Obviously, it will
not only be the targeted gene that is investigated, but the entire linked fragment, containing thousands of polymorphic nucleotides affecting protein structure and expression. The optimal solution is, of course, to use a mouse that is genetically identical to the used ES cell. There are now ES cells available from different strains, derived from substrains of 129, Balb/c, DBA/1 or C57Bl6/N, although the most commonly used strain is still 129. Remarkably, it has not been possible to make ES cells from the most commonly used standard strain, i.e. C57Bl6/J, instead the existing ES cells said to be from B6 are contaminated with other strains. For example, the commonly used Bruce ES cell selleck 9, believed to be derived from B6, differs from C57Bl6/J by 6.4% of 10 000 investigated single nucleotide polymorphisms (SNPs) (Holmdahl et al., unpublished data). Recently, ES cells from the C57Bl6/N background 10 have been established but it must be remembered that the C57Bl6/N mouse differs significantly both genetically Ulixertinib purchase and phenotypically from, for example, the C57Bl6/J strain
10, possibly due to contaminating genes from the Swiss mouse. In most cases, however, it is not possible to use mice with ES cell identity. Such experiments will not be conclusive but are nonetheless valuable if supporting functional evidence is provided or if the phenotype is qualitative rather than quantitative; however, it is reasonable to expect that in such cases the borders of
the linked fragment are reported to provide the reader sufficient information to judge the results. Genotyping the fragment is standard technology today, and it is possible to have this done as a service. However, there are additional pitfalls. A major problem in many publications concerns the genetic background of the proband mice compared with that of control mice, a problem that is occasionally exposed by way of a debated controversy 11, 12. Backcrossing a targeted gene to the control mouse background even with ten generations of backcrossing, which seem to be the informal standard of today, does not necessarily clean up the genetic background. Small fragments may still remain due, for example, to selection of breeding performance or just by chance. 2-hydroxyphytanoyl-CoA lyase We have screened more than twenty 10n backcrossed strains with a specifically designed 10k SNP chip 13 and found that almost half of these strains still contained detectable fragments originating from the donor. Even more disturbing is that the control strain used in many published papers is not in fact identical to that used for the backcrossing. In these cases, the control strain is selected from a parental colony in the same animal house or, worse, from another animal house or from a commercial supplier; the selected strain may only share the genealogic name of the strain.