Homing assay.

In this assay, donor and target constructs were placed at the same φC31 insertion site on homologous chromosomes (the donor and target chromosomes marked black and blue respectively). The target construct contains a GFP open reading frame (ORF) driven by an eye-specific promoter where the GFP ORF is split with an in-frame homing endonuclease recognition site (represented by adjacent green boxes). Transgenics bearing an intact target construct therefore exhibit GFP fluorescence in the eye. The donor construct has a homing endonuclease transcription unit is inserted into the HEG recognition site disrupting the GFP ORF and abolishing GFP fluorescence in the eye (loss of fluorescence represented by the GFP ORF being filled in white). Most constructs also include an RFP marker to allow the HEG insert to be tracked. Expression of the HEG in the germline causes cleavage of its recognition site in the target construct and subsequent repair leads to a number of different outcomes that can be differentiated by fluorescence and phenotypic markers as shown in the figure. The donor and target chromosomes are distinguished either with the linked cu marker (applicable with males only because of recombination) or a very closely linked mini-white marker within the donor construct (which is applicable to both sexes). It should be noted that NHEJ repair results in loss of GFP fluorescence in approximately two-thirds of cases only. The remaining third of NHEJ lesions can only be distinguished from unmodified targets by PCR and cleavage with I-SceI.