New tricks for protein trap lines in Drosophila

Protein trap lines in various organisms have mainly been used to study the expression pattern of the tagged gene products. In addition to this approach we show that protein trap lines can effectively be used to perform high stringency loss of function experiments and to probe the interactome of the tagged protein, significantly expanding the experimental versatility of existing protein trap lines.

HFSP Long-Term Fellows Ralph Neumüller and Frederik Wirtz-Peitz and colleagues
authored on Thu, 12 April 2012

A method commonly used for visualization of endogenous proteins at the sub-cellular level without the need for antibodies or dyes is the “protein trap” approach.Protein trapping allows for fluorescent protein- and/or epitope-tagging of endogenous proteins via transposon-based insertion of a construct that supplies the open reading frame (ORF) of a moiety such as green fluorescent protein (GFP) in-frame with an endogenously encoded ORF. This approach has been used in several species including Drosophila where large collections of protein trap lines have been generated.

We reasoned that besides expression analysis, the GFP sequence could also be used to interfere with gene function of the fused gene by targeting GFP at the mRNA level using transgenic RNA interference (RNAi). This strategy (tag-mediated loss of function) would be advantageous to the use of classical RNAi approaches, in which gene specific sequences are targeted, as a single optimized RNAi reagent can be used to analyze all GFP-tagged proteins. We generated multiple small hairpin RNA (shRNA) transgenic fly lines targeting GFP that can independently be used to induce and validate tag mediated loss of function phenotypes. To test this approach, we screened homozygous viable protein trap lines that show strong GFP expression in the Drosophila female germline stem cell lineage and used tag mediated loss of function to reveal a role for the gene spt6 in the maintenance of germline stem cells as well a role for the genes Cp1 and Pabp2 in germline cell survival.

Besides performing high stringency loss of function studies using the GFP tag, we wanted to explore if GFP protein trap lines lend themselves to high stringency protein-protein interaction studies. The use of GFP traps or GFP-tagged genomic rescue constructs allows the purification of endogenously expressed protein complexes using a single validated antibody reagent. We used recombinant anti-GFP nanobodies to demonstrate that protein trap lines can be used to interrogate the interactome of these proteins at high stringency.

In summary, we established an experimental framework that allows important aspects of gene function to be studied, such as localization, loss of function phenotypes and biochemical interactions by using protein trap lines and highly optimized reagents for functional interference and biochemical purification.

Reference

Stringent analysis of gene function and protein-protein interactions using fluorescently tagged genes. Neumüller RA*, Wirtz-Peitz F*, Lee S, Kwon Y, Buckner M, Hoskins RA, Venken KJ, Bellen HJ, Mohr SE, Perrimon N.  Genetics. 2012 Mar;190(3):931-40. Epub 2011 Dec 14. PMID:22174071. Published online before print December 14, 2011, doi: 10.1534/genetics.111.136465
(*equal contribution)

Pubmed link