PMC

Cell survival of PEF derived from wt (•) and APNG ko (■) mice after treatment with MMS, temozolomide, BCNU, or mitozolomide.

Release of 3-meA (•) and 7-meG (■) from a ³H-methylated calf thymus DNA substrate by testis extracts prepared from wt, APNG null heterozygote, and APNG ko mice.

Numbers of leukocytes in peripheral blood from wt (•) and APNG ko (■) mice following treatment with 30 mg of BCNU/kg. Data are presented as means ± standard errors for groups of 17 mice of each strain.

Induction of micronuclei in PCE of wt and APNG ko mice following treatment with increasing amounts of MMS or mitozolomide. Data are presented as means ± standard errors for groups of at least four animals per point.

Induction of hprt mutations in splenic T lymphocytes from wt and APNG ko mice after treatment with 150 mg of MMS/kg. Control groups contained four animals, and treated groups contained a minimum of eight animals per group (P < 0.009 for treated wt mice versus treated ko mice).

Persistence of 7-meG and O⁶-meG in liver sections from APNG wt and ko mice following treatment with 60 mg of MNU kg⁻¹ (a) or 6 mg of NDMA kg⁻¹ (b). (a) Panels A and B, 7-meG staining in wt liver 24 h and 7 days after MNU treatment, respectively. Panels C and D, 7-meG staining in ko liver 24 h and 7 days after MNU treatment, respectively. (b) Panels A through D, wt liver; panels E through H, ko liver. Panels A and E show 7-meG staining, and panels B and F show O⁶-meG staining, 24 h after NDMA treatment. Panels C and G show 7-meG staining, and panels D and H show O⁶-meG staining, 7 days after NDMA treatment. cv, central vein; pt, portal tract.

(A) Strategy for the disruption of the murine APNG gene. The genomic structures of the wt and mutated genes, together with the gene-targeting construct, linearized at a unique NotI site, are shown. For the targeting construct, a 3.4-kb NotI-XhoI genomic fragment was ligated upstream of a pgk-neo cassette and a 0.9-kb BamHI-EcoRI fragment was placed between the neo and pgk-HSV tk expression cassette. Upon homologous recombination a 2.5-kb fragment of the gene containing exons 1 and 2 is replaced by pgk-neo, and HSV tk is deleted. Identification of correctly targeted ES cell clones was achieved in the first instance by PCR using oligonucleotides complementary to sequences in exons 2 and 3 and to neo (open arrows), and then by Southern blotting following XbaI digestion of genomic DNA. By using a 1.4-kb XbaI-NotI fragment external to the targeted region, a 5.2-kb band indicates the presence of the wt gene, and a 6.5-kb band indicates the presence of the mutant gene. Restriction enzyme sites: B, BamHI; E, EcoRI; H, HindIII; N, NotI; Xb, XbaI; Xh, XhoI. (B) Southern blot analysis of XbaI-digested tail DNA from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mutant mice, showing fragments of the expected sizes after hybridization to the probe. (C) Multiplex RT-PCR for APNG and ATase on RNA prepared from testes of wt mice (+/+; lanes 1 and 2) and mice heterozygous (+/−; lanes 3 and 4) or homozygous (−/−; lanes 5 and 6) for the mutant APNG gene. Lanes 1, 3, and 5, 1 μl of cDNA; lanes 2, 4, and 6, 2 μl of cDNA; lane 7, no cDNA; lane 8, DNA ladder.