Figure 1.

Pattern of cleavage and repair generated with Adeno-HO nuclease. A) Diagram of the integrated HO target cassette, including a representation of the HO cleavage site, position of the primers used, and putative positioning of nucleosomes and secondary cleavage sites. B) Kinetics of cleavage and repair (hours post-infection [PI] with adeno-HO) in unselected and puromycin reslected cells. For these experiments, 10,000 cells were infected at an MOI of 200 in 96-well plates, and the DNA was isolated at the indicated times with Wizard genomic DNA isolation kit (Pormega). The product of the original target (amplicon T7ST/Puro 1) is indicated for cells at the start of infection as "puro". Other intermediate PCR bands are indicated by lines. FABP is a PCR product from a single-copy gene on mouse chromosome 6. Wortmannin (WMN; 30 μM) was added to the cells in the bottom panel throughout the time course post-infection. The products from 4h time point of the bottom panel were separated on a new gel to the right, using more material and a longer gel run. The two PCR reactions (Puro and FABP) were run in parallel with the same thermocycle settings and the products were mixed before loading each lane. C) RT-PCR for semi-quantitative analysis of the expression of puro mRNA. First-strand cDNA was produced with oligo-dT from 5 μg of total RNA from unselected and reselected cells. Puro mRNA and Yes mRNA were amplified in parallel for estimation of their relative expression.