Ethics of Research involving animals
Genetic studies
Selective breeding
5.16 Selective breeding has been used for many decades to create more productive, or higher yielding farm animals, and for the breeding of animals with particular features and characteristics, including some companion animals and ‘show’ animals. It has also been used in medical research to investigate basic biological processes. Many mouse mutants have arisen spontaneously in colonies maintained specifically for experimental purposes. Some of these have been used as models of human disease, including diabetes, obesity and neurodegenerative diseases.9
‘Forward genetics’
5.17 Other techniques seek to deliberately change the genetic complement of animals, in order to observe the consequences of these alterations. Classical genetic experiments (also called ‘forward genetics’) are performed by inducing random mutations. The animals are treated with mutagens such as X-rays, chemicals that alter genetic information or viruses that insert DNA into the host genome. Offspring are screened for abnormal features in development, physiology or behaviour. The advantage of this approach is that when a mutated gene is found, it is likely to be important for the feature that is abnormal in the mutant. The mutant gene can then be identified, by comparing gene sequences from the mutated animal to those from normal animals. This procedure has become much more straightforward since the genomes of a number of animals have been mapped and sequenced.
5.18 Until recently, these studies were mainly carried out in fruit flies and nematode worms, organisms which are small, low cost and have rapid generation times. These are crucial features for large-scale genetic studies that involve many thousands of animals. Genetic screens in flies and worms have contributed to many important advances in our understanding of animal development. Many of the genes identified were later shown to be common to all animals, including humans, and they often function in very similar ways. The conserved functions of particular genes have been demonstrated by transferring them, for example, from humans to worms or flies, and showing that they function in the same way.
This research has revealed a remarkable degree of conservation of genetic information during evolution. More recently, large-scale genetic screens have been carried out using zebrafish and mice, primarily to discover the genes responsible for a particular developmental or physiological process. The welfare implications of such experiments are difficult to predict and, depending on the genes involved, could range from no adverse affects to severe developmental abnormalities and disability (see paragraph 5.13).
‘Reverse genetics’
5.19 Another genetic approach, called ‘reverse genetics’, is mainly applied to mice. Researchers can alter a specific gene of unknown function either by over-expression (in transgenic mice), elimination (in knock-out mice) or replacement with an altered form of the gene (in knock-in mice). The genetic change is then passed on from generation to generation in the new, genetically engineered mouse strain, in which the function of the gene under study can be analysed.
5.20 In order to over-express a gene, DNA is injected into the nucleus of a fertilised egg, which is then implanted into the uterus of a surrogate mother. A gene might also be eliminated (knocked out) or altered (knocked in) in ES cells, which are then injected into an early mouse embryo so that the cells derived from the modified ES cells develop into the tissues of the developing mouse. If cellular descendents of the ES cells form germ cells (sperm or eggs), these chimeric mice will produce offspring that have the eliminated or altered gene. Further breeding will produce some mice in which the gene has been completely eliminated or in which only the altered form of the gene is present (see Box 5.6).
5.21 A specific gene can also be altered, over-expressed or deleted in particular cell types or at specific times, providing even more precise ways of studying gene function in animals. There are between 22,000 and 25,000 genes in the mouse genome, and several hundred have already been specifically eliminated in mice. In principle, all of the remaining genes could be deleted in further studies, alone or in combination with other genes. Not all of these procedures would result in viable offspring, as the elimination of some genes would lead to the death of the developing embryo. However, more sophisticated techniques have been developed, such as producing ‘conditional knock-out’ animals, in which the gene deletion is only triggered for experimental purposes or in specific tissues.10
5.22 The welfare implications for animals used in these kinds of experiments cannot be predicted because it is not known beforehand what type of defect may be produced by the genetic modification (see paragraph 4.57). As we have said, licences require that research is stopped and animals are killed humanely if defined thresholds of pain or suffering are exceeded (paragraphs 5.13 and 12.21). Although many of the mice created have no obvious abnormality, others have severe developmental defects. For example, mice in which a growth factor receptor gene was knocked out had severe abnormalities including skeletal defects and profound deafness.11 The methods by which GM animals are produced also have the potential to be painful and distressing (paragraph 4.58). Large numbers of animals are used to produce a single GM strain due to the relatively low efficiency of the methods used to achieve genetic modification.
Usually, the majority of the animals that are produced do not have the desired genetic traits and are usually euthanised (see Box 5.6). More efficient methods would be desirable. Many strains of GM animals are expected to be established in the future. For example, it has been predicted that 300,000 new genetic lines of mice could be created over the next two decades.12
Box 5.6: Common techniques for creating transgenic animals
| Pro-nuclear injection In the 1980s the first transgenic animals were created by pro-nuclear injection, which allowed only random introduction of new DNA sequences into the genome.* DNA is injected into a fertilised egg that is then transferred to a recipient female. Only a small proportion of the injected eggs will produce a firstgeneration (‘founder’) transgenic animal containing the gene of interest. Therefore the resulting offspring need to be selectively bred in order to obtain a line of animals all with the desired traits. This method has been used in mice, rats, pigs, sheep, cattle and goats. The efficiency is low as approximately three to five percent of the animals born as a result carry the transgene.* * See Clark J and Whitelaw livestock Nat Rev Genet 4: † Roslin Institute (2002) Somatic Efficiency, available at: http://www.roslin.ac.uk/public/ 25 Apr 2005. |
Study of protein and cellular function
5.23 Genetic modification can also be used to produce mice that express a fluorescent form of a particular protein under study. This intervention allows researchers to observe the location of specific proteins in living cells and to analyse their activity. The cells expressing these fluorescent proteins can be readily visualised in tissue using a fluorescence microscope and purified using a fluorescence-activated cell sorter. Fluorescent proteins themselves are not known to cause adverse welfare effects. Mice can also be engineered to express a toxic protein in a specific cell type so that cells of this type can be eliminated by the body. This technique is used as an effective way of determining the normal function of a particular type of cell.13 Adverse effects on the animal would depend on the cell type that is eliminated.
Footnotes9 See Schuler AM and Wood PA (2002) Mouse models for disorders of mitochondrial fatty acid ß-oxidation Inst Lab Anim Res 43: 57–65.
10 For a review, see Cohen-Tannoudji M and Babinet C (1998) Beyond ‘knock-out’ mice: new perspectives for the programmed modification of the mammalian genome Mol Hum Reprod 4: 929–38.
11 Colvin JS, Bohne BA, Harding GW, McEwen DG and Ornitz DM (1996) Skeletal overgrowth and deafness in mice lacking fibroblast growth factor receptor 3 Nat Genet 12: 390–7. 12 Abbot A (2004) Geneticists prepare for deluge of mutant mice Nature 432: 541.