Mouse models feature: How to make a mutant mouse

By Graeme O'Neill
Friday, 11 October, 2002

There are two basic types of rodent model for investigating inherited genetic disorders or health problems in humans -- the 'knockout' and the 'knock-in' mouse.

In the former, the gene of interest is completely inactivated, so its normal function in the animal, or in specific tissues or organs, can be inferred from the differences between the 'knockout' strain and normal mice of the same strain. In knock-in mouse strains, the gene of interest is merely edited to introduce a specific mutation known to cause the inherited disorder in humans. Alternatively geneticists may completely replace a normal mouse gene with a mutant form of the corresponding human gene.

Certain mutations in vital developmental genes are lethal because they have global effects on the developing embryo. Geneticists can circumvent the problem by making 'conditional lethal' mutant strains, in which the mutation is effectively postponed until the mouse matures -- and then only affects specific target tissues such as the immune system, heart, liver or brain.

A DNA construct is created, consisting of the mutation and its flanking DNA sequences, linked to a gene that confers resistance to the antibiotic neomycin. Microinjected into mouse embryonic stem (ES) cells, the construct seeks out and 'overwrites' the corresponding segment of the normal gene, installing the mutation -- along with the neomycin resistance gene. Adding neomycin to the nutrient medium kills off any ES cells in which the construct has failed to install; only the successfully transformed cells survive.

The mutant ES cells are then mixed with the normal ES cells of a blastocyst -- the small cluster of rapidly dividing cells that will become a mouse embryo. With luck, some of the mutant cells will become germline cells in the new embryo -- the specialised reproductive cells that give rise to eggs or sperm.

Melbourne company IngenKO has created a chimaeric mouse. The transformed ES cells come from a mouse strain with agouti fur, which is a different colour to the mouse strain that provides the blastocyst. The fur of chimaeric embryo that develops from the mixed cells has a tortoiseshell pattern; if the reproductive region features agouti fur, the individual's sperm or eggs will carry the mutation.

Because the normal gene masks the effect of the mutation, male and female chimaeras must be mated to create offspring with the 'double whammy' mutation -- these mice will display the phenotypic effects of the mutation. These first few mutant offspring must then be interbred to establish a colony of the knockout or knock-in mice for research -- it takes a minimum of three generations, and nine months to reach this take-off phase.

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