed for numerous tagged proteins (Venken et al., 2011; Nagarkar-Jaiswal et al., 2015).10.7554/eLife.08469.006Figure 3.Expression of EGFP tagged protein in various tissues.


A genetic toolkit for tagging intronic MiMIC containing genes.

Nagarkar-Jaiswal S, DeLuca SZ, Lee PT, Lin WW, Pan H, Zuo Z, Lv J, Spradling AC, Bellen HJ - Elife (2015)

Expression of EGFP tagged protein in various tissues.(A) Colocalization of GFP tagged proteins with gene specific antibodies: anti-GFP signals (green) show colocalization with anti-Eys (red) in inter-rhabdomere space in adult eye (top panel) and anti-Delta (blue) in L3 eye imaginal disc in bottom panel. Scale bars, 2 μm and 5 μm. (B) Examples of EGFP expression pattern in different tissues from third instar larvae: brain; l(2)gl (A), Dl (B), twins (tws) (C), Sap-r (E), Rgk3 (F), and Hrb98DE (G), wing imaginal disc: kay (D), hindgut: CG10086 (H), and cells of the cuticle: CG5656 (I). Scale bars 100 μm. (A–I, except G), 25 μm (G). Eys, eyes shut.DOI:http://dx.doi.org/10.7554/eLife.08469.006
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499919&req=5

fig3: Expression of EGFP tagged protein in various tissues.(A) Colocalization of GFP tagged proteins with gene specific antibodies: anti-GFP signals (green) show colocalization with anti-Eys (red) in inter-rhabdomere space in adult eye (top panel) and anti-Delta (blue) in L3 eye imaginal disc in bottom panel. Scale bars, 2 μm and 5 μm. (B) Examples of EGFP expression pattern in different tissues from third instar larvae: brain; l(2)gl (A), Dl (B), twins (tws) (C), Sap-r (E), Rgk3 (F), and Hrb98DE (G), wing imaginal disc: kay (D), hindgut: CG10086 (H), and cells of the cuticle: CG5656 (I). Scale bars 100 μm. (A–I, except G), 25 μm (G). Eys, eyes shut.DOI:http://dx.doi.org/10.7554/eLife.08469.006
Mentions: To ensure that the expression pattern and protein distribution correspond to the endogenous protein, we costained two tagged lines with GFP for which specific monoclonal antibodies are available: Eyes shut (Eys) (mAb 21A6,) and Delta (Dl) (mAb C594.9B) (Figure 3A). In both cases, the protein recognized by the mAb colocalizes with the GFP and match the described expression patterns (Das et al., 2013; Haltom et al., 2014). Note, however, that the GFP tagged Eys protein is present in the cytoplasm of the photoreceptors and the inter-rhabdomere spaces (IRS) of the photoreceptors, whereas the mAb against Eys mostly localizes to the IRS (Figure 3A). These data are in agreement with what we previously observed for numerous tagged proteins (Venken et al., 2011; Nagarkar-Jaiswal et al., 2015).10.7554/eLife.08469.006Figure 3.Expression of EGFP tagged protein in various tissues.

Bottom Line: These modified genes permit numerous applications including assessment of protein expression pattern, identification of protein interaction partners by immunoprecipitation followed by mass spec, and reversible removal of the tagged protein in any tissue.At present, these conversions remain time and labor-intensive as they require embryos to be injected with plasmid DNA containing the exon tag.We document the efficiency and tag 60 mostly uncharacterized genes.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Baylor College of Medicine, Houston, United States.

ABSTRACT
Previously, we described a large collection of Minos-Mediated Integration Cassettes (MiMICs) that contain two phiC31 recombinase target sites and allow the generation of a new exon that encodes a protein tag when the MiMIC is inserted in a codon intron (Nagarkar-Jaiswal et al., 2015). These modified genes permit numerous applications including assessment of protein expression pattern, identification of protein interaction partners by immunoprecipitation followed by mass spec, and reversible removal of the tagged protein in any tissue. At present, these conversions remain time and labor-intensive as they require embryos to be injected with plasmid DNA containing the exon tag. In this study, we describe a simple and reliable genetic strategy to tag genes/proteins that contain MiMIC insertions using an integrated exon encoding GFP flanked by FRT sequences. We document the efficiency and tag 60 mostly uncharacterized genes.

No MeSH data available.