Cautionary insights on knockout mouse studies: the gene or not the gene?

Abstract

Gene modification technologies play a vital role in the study of biological systems and pathways. Although there is widespread and beneficial use of genetic mouse models, potential shortcomings of gene targeting technology exist, and are not always taken into consideration. Oversights associated with the technology can lead to misinterpretation of results; for example, ablation of a gene of interest can appear to cause an observed phenotype when, in fact, residual embryonic stem cell-derived genetic material in the genetic background or in the area immediately surrounding the ablated gene is actually responsible. The purpose of this review is to remind researchers, regardless of scientific discipline, that the background genetics of a knockout strain can have a profound influence on any observed phenotype. It is important that this issue be appropriately addressed during data collection and interpretation.

Figure 1

Schematic of approaches that address potential genetic confounds associated with knockout gene characterization. The basic method for developing a knockout mouse is illustrated in the left hand side of the figure. 129-derived ES cells are typically used in the generation of gene-targeted mice. The targeting vector containing the mutant gene of interest is electroporated into the ES cells, where the mutant gene integrates into the genome by homologous recombination. ES cells carrying the mutation are then injected into blastocysts, which are in turn injected into pseudopregnant females. The resulting chimeric mice undergo selective breeding. As there are potential problems with determining whether an observed phenotype is due to the ablated gene or other 129 genes (see text for details) a number of approaches to combat this problem have been developed. Six approaches are represented here. First, the null mutation can be maintained on the same 129 genetic background as the ES cells, thus producing a coisogenic strain (Approach #1). Second, a knockout/congenic strain can be generated by backcrossing the knockout mouse to B6 for more than 10 generations (Approach #2). This process maintains the null mutation on a primarily B6 background, but multiple regions of 129 ES cell-derived genetic material remain. The role of the ablated gene in the observed phenotype(s) may be clarified by employing SNP-based genotyping throughout the genome (Approach #3), quantitative trait loci mapping (Approach #4), or phenotypic rescue (Approach #6). Finally, the use of B6 rather than 129 ES cells completely eliminates the 129-background gene issue (Approach #5).