Figure 4.

Crosses to measure DSB repair pathway usage. Rr3 and the I-SceI endonuclease are shown on chromosomes 2 and 3, respectively, as an example, but other versions of these crosses can be used equivalently. Mosaicism for DsRed expression is indicated in the diagram for two of the genotypes. In practice, however, these mosaics are usually indistinguishable from full-bodied DsRed flies. Thus, mosaics and full-bodied DsRed offspring are classified together as “red” in our scoring. Dominant markers, not indicated in the diagram, enable classification of G1 offspring as to whether they received Rr3 and/or endonuclease. In some versions of these crosses, the endonuclease transgene resides on the X chromosome, allowing us to use the sex of the offspring for this purpose. The female G0 parents, not drawn, are endonuclease-free for the example shown. However, for situations where the I-SceI endonuclease is not transmitted from the G0 male or when it is not expressed somatically, it is necessary to use endonuclease-bearing G0 females for a portion of the crosses to score for NHEJ. (A) Cross 1 is used to measure SSA and NHEJ under conditions in which no template for conversion is available on the homolog. No PCR tests of the G1 offspring are needed. (B) Cross 2 measures conversion in addition to SSA and NHEJ. It is necessary to perform PCR tests of nonred (endonuclease-bearing) G1 offspring to distinguish between NHEJ and conversion. These PCR tests utilize a primer specific for Rr3EJ1. The loci designated “a” and “b” are arbitrary visible markers used to score for recombination. They can be located anywhere to the left and right (respectively) of Rr3. The G0 females carry the needed alleles to allow scoring of recombinants.