Direct separation of in vivo and in vitro am3 revertants in transgenic mice carrying the phiX174 am3, cs70 vector

Target genes in most transgenic systems have higher spontaneous mutation fr equencies than do endogenous mammalian genes. Spontaneous mutations in tran sgenes predominantly arise from three sources. (1) mutations fixed in the a nimals, (2) mutations arising from replication errors caused by damage to t he DNA that may have occurred in vivo or in vitro and then was fixed during amplification of the vector in vitro, and (3) mutations arising during rep lication of non-revertant phages in non-permissive bacteria. An assay based on single bursts was developed to directly distinguish between the in vivo and in vitro origins of revertants. The sire of the aliquot is determined by mutant frequency and is adjusted so that ideally no more than 10 to 20% of the aliquots contain a bacterial cell transformed with a mutant phage. M utations are detected as revertants of an amber mutation (am3) in phiX174 a m3, cs70. The minimum burst size of non-revertant phiX174 am3, cs70 From sp lenic DNA on a permissive bacterial strain was larger than 30 plaque-formin g units (pfu). Based on this observation, a burst size of 31 plaque-forming revertants was chosen as the minimum burst size of a fixed mutation. The s ingle burst assay was tested on DNA from spleens of animals that were treat ed with 150 mg/kg 1-ethyl-1-nitrosurea. Only the fraction of aliquots with single bursts of revertants (> 30) increased in the treated animals compare d to the controls. In contrast, there was no difference between treated and control animals For revertant frequencies calculated for burst sizes less than or equal to 30 pfu. Among the spontaneous mutations, only 30% were cau sed by mutations fixed in animals (i.e. burst size > 30 pfu). Total average revertant Frequency measured in DNA from treated animals was less than two fold more than the average spontaneous frequency (P = 0.048). When frequenc ies were based on burst sizes > 30, there was a 4.6-fold increase among tre ated animals compared with controls (P = 0.026). The single burst-assay res ulted in a more sensitive test for mutagenicity because it eliminated noise from invitro mutations. Published 2001 Wiley-Liss, Inc.(+)