Knockout Mouse - Limitations

Limitations

The National Institutes of Health discusses some important limitations of this technique.

While knockout mouse technology represents a valuable research tool, some important limitations exist. About 15 percent of gene knockouts are developmentally lethal, which means that the genetically altered embryos cannot grow into adult mice. This problem is often overcome through the use of conditional mutations. The lack of adult mice limits studies to embryonic development and often makes it more difficult to determine a gene's function in relation to human health. In some instances, the gene may serve a different function in adults than in developing embryos.

Knocking out a gene also may fail to produce an observable change in a mouse or may even produce different characteristics from those observed in humans in which the same gene is inactivated. For example, mutations in the p53 gene are associated with more than half of human cancers and often lead to tumours in a particular set of tissues. However, when the p53 gene is knocked out in mice, the animals develop tumours in a different array of tissues.

There is variability in the whole procedure depending largely on the strain from which the stem cells have been derived. Generally cells derived from strain 129 are used. This specific strain is not suitable for many experiments (e.g., behavioural), so it is very common to backcross the offspring to other strains. Some genomic loci have been proven very difficult to knock out. Reasons might be the presence of repetitive sequences, extensive DNA methylation, or heterochromatin. The confounding presence of neighbouring 129 genes on the knockout segment of genetic material has been dubbed the "flanking-gene effect". Methods and guidelines to deal with this problem have been proposed.

Another limitation is that conventional (i.e. non-conditional) knockout mice develop in the absence of the gene being investigated. At times, loss of activity during development may mask the role of the gene in the adult state, especially if the gene is involved in numerous processes spanning development. Conditional/inducible mutation approaches are then required that first allow the mouse to develop and mature normally prior to ablation of the gene of interest.

Another serious limitation is a lack of evolutive adaptations in knockout model that might occur in wild type animals after they naturally mutate. For instance, erythrocyte-specific coexpression of GLUT1 with stomatin constitutes a compensatory mechanism in mammals that are unable to synthesize vitamin C.