Gene targeting is frequently used in the investigation of molecular mechanisms of brain function and behavior. However, one pitfall of the technology is hybrid genetic background. Most null mutant mice generated using conventional gene targeting methodology originate from embryonic stem cells derived from a mouse strain called ‘129’ or one of its substrains. These mice, however, suffer from numerous abnormalities, and thus the mutation is crossed to a better host strain, such as C57BL/6. As a result, null mutants are of hybrid origin, and although they are good hosts for the null mutation, their phenotypical characteristics are influenced by the contribution of the alleles of their parents.Particularly important is the fact that the locus of the null mutation is flanked by long stretches of 129-type DNA. Unfortunately, this DNA and its potential interaction with the effect of the null mutation make it difficult to determine whether phenotypical changes observed in the mutants are due to the presence of the null mutation, the presence of the 129-type alleles, or to a combination of the two. Now, Dockstader and van der Kooy warn [1xMouse strain differences in opiate reward learning are explained by differences in anxiety, not reward or learning. Dockstader, C.L. and van der Kooy, D. J. Neurosci. 2001; 21: 907–9081See all References][1] that contribution of 129 alleles might make the null mutant mouse more anxious, a trait that can interfere with performance in a large number of behavioral paradigms, complicating analysis of the behavioral effects of the null mutation.The authors examined the rewarding effects of psychoactive drugs in the 129/SvJ and C57BL/6 mouse strains using a two-chamber, place preference paradigm. C57BL/6 mice preferred the ‘rewarded’ chamber, in which they previously received morphine injection, to the non-rewarded one when tested after completion of four conditioning trials. Furthermore, they made their choice irrespective of whether they received morphine or saline on the test day. Interestingly, however, 129/SvJ mice trained with morphine reward showed preference for the rewarded chamber only when they were under the influence of morphine during testing, and not when they received saline. Further experiments revealed that the inability of 129/SvJ mice to exhibit morphine-rewarded place preference without receiving morphine before testing could be fully reversed by pre-test injection of diazepam and pentobarbital, but not by other drugs, such as naloxone or cocaine.Analysis of locomotion demonstrated that the above pattern of results was not due to motor alterations. Furthermore, it is known in the literature that the stimulus effects of diazepam and pentobarbital do not generalize to morphine. Thus, the authors concluded that all three of these were effective because of their anxiolytic effects. In summary, the authors not only demonstrated a robust genotype–drug interaction but also revealed a crucial problem that must be born in mind: mice of 129 strain origin might suffer from elevated anxiety, a behavioral trait that could represent a serious confounding factor in gene targeting experiments.