Ethics of Research involving animals
Research tools and techniques
Production of antibodies
5.24 Antibodies are proteins that are widely used in many areas of biomedical research, as well as in clinical medicine. They are highly useful tools, as each antibody type recognises the specific ‘foreign’ molecule (antigen) against which it was produced. Antibodies of a particular type can therefore be used to identify, localise, quantify or purify an antigen. For example, antibodies might be labelled with fluorescent dyes and then used to locate specific molecules by fluorescence microscopy of tissues in vitro (i.e. in a tissue sample in the laboratory). They can also be labelled with enzymes and used to quantify specific molecules in blood or other fluids or tissues, as for example in the common pregnancy test. Antibodies are also used to purify cells or molecules by attaching them to magnetic beads. The antibodies bound to the cells or molecules of interest can then be ‘attracted’ out of solutions.
5.25 Antibodies are made by B lymphocytes, which develop in the bone marrow. In order to produce antibodies against an antigen of interest, an animal (usually a mouse, rabbit, sheep or goat) is administered with the antigen one or several times (immunised), together with a stimulant (an adjuvant), and the antibodies that are activated in response are then collected from the blood. Adverse effects depend on the dose, frequency of injections and the use of adjuvants, which can lead to irritation and the formation of an abscess. Immunisation can also occasionally lead to a severe allergic reaction (anaphylaxis), which can be fatal. If animals are used for the production of purified monoclonal antibodies (the ascites method), then serious adverse effects can occur. This procedure is rarely used in the UK, although antibodies made by this method may be imported from abroad.14
Animal cloning
5.26 The term cloning refers to the process of creating an identical copy of a gene, cell or a whole animal. Two types of cloning need to be distinguished: reproductive and therapeutic. The former is used to produce an animal that is virtually genetically identical15 to the predecessor from which it was cloned (see Figure 5.1 and Box 5.6).
5.27 The main purpose of developing reproductive cloning techniques is to facilitate the targeted genetic modification of animals.16 Research also seeks to explore their potential for novel medical applications such as providing organs for xenotransplantation (see paragraph 1.18). In addition, cloned animals could be used to rapidly increase the number of animals of a genetically identical strain and therefore might replace repeated inbreeding (paragraph 5.8). Cloned animals are being used to study age-related changes in cells, including cancers, and some people are hopeful that the approach could help to conserve endangered species. A range of other purposes are possible in principle, such as the breeding of champion racehorses, the replacement of deceased pets or ‘pharming’ (see paragraph 5.31).
Figure 5.1
| Figure 5.1 Reproductive cloning of a sheep using nuclear transfer* Dolly the sheep was produced using nuclear transfer. If the embryo is used to make ES cells for research rather than a new individual, the procedure is called ‘therapeutic’ cloning (see paragraph 5.30). |
5.28 The first animals to be cloned from the nuclei of adult somatic cells were amphibians. This significant work using tadpoles in the 1970s showed that somatic cells (and not only reproductive cells) contained all the information required to develop into the organism.17 In 1996, Dolly the sheep was the first mammal to be cloned from a cell from an adult animal, and the event attracted worldwide media attention (see Figure 5.1). Other animals that have now been cloned include the mouse, rat, cow, goat, pig, cat, rabbit, mule and horse.18 Certain cloned animals have also been ‘re-cloned’ to produce a second generation of clones.19 Scientists are using these animals to study the longer-term effects of cloning, in order to assess any possible developmental abnormalities and welfare implications.
5.29 Reproductive cloning of animals raises a number of concerns. The method is currently highly inefficient, requiring repeated attempts to remove eggs and implant embryos to obtain even a single viable clone. The cloning of Dolly the sheep, for example, required the production of 277 fused embryos. Of this number, 29 cloned embryos were transferred into surrogate ewes, from which one pregnancy resulted.20 More recently, 358 eggs fused with skin cells from a cloned animal yielded two re-cloned bulls, one of which died shortly after birth. Attempts to create a third generation of clones failed after 248 embryos were fused, six of which resulted in pregnancies, but all failed to develop into viable calves.21 Cloning also has implications for animal health. Large offspring syndrome, in which the animals are too large for normal birth, occurs frequently, and cloned animals may also show signs of early aging. Dolly the sheep was euthanised in March 2003, six years after her birth, after suffering progressive lung disease and arthritis. These conditions are not uncommon in sheep of this age, and it is uncertain whether cloning was a factor.
5.30 The term therapeutic cloning is used to refer to the technique of producing ES cells that are genetically identical to the donor of the nucleus. ES cells, isolated from developing embryos, have the unique potential of being able to develop into different types of cells and to reproduce indefinitely. Therapeutic cloning could improve the prospects for the development of cell replacement therapy in humans. Genetically foreign cells (from another person) would be rejected unless the immune system was suppressed with powerful pharmaceuticals that may need to be taken for many years. However, if ES cells were produced from a cloned embryo made with the nucleus from one of the patient’s own cells, they will be almost genetically identical. Cells and tissues made from these ES cells would not be rejected if transplanted into this patient (see paragraph 5.8). Advocates of this technique, currently being used in research with animals, hope that it could be used to treat patients suffering from conditions such as Alzheimer’s disease and Parkinson’s disease (see paragraph 5.10). Some preliminary work with cloned human embryos in the first few days of development has recently been licensed in the UK.22
‘Pharming’
5.31 The term ‘pharming’ refers to the production of pharmaceuticals in plants or animals. Although, strictly speaking, pharming does not fall within the category of basic research, given its potential applications we consider it here as research in the area is still in its infancy. In plants, pharming generally involves the genetic modification of a crop plant in order to produce substances which can be extracted and processed into refined compounds. In animals, a potential pharming technique involves the transfer of human genes that encode specific therapeutic proteins. If the method is successful, the proteins which would be produced in milk, eggs or blood could be isolated for further processing. Sheep, goats and cows are used the most frequently in research on pharming as they produce relatively large quantities of milk. The production of these therapeutic proteins by other means can be technically difficult, expensive and time-consuming.
5.32 Clinical trials to test pharmed medicines have been initiated. The company PPL Therapeutics produced alpha-1 anti-trypsin (AAT), a treatment for emphysema and cystic fibrosis which was used in trials at hospitals in Europe, Canada, Australia and New Zealand. It was initially hoped that genetically engineered AAT would be on the market by 2007 but the project ceased in 2003. The European Medicines Agency (EMEA) is currently reviewing a Market Authorization Application for the pharmed pharmaceutical ATryn (human anti-thrombin).23 It was developed to treat patients with hereditary anti-thrombin deficiency, a condition resulting in vulnerability to deep-vein thrombosis. The human gene for the required protein was inserted into an egg cell from a goat and activated only in udder cells so that it was possible to extract it from the goat’s milk (see Box 5.6).24 A number of other companies are also developing transgenic animal proteins.25
5.33 With regard to implications for animal welfare, there is some uncertainty as to whether the GM process may cause unexpected side effects. Genes may not always be expressed in the intended tissues or at appropriate levels, since insertion of microinjected DNA into the genome can be random (see Box 5.6). Advances in the process are aiming to overcome this problem, for example by designing the inserted DNA to ensure that it is only expressed in the intended tissue (a technique used in the production of ATryn). There are also concerns that the pharmed proteins might cause a toxic reaction.
Footnotes13 The specific use of GM animals as disease models is discussed separately (see Chapter 7).
14 No procedures were performed during 2003 in the UK using the ascites model. See Home Office (2004) Statistics of Scientific
Procedures on Living Animals Great Britain 2003 (London: HMSO).
15 A very small fraction of DNA (16,500 base pairs out of a total of 3,000 million base pairs in the human genome) is external to the nucleus, and therefore comes from the donor egg rather than the donor nucleus. 16 See Clark J and Whitelaw B (2003) A future for transgenic livestock Nat Rev Genet 4: 825–33.
17 Gurdon JB, Laskey RA and Reeves OR (1975) The developmental capacity of nuclei transplanted from keratinized skin cells of
adult frogs J Embryol Exp Morphol 34: 93–112.
18 See UN Educational, Scientific and Cultural Organization (2004) Human Cloning (France: UNESCO).
19 Kubota C, Tian XC and Yang X (2004) Serial bull cloning by somatic cell nuclear transfer Nat Biotechnol 22: 693–4.
20 Wilmut I, Schnieke AE, McWhir J, Kind AJ and Campbell KHS (1997) Viable offspring derived from fetal and adult
mammalian cells Nature 385: 810–3.
21 Kubota C, Tian XC and Yang X (2004) Serial bull cloning by somatic cell nuclear transfer Nat Biotechnol 22: 693–4.
22 Human Fertilisation and Embryology Authority (HFEA) (2004) Press release HFEA grants the first therapeutic cloning licence
for research, available at: http://www.hfea.gov.uk/PressOffice/Archive/1092233888. Accessed on: 24 Apr 2005; HFEA (2005)
Press release: HFEA grants embryonic stem cell research licence to study motor neuron disease, available at:
http://www.hfea.gov.uk/PressOffice/Archive/1107861560. Accessed on: 24 Apr 2005.