Many of the inbred mouse strains employed in behavioral neuroscience exhibit differences in phenotypes ranging from fear regulation to spatial learning to alcohol preference (Belknap et al., 1993; Owen et al., 1997; Bothe et al., 2005; Hefner et al., 2008). The patterns, although not always the degree, of some phenotypic variations are quite stable across time, across studies, and across laboratories (Wahlsten et al., 2006). The good news is that these strain differences offer a tractable and potentially very fruitful means to uncover genetic influences on behavior. The tougher news to swallow relates to many of the extremely powerful mutant mouse tools the field has become heavily invested in. This issue is that the genetic background of these mutants can be a major, and oftentimes unpredictable, determinant of not only the penetrance, but also the direction of behavioral results obtained with, for example, transgenics and targeted gene mutants (Holmes and Hariri, 2003). Those critical of the field might point to this as more evidence of behavioral studies being unpredictable and “soft.” On the contrary, in so-called “harder” sciences such as oncology (Krentz et al., 2009) and immunology (Bygrave et al., 2004), the profound influence of genetic background is accepted as being part and parcel of the gene× gene interactions (epistasis) that underlie complex phenotypes.

How to get a handle on this issue? A good starting point is the careful characterization and comparison of strain phenotypes. Fortunately, much work has been done and discussed in this regard across various behavioral domains. An issue that has not been so well broached in the literature is whether substrains within a single strain also exhibit variation. While we often use shorthand such as “B6” and “129” to describe strains, there are in fact multiple substrains of many of the more commonly used inbred strains. The “129” family is particularly diverse, with numerous substrains across four separate genetic lineages, and with known phenotypic differences (eg, Simpson et al., 1997; Bothe et al., 2004; Camp et al., 2009). Diversity within the 129 substrain can become an issue when trying to sort through the “flanking gene” problem that results when genes from a 129 embryonic stem cell donor get passed along with a targeted mutation (Gerlai, 1996). However, there are also multiple substrains of inbred strains more commonly used as genetic backgrounds for backcrossing, including the ever popular C57BL/6 (Figure 1).