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What does an insertion at 40D mean?

PCR genotyping has shown that some KK lines have an insertion of the RNAi hairpin construct at the 40D landing site, located in the 5’ UTR of the Tiptop (tio) gene. Lines with an insertion at 40D may cause non-specific phenotypes when crossed to some Gal4 drivers, which are due to the insertion site and not due to expression of the RNAi hairpin (see Green et al, 2014 and Vissers et al, 2016).

KK lines with an insertion at 30B (as well as all GD and shRNA lines) are NOT problematic. Lines with an insertion at the 40D site will ONLY be a problem in specific cases where an assay is affected by misexpression of the tio gene.

To test for non-specific phenotypes, we suggest crossing your Gal4 driver of interest to the 40D-UAS control line (VDRC_ID 60101) and checking whether the F1 progeny demonstrate a dominant phenotype. The 40D-UAS control line contains Gal4-responsive UAS repeats but no functional RNAi coding sequence at 40D and no transgene insertion at 30B and enables you to easily test, in a single cross, whether your genetic screen of interest will be affected by ectopic tio expression.

If your assay is not affected by tio misexpression, you can use all ~ 10,000 KK lines with confidence, but if you see an effect, then KK lines with an insertion at 40D (~ 30% of all KK lines) will give misleading results in that particular screen.

For more detail see here under “KK library - landing site information”.