Mice are the most widely-studied mammalian model organism, but not all lab mice are alike. Most varieties, or strains, of mice used in science originate from mice kept as pets by enthusiasts at the end of the 19th century. Scientists in many fields such as immunology prefer to work using a strain called C57BL/6 (known as “Black 6”) – this was also the strain chosen for the mouse reference genome. However, making knockout mice (mice in which a specific gene is disrupted) has historically been done using mice from strain 129. This is because this strain is the easiest to isolate embryonic stem (ES) cells from, the starting point for making a knockout.
This leads to an inconvenient situation: mutating a gene is easiest in 129, but it is better to study it using Black 6. So a lot of time is spent breeding 129 mice with Black 6 for several generations, until a mostly Black 6 mouse (in terms of its genes) carrying the mutation is obtained to study. The opportunity to change this arrived with the International Knockout Mouse Consortium, which aims to make ES cells with a mutation in each of the 20,000 mouse genes. For such a large scale project, we were keen to do this directly in Black 6 ES cells, to reduce the amount of breeding required.
An extensive search at the start of the project found a good, robust Black 6 ES cell line from colleagues at the University of California, which we tested and found to be as good as the 129 lines. Being able to make knockout mice directly in Black 6 saves at least five generations of breeding, but there was one more problem. When breeding mice from 129 ES cells with Black 6, the different coat colours can be used to check that the genetically modified ES cell DNA is being passed to the next generation, since the brown agouti colour of 129 is dominant to black. However, with the new cells, the coat colours are the same, so a separate cross with an albino mouse would need to be done to check that the ES cell genes are being inherited.
We decided to alter the genes in the new ES cells so they coded for an agouti coat not black. The new cells have all the advantages of 129 ES cells, including the coat colour, but with a Black 6 genetic background – so no extensive breeding is necessary. This will allow mice made from the cells to be generated and analysed using as few animals as possible – saving hundreds of thousands of mice over the course of the project. We were very pleased to receive a “Highly Commended” award from the NC3Rs for this work.
Dr Stephen Pettitt, Institute of Cancer Research