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Once transgenesis became available, it was possible to insert or inactivate one or several genes to understand their functions in vivo. Today, there is an important focus on all sorts of transgenic animals because they are thought to provide models to study the fine molecular mechanisms underlying diseases. Ethically, the production of these mutants requires millions of mice, which are discarded either because they do not present the right phenotype (e.g. specific characteristic) or because they are not viable.
Actually, many of these transgenic animals do not survive long after birth because the physiological or anatomical defects are too serious. Among the normal mice, the monsters and the dead, a minute percentage of mice will be selected. Companies, such as Jackson Laboratories, have specialized in creating and distributing these genetically modified rodents to respond to the needs of investigators. According to scientists, genetic manipulation should fill the knowledge gap in spite of two decades already of creating transgenic mice.
Now, biomedical science is building huge animal facilities, some of them the state of art with robotic washers and holding thousands of cages. New facilities open to accommodate thousands of mice. Those mice may have or not some academic or medical interest but the number of possible strains is enormous and could reach up to 60 millions animals. If you consider that 200 female animals are needed to receive the hundreds of frozen embryos necessary to ensure the preservation and reliability of one particular strain, obviously there are problems of logistics coming up. It takes a lot of room, and many technicians to run this sort of activity.
Some researchers receive grants to improve their animal facility. For instance, the Institute of Neuropathology at the University of Zurich spends more than 400,000 euros (US$337,000) a year, just to maintain its mouse colony. The NIH invests millions of dollars per year to sponsor the collection and distribution of mutant mice. Since a series of frustrating results delaying and confusing further our understanding of human genetics is predictable, the fever of mouse genetics will cool down and disappear. Actually, it is already proven costly and new methodologies have already sprung up.
In tune with the hot air created by the use of these genetically modified mice, Kathleen Murray director of transgenesis services at Charles River Laboratories, the world's largest supplier of lab animals has this to say about trangenic mice: "There is not a single genetically manipulated mouse that has been used yet to produce a drug that cures a disease. It will be a combination of multiple [mice] that all have data that complete the picture."(1)
Initially, mice started to be used extensively early the last century to study genetics, and inbreed mice were selected to maintain homogenous strains whose features facilitated experimental monitoring and interpretation of results. Consanguineous males and females were generated, but also mice having no immune system or immune deficient (e.g. Nude or Severe Combined Immunodeficiency mice respectively) because of a lack of T and B-cells that belong to the immune system. They were used in cancer immunology and studies of graft transplants and helped understand the biology of mice in response to vast range of injuries (e.g. irradiation or mutations induced by chemicals).
From a conceptual point of view, these mice have helped establish principles, oriented research into different directions and allowed the development of technologies impossible to establish in humans for ethical reasons or difficult to establish in other species because of the high cost involved. But the overall utility of mice to understand the human biology, despite the agitation created during the last century, and the more recent one, still remains dubious and not convincing.
Unfortunately, it is not easy to know this fact because a great deal of publicity biased the fact thatany relative or complete success of an animal study means nothing in terms of therapy and benefits to people. Unless a trial is under way to confirm or invalidate the findings in human subjects, animal research, namely on mice, looks pretty in scientific papers and makes people talk about it. However, such research brings up principles often inaccurate and even dangerous when extrapolated to the human species.
Such is the law of nature and from the bench to the bed of the patients, there is a wide gap that research on rodents does not fill.
There is a growing body of evidence to prove it.
In the early 1980s, the use of transgenic animals started to soar and was expected to fill the gap. However this technology, far from adhering to the 'Three Rs commitment' so as to reduce and replace the use of animals, has not yielded important insights in the biology of humans, despite the huge collection of data on mice and the increasing number of experiments in the making. Between 1990 and 1994, the number of transgenic animals used in scientific procedures increased by 300% in the U.K. alone.
There are two methods to produce transgenic animals. One is transgenesis, in which a foreign gene is introduced into a fertilized mouse egg obtained from killed female mice. Injected embryos are placed in a foster female uterus and transgenic newborn mice are screened to find the gene of interest. The second method is gene targeting in which embryonic stem cells (ES cells) are genetically altered by microinjecting some extra genes into the cavity of an egg (e.g. a blastocyst composed of an aggregate of cells after 3.5 days of gestation) that is placed in the uterus of female mice. The cells divide and spread to all tissues of the embryos. Then, the offspring is crossed with wild type mice in order to obtain the homozygous mutants possessing two copies of the mutated gene.
By using these methods, it is possible to add a foreign gene, a mutated gene or to delete a host gene so that the gene is either expressed or repressed. Although the technological challenges were impressive at that time, the view that we owe to mice transgenesis is balanced by the fact that transgenesis in mice is only instrumental to understand human genetics.
Today pigs are also transgenic to serve as organ banks. As judged by the plethora of studies involving transgenic mice, scientists have barely asked the question as to whether the creation of transgenic animals is acceptable regarding the degree of harm to millions of individual, impaired, sick and moribund animals. In particular, the number of wasted animals is immensely high, up to 90% of the offspring are discarded because needless.
How to relate a targeted susceptibility gene whose alteration may produce no change in animal behavior or physiology to a function? If it produces a change, how significant and relevant is the change compared to the human situation? To answer these questions can create a great deal of confusion; a single gene can harbor various mutations, be the cornerstone of different biochemical pathways that recruit many molecules, and not necessarily the same pathways and molecules involved in humans. Transgenesis is like finding a needle in a haystack, and is a good example of a 'shoot in the dark' strategy.
Of course, the creation of transgenic animals fails to take into account the extent to which a disease is caused by environmental factors and overlooks the problems related to consanguinity and unknown genetic alterations with no visible associated phenotype. These difficulties would command to use transgenic animals to answer very specific questions when no alternative is found. Instead, it has become an automatic tool for those of the researchers in quest for the sort of professional agenda that keeps them in the race.
In the labs, scientists may delete or amplify a gene to see what it does, hoping it does something in the way that matches their hypotheses? Since mice have evolved differently over millions of years, they barely express what is observed in patients. This is not new and the literature shows it; nevertheless the present fashion is clearly favorable to any genetic manipulation for whatever the question is. Sadely, millions of mice have become the test tubes for a great deal of nothing.
Some studies have considered harmful consequences of transgenesis for farm animal welfare.(2)There is evidence that the manipulations imposed in the context of animal transgenesis are not without risks in terms of animal health and welfare. There are factors posing a risk for the welfare of transgenic animals including: 1) Integration of a transgene within an endogenous gene with possible loss of host gene function by insertional mutations, 2) inappropriate transgene expression and exposure of the host to biologically active transgene-derived proteins, and 3) in vitro reproductive technologies employed may result in an increased incidence of defects such as difficult parturition, but also fetal and neonatal losses and the development of unusually large or otherwise abnormal offspring (e.g. large offspring syndrome).
The ethical and scientific problems involved with the generation of multiple forms of transgenic animals do not slow down their use. Especially in neuroscience, different approaches have focused on the use of transgenetic animals to study the molecular and cellular mechanisms underlying neurodegeneration.(3) Since other animal models have little value or have only contributed to add scattered bits and pieces of information, manipulating one or several genes in a mouse, thought to be similar to the human gene(s), should provide a way to change the genetic make up of this mouse. Therefore, it should advance our understanding of the diseases. Again, mice differ in terms of biochemistry and physiology because Mother Nature has been working very hard over the last millions of years to ensure that humans differ from mice.
What follows provides some explanations:
Mice over-expressing G37R mutation-in which G coding for amino acid glycine is mutated to R coding for arginine at position 37 of the protein-of the copper-zinc superoxide dismutase (SOD1), an enzyme that turns toxic oxygen species into peroxides, was engineered to mimic the amyotrophic lateral sclerosis (ALS) characterized by the degeneration of the motor neurons in the brain and spinal cord. ALS affects 4 to 6 individuals per 100,000 people and most of the affected patients die within 5 years after the onset of the disease. Also, 20% of patients carried a mutation in the SOD1 gene, on chromosome 21. This discovery prompted the hypothesis that pathology was due to an excess of oxidative stress.
On the one hand, the transgenic mice developed severe and progressive motor neuron loss, and presented membrane-bound vacuoles in the axons, dendrites and cell bodies, some patholological aspects not common in the human disease. On the other hand, when the gene was suppressed the enzyme, which is important to discard toxic superoxides, no longer operates. Nevertheless, knockout mice having lost the SOD1 gene developed normally and do not show motor defects, thus suggesting a gain rather than a loss of function. Given that 80% of patients do not bear a mutation, SOD1 alteration cannot be the only cause of the motor neuron disorders and other factors are involved.
Many different mutants have been created, reproducing some traits of ALS, and tested but the mechanisms for mutant SOD1-mediated familial ALS remain unknown and the information collected thus far from animal studies have contributed little to our understanding of the human disease. The current treatments, based on glutaminergic inhibitors such as riluzole, have been tested on these transgenetic mice but no effect on the disease onset was observed and rather survival was prolonged.
Other attempts were made to look at the role of molecules called neurotrophins-including Brain Derivated Neurotrophin Factors (BDNF) and Ciliary Neurotrophic Factor (CNTF)-in the survival of neurons by blocking the expression of their genes, and the animals developed either progressive loss of motor and sensory functions respectively. Dependence on these factors can be tested in vitro and it is suggested, based on a recent clinical trial, that CNTF does not benefit humans in contrast to observations in animals.(4)
Species, cell types, sex, and genetics account for the variations of animal experimental results. Thus, mice, like other animal species cannot mimic all critical aspects of human pathologies. Other spontaneous forms of motor neuron disorders exist in dogs and horses, but they are not popular due to the cost of maintaining colonies of such deficient animals. In addition, all the species differ from humans in terms of their corticospinal tract, which carries motor information from the cortex to the spinal cord, uniquely and specifically altered in ALS. It is interesting to note that non-human primates do not develop inherited or naturally motor neuronal diseases. For all these reasons, no animal model mimic ALS and research on animals has yielded unsatisfactory results to lay the ground for clinical trials.
Huntington's disease (HD) is a neurological disorder associated with progressive chorea, rigidity and dementia that manifests itself in midlife and is characterized by selective neuronal loss. A critical gene encodes the ubiquitous protein called huntingtin containing between 8 to 35 glutamine repeats in healthy individuals. Glutamine is an amino acid, and each amino acid is the elementary unit of any protein of the body. Neither heterozygous nor homozygous HD-like knockout mice (i.e. one or two copies of the gene is inactivated) recapitulated the neuropathological features of the disease. This finding led some scientists to suggest that the disease was not due to a loss of function but rather to a gain in function although this is a speculative hypothesis. At the present time, there is no cure for the disease.
In the case of spinal cord ataxia, characterized by progressive neuronal loss within the cerebellar cortex and the brainstem, knockout mice by deletion of the SCA1 gene encoding ataxin-1 did not show any evidence of ataxia or neurodegeneration. The study of another genetically modified mice K772T (so-called because T coding for amino acid threonine is substituted for K coding for lysine at position 772 of the protein) showed that without nuclear distribution of ataxin-1, containing 82 CAG nucleotide repeats in the gene, the mice did not exhibit ataxia (there are four different nucleotides forming the molecule of DNA and symbolized by four letters A, T, G and C. Each combination of three of these nucleotides will code for one amino acid). This finding led the scientists to suggest that nuclear distribution of ataxin-1 was critical for the development of the pathogenesis. There is no cure for spinal cord ataxia.
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1. Brendan A. Maher. "Test Tubes With Tails." The Scientist, February 2, (2002)
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2. Van Reenen C.G. et al. Institute for Animal Science and Health (ID-Lelystad), Division of Animal Sciences, The Netherlands. "Transgenesis may affect farm animal welfare: a case for systematic risk assessment." Journal of Animal Science 79(7): 1763-79 (2001)
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3. Han-Xiang Deng and Teepu Siddique. "Transgenic mouse models and human neurodegenerative disease." Archives of Neurology, vol. 57 dec. (2000)
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4. Christelle Monville and Marc Peschanski. "Le CTNF, une cytokine gliale au secours des neurones" Médecine Sciences 17: 568-76 (2001)
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