molecular B(io)LOG(y)

One would assume that KO mice harbour only the mutation that was introduced by homologous recombination in ES-cells. However, this is unlikely. Depending on the genetic background (129SV, C57BL/6, Balb/c etc.) mice with the very same mutation often display phenotypes of different severity which may range from lack of phenotype to lethality. This is due to allelic differences in other loci that arose through genetic drift during inbreeding and were fixed in the course of the generation of the different inbred lines. Thus, beside the mutation introduced by the investigator a knock out line may harbour additional genes that are inactivated.

There are not too many cases in which the causes of different phenotypes in different genetic backgrounds have been elucidated. E.g., Sod2-/- mice, which are deficient in the mitochondrial form of superoxide dismutase (MnSOD), have a short survival time in C57BL/6J while survival time in normal in DBA/2J. QTL mapping showed that the differences are due to a defective nicotinamide nucleotide transhydrogenase (Nnt) in C57BL/6J mice (Huang TT et al. (2006): Genetic modifiers of the phenotype of mice deficient in mitochondrial superoxide dismutase. Hum Mol Genet 15(7):1187-1194). Thus, one has to be aware, especially when backcrossing to another genetic background, that certain substrains of inbred lines harbour additional mutations.
One such example is the C57BL/6 substrain C57BL/6S bred at Harlan which in contrast to the parental Jackson strain lacks a functional alpha-synuclein (Specht CG and Schoepfer R (2001): Deletion of the alpha-synuclein locus in a subpopulation of C57BL/6J inbred mice. BMC Neurosci 2:11).
Another example is the deletion of the Disc1 gene in all 129 sublines and ES cell lines derived from these strains. (Clapcote SJ and Roder JC (2006): Deletion polymorphism of Disc1 is common to all 129 mouse substrains: implications for gene-targeting studies of brain function. Genetics 173(4):2407-2410)

While mutations in inbred lines that modify a phenotype caused by targeted mutations can in principle be identified through backcrossing and SNP analysis other mutations are currently invisible because they reside in regions of the genome that are identical in all mouse inbred lines (aka blind spots). These regions have been recently described by Frazer and co-workers (Frazer KA et al. (2007): A sequence-based variation map of 8.27 million SNPs in inbred mouse strains. Nature 448(7157):1050-1053; subscription required)and Yang et al. (Yang H et al. (2007): On the subspecific origin of the laboratory mouse. Nat Genet 39(9):1100-1107; subscription required).
One may speculate that these regions are maintained identical because they harbour alleles advantageous for inbreeding and caged life. Recently a colleague told me that all inbred mouse strains contain about 50 knocked out genes that are believed to be necessary for inbreeding. It is unclear though, if these genes reside within the blind spots. Irrespective of the location of mutated genes identical in all inbred strains such genes are fixed through selection rather than genetic drift. Selection, though unintentional, indeed takes place during breeding. E.g., breeders may select for bigger litter size and lower aggressiveness or against poor fecundity etc.
It will be interesting to see if the regions homologous to the blind spots will develop in the same direction during the collaborative cross for the generation of recombinant inbred lines that is based on five inbred strains (C57BL/6J, A/J, NOD/LtJ, NZO, and 129S1/SvIm) and three wild derived mouse lines (WSB/Ei (M. musculus domesticus), PWK/Ph (M. musculus musculus), CAST/Ei (M. musculus castaneus)) (Chrchill GA et al. (2004):The Collaborative Cross, a community resource for the genetic analysis of complex traits. Nat Genet 36(11):1133-1137).

Another source for additional knocked out genes are the ES cells used for gene targeting because they acquire new mutations all the time. Normally, these mutations should remain heterozygous within the ES cells. However, since generation of a homozygous knock out mouse line requires brother/sister mating such mutations may be fixed in the mouse line, especially if the mutation is in close neighbourhood of the targeted allele.

While mutations in regions identical in all inbred strains can not be eliminated by breeding mutations specific for certain substrains and mutations that occurred in ES cells can be eliminated by repetitive crossing to a wildtype strain without this mutation. Thereby the frequency of the mutation in the colony is lowered and the unwanted mutation gets eradicated. Unfortunately, as I described earlier this only works if the mutation is on a different allele as the targeted mutation or if the two mutations are separated by several cM on the same chromosome.

Edit: Tags updated October, 16