Cre-mediated conversion to Ate1-null genotype in different mouse tissues.

(A) PCR-based genotyping of tail DNA to detect the Cre-mediated Ate1floxAte1 conversion of the functionally active flox-on (Ate1flox) allele to the null Ate1 allele in a 27-day old Ate1flox/−;CaggCreER mouse immediately after the fourth (daily) intraperitoneal (IP) injection of tamoxifen (TM+), or in the absence of TM treatment (TM-). Upper panel: the 512 bp DNA fragment characteristic of the flox-on (Ate1flox) allele and the 472 bp DNA fragment characteristic of either wild-type or the previously constructed [10] unconditionally null Ate1 allele, using primers CB156 and CB157 (Table 4). Lower panel: the 470 bp DNA fragment characteristic of the Cre-produced flox-off (Ate1) allele, with primers CB110 and CB157 (Table 4); and the 324 bp DNA fragment (control), amplified from the IL-2 gene using primers IMR42 and IMR43, in the same PCR reaction. (B) The Cre-mediated Ate1floxAte1 conversion, detected by PCR (as described in panel A) in genomic DNA isolated from the indicated tissues immediately after the fourth (daily) IP injection of tamoxifen in a 24-day old Ate1flox/−;CaggCreER mouse. (C) Relative in vitro arginylation activity (cpm/reaction) in extracts of the indicated tissues from a wild type mouse (Ate1+/+) (black bar), a heterozygous mouse (Ate1+/−) (blue bar), and an Ate1−/− mouse (the latter mouse was initially Ate1flox/−;CaggCreER) (red bar) from the same litter 76 days after TM treatment. A white bar on the right indicates the relative arginylation activity obtained with purified recombinant mouse Ate1 (denoted as “rAte1”) that had been expressed in S. cerevisiae. Shown here are “cpm/reaction” after subtracting “cpm/reaction” in the null-control (“buffer alone”) sample. The control incorporation was approximately equal to that observed in extracts from spleen and thymus. In other words, the assay configured as described in this panel and in Materials and Methods was not sensitive enough to robustly detect the arginylation activity in extracts from spleen and thymus. (D) Relative in vitro arginylation activity (cpm/reaction) in the whole brain, cerebellum, and hippocampus harvested from wild type mice (Ate1+/+; n = 3), heterozygous mice (Ate1+/−; n = 3), and Ate1−/− mice (specifically, Ate1flox/−;CaggCreER mice; n = 3) mice 40 days after TM treatment. Standard deviations are indicated. (E) Relative in vitro arginylation activity (cpm/reaction) in testis extracts from Ate1+/+ mice (n = 3) and Ate1−/− mice (specifically, Ate1flox/−;CaggCreER mice; n = 3) ∼130 days after TM treatment. Standard deviations are indicated.

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