Fig. 4

(a) Stability of genomic insertion in A. deanei and (b) knock-out of the amastin genes does not affect growth of A. deanei. (a) Ten clones of the Δ-δ-Ama^Neo SKO were grown in the absence of G-418 for a period of two months. The gDNA was then extracted and the presence of neo tested by PCR. A single band at 3.45 kb corresponding to the δ-amastin gene can be seen in the wild-type cells (WT), while an additional band at 3.65 kb corresponding to the insertion of neo appears in all 10 Δ-δ-Ama^Neo clones examined (lanes 1-10). (b) Cells from mid-log phase cultures of the Δ-δ-ama^Neo and Δ-γ-ama^Hyg SKOs and the Δ-γ-ama^Hyg/Δ-γ-ama^Neo DKO cell lines were diluted to 1 × 10⁴ cells/ml in fresh growth medium supplemented with 500 μg/ml of the corresponding drug(s) and cells were counted every day. Values represent an average and standard deviation of three independent experiments