The phiC31 RNAi library (KK) was created using the phiC31 integrase system to target RNAi transgenes to a specific landing site on the second chromosome. This insertion site (P{attP,y⁺,w^3'}VIE-260B) was selected based on its low level of basal expression and consistent high level of GAL4-dependent expression across a range of different tissues. The RNAi transgenes consist of inverted repeats that were designed to reduce the risk of off-target effects.

The host strain used for transgene integration actually has two landing sites: the annotated site in the 5' UTR of tio at R6:2L:22 ,019,296 (P{attP,y⁺,w^3'}VIE-260B), and a previously non-annotated site at R6:2L:9437482 (P{attP,y⁺,w^3'}VIE-260B-2). The previously non-annotated site seems to be the main integration site, occupied in all 39 lines tested. The originally annotated site was occupied in only 9 of 39 lines tested.

Of 39 VDRC KK lines tested, the 9 lines with integrations at the originally annotated landing site ( 2L:22019296 ) were all associated with a non-inflating wing phenotype using the P{GawB}elav^C155 driver, and pupal lethality using the P{Act5C-GAL4}17bFO1 driver. This is presumably due to overexpression of the nearby tio gene.

Researchers are encouraged to validate results using KK lines. PCR diagnostics can be used to identify strains with integration at the problematic landing site, and recombination can be used to 'clean' lines with problematic integrations.