Figure 1.

Copy number selection for recovery of homozygous mutants. (A) Mechanism of LOH in Blm-deficient cells. Blue and red chromosomes represent homologs. Crossing over between homologous chromosomes is normally suppressed in mitosis (top). In the absence of Blm, crossovers can occur in G2 phase, forming recombinant chromatids. If these segregate to different daughter cells, LOH occurs distal to the point of crossover. If the cells carry a heterozygous mutation (represented by the star), this becomes homozygous after LOH, and the copy number of the mutation increases from one in the starting cell to two in the homozygous daughter. (B and C) Design of vectors for copy number-based selection. The vectors DHSV (B) and TNN (C) are shown integrated into an intron, with grey boxes representing exons of the disrupted gene; note that TNN can trap genes transcribed in either direction. SA, splice acceptor; PB5 and PB3, PB repeats.