1 Introduction
Animals have been used not only to study the basic principles of life, but also to better understand the anatomy, and physiology of living organisms. Moreover, animals play a central role in education, pharmacology and research. The ability to conduct experiments in controlled situations, and to mimic the biological conditions of human disease in animals led to the development of scientific methods and originated in the concept of animal models of human diseases. The model organism is a species, different from humans, which we intensively investigate to gain knowledge about specific biological processes and to understand wider, general laws that rule over organisms
2 Why Does a Particular Animal Become a Research Model?
The main reason is availability and the ease of maintenance. For example, mice, chickens, frogs and fruit flies (Drosophila melanogaster) (Figure 14.1) are widely used just because they are easily accessible and simply cheap. The anecdote regarding one of the frog species, called the African clawed frog (Xenopus), is a nice example showing why a particular animal becomes a research model. Once upon a time, scientists discovered that the urine of pregnant women, containing hormones, can induce the development of Xenopus ovaries. Therefore, between the 1940s and 1960s thousands of frogs started to act as living pregnancy tests all around the world. The Xenopus, abundant in laboratories and easy to maintain, can be utilized in other kinds of experiments, trials and research, and as a consequence became a useful animal model. In general, the amphibians turned out to be great models in a variety of research and took a profound place in scientific areas such physiology of musculoskeletal, cardiovascular, renal, respiratory, reproductive, and sensory systems (Burggren & Warburton, 2007; Kiełbwna & Migocka-Patrzałek, 2017).
To be used as a model, the species must meet certain criteria that enable the feasibility of experimental procedures. From the researcher’s point of view, the high rate of reproductivity and relatively short life cycle (the time between being born and the ability to reproduce) is a very valuable feature. In the case of mice, having an accelerated lifespan, we can assume that one mouse year equals about 30 human years. Therefore, their whole life cycle could be examined within only two or three years. It is beneficial, especially in genetic research, in which crossing individuals to obtain several, subsequent generations in a relatively short time, is crucial. The financial feasibility and previous history of research with the use of a particular species are also useful. Such experimental data allows for research outcome comparison with the other
Fruit fly (Drosophila melanogaster). Fruit flies are one of the most popular model organisms. With the low cost of husbandry, short generation time, and variety of molecular tools for genetic engineering, flies become a useful model in biomedical experiments
Choosing an animal model for a particular purpose is a complex decision, involving scientific and practical considerations. Many species are used in biomedical research, such as yeasts, insects, nematodes (Caenorhabditis elegans), fish (Danio rerio – zebrafish), frogs and many mammals, such as mice, rats, dogs, cats, pigs and even monkeys. Several other issues should be also taken into account during experiment planning, such as the availability of adequate techniques and methods, including equipment, and molecular tools (such as specific antibodies, etc.) (Andersen & Winter, 2019; Dubińska-Magiera et al., 2016; Ericsson et al., 2013; Robinson et al., 2019).
3 Animal Models of Disease Can Be Divided into Two Categories, Spontaneous and Induced
The use of animals as a model of human diseases had increased dramatically in the twentieth century. The beginnings of detailed biological analysis of particular disorders were possible because animals sometimes suffer from the same
On the other hand, nowadays, we have learned how to induce particular pathological conditions in animals to create disease models. Such induction could be a combination of physical and chemical factors, or targeting genetic manipulation. The induced models are based on healthy animals, in which the pathological disorder to be examined is experimentally induced. Examples include partial hepatectomy to study liver regeneration, or studies of cow’s milk allergy in mice by immunizing them with tiny doses of cow milk protein (reviewed in: Hau, 2008). Animals can also carry out a mutation in certain, important genes and suffer from genetic diseases, similar to humans. A genetic disease is a disorder caused by an abnormality in an individual’s genome. The genetic disease may be caused by a mutation in a single gene but can be also caused by different factors such as a combination of environmental factors and mutations in multiple genes. The genetic disease could come also from chromosome alterations or mutations in the non-chromosomal DNA of mitochondria (Dubińska-Magiera et al., 2016; Plantié et al., 2015). Mutants are extremely rare in nature, therefore, the acceleration of research conducted on animal models of human diseases took place when mutagenesis techniques became available. Effective mutagenesis tools were needed to make genetic approaches functional and practical. The fact that X-rays could induce mutations in mice was known from 1923. Then in 1980 extensive, chemical mutagenesis studies were continued using the alkylating chemical called ENU (N-ethyl-N-nitrosourea). Next, in 1990, the human genome sequencing project began, aiming for the identification and mapping of all of the genes in the human genome. The project progress was unexpectedly fast and created an urgent need for knowledge regarding particular gene’s functions. Therefore, approximately in the same period, the idea of a large-scale mouse mutagenesis
The key moments in mice mutagenesis. The aim of mutagenesis is to establish an animal model of human genetic diseases. The progress in genetic engineering starts from the discovery that X-rays can induce mutagenesis. Then the chemical compound ENU (N-ethyl-N-nitrosourea) was used to create mutations. With the technique development, many large-scale programs started in 1997, with the culmination point in 2007, when the International Knockout Mouse Consortium (IKMC) was established, with the aim to provide knockout and conditional mouse strains for all mouse genes. Such acceleration was possible because the whole mouse genome was sequenced in 2022. Starting from 2010 new techniques, allowing for precise mutagenesis were discovered. In the beginning, the ZNF s and TALENS were developed. Then, the CRISPR-Cas9 technique was shown to be a real scientific breakthrough, appreciated by the Nobel Prize awarded in 2020 to Emmanuelle Charpentier and Jennifer Doudna (according to: Chenouard et al., 2021; Gondo et al., 2009)
The way from primary, spontaneous animal models of human diseases to directed mutagenesis, allowing for the customization of genetic modifications,
4 The Efforts toward Understanding the Disease’s Genetic Source
We, humans, share the vast majority of genes with animals such as mice, zebrafish, and even fruit flies. The similarities between human and mouse genomes allow us to assume that whenever candidate genes for human diseases are found, their homologs usually are present in, e.g., the mouse genome. On the other hand, we can mutate genes with unknown functions and observe the phenotype. Such an approach guides us to the discovery of genes, which disruption can lead to diseases. As an example, we can mention the research results published by (Dickinson et al., 2016) regarding the high-throughput discovery of novel developmental phenotypes. The scientists identified some disorders and changes in phenotypes in different developmental stages, which were the results of mutations in many previously uncharacterized genes (Groza et al., 2023; Muñoz-Fuentes et al., 2018). The effort of several international projects aiming to investigate the function of mouse genes by mutagenesis, and following molecular analysis and phenotyping, greatly expand our knowledge. This approach creates the possibility of using the mouse as a valuable organism to interpret the information coded in the human genome. For example, the phenotypic analysis shows that mutation in the transmembrane protein 132A gene, Tmem132a, leads, among others, to abnormalities in the animal spinal cord structure and narrower limbs (Rosenthal & Brown, 2007). Such information could make the identification of genetic disease sources easier. Also, can help in the therapy and treatment of such diseases, by creating accurate models of human disease for insightful and detailed studies.
5 Ways to Interpret the Myriad of Biological Data
To gain a wide perspective of the particular gene mutation consequences, we have to correlate the information obtained from experimental analysis with clinical data. Particularly taking into account the sequencing data from patient genomes. Such data become more and more available since nowadays whole exome and genome sequencing are often incorporated into personal health care. To achieve this goal, in the USA the Undiagnosed Disease Network (UDN) was established. The UDN brings together clinical and research data to learn
Understanding the impact of gene variants would help us to increase the efficiency of diagnosis. To find a “good candidate gene for disease” the scientists have to combine and compare several sets of data, from different resources. This information can be gained from genetic data sets, but also molecular, morphological, and phenotypic data by the use of genetic animal models. The one of approaches called the MARRVEL (Model Organism Aggregated Resources for Rare Variant Exploration2) aims to combine public genetic resources to analyze and choose the gene variants, which most likely are the cause of certain human diseases. Such variants became a candidate for study in model organisms, for in-depth analysis of pathogenic mechanisms. To facilitate this process and gather all the data in one database, MARRVEL extracts data from clinical and scientific public databases and aligns the variants with model organism genetic sequences. Additional biological and experimental data can be gained from public databases regarding particular organisms. For example, FlyBase3 is the most popular database describing fruit fly molecular data, and ZFIN4 contains information concerning zebrafish.
The whole MARRVEL approach starts with a patient, who has a disease symptom with a potential genetic background. The results of Whole Exome Sequencing or Whole Genome Sequencing leads to a long list of candidate gene variants that can cause disease in this particular case. The data from different sources help to assess how likely a particular variant can be a reason for such a genetic disorder. Then the scientist checks if a particular variant has its homolog (counterpart) in the model organism. Sometimes, there is experimental data regarding this variant, which facilitates its assessment. Having such information, scientists are often able to prioritize the variants’ pathogenicity. If there is no data, gene variants can be introduced into the model organism by genetic engineering. The animal genetic model is observed, tested and analyzed with the hope of finding similar phenotypic features, to those observed in the patient. This validation can show if the variant is pathogenic or not. Further, it can help to find appropriate treatment.
6 How Can Fruit Fly Help You?
To visualize the importance of this huge international scientific effort, we can use the true story as an illustration. Let’s imagine a patient, a small girl, with severe disease symptoms, yet undiagnosed. Bristol was born as a healthy baby, but during the first three months, her parents noticed that her development was not as fast as it should be. Her body was floppy and flexible, she was not able to lift her head, and sometimes her left eye turned inwards. She was also delayed in further progress such as crawling, standing and walking. She cannot smile and seems to be not sensitive to pain. For example, she did not cry when she fell or get her vaccine shots. This girl was diagnosed with congenital hypotonia, a symptom that can be caused by different neurological or non-neurological conditions. To find a reason for Bristol hypotonia, for five years she underwent tests checking for cerebral palsy, Down syndrome, autism, muscular dystrophy and many others, all of which were negative. Finally, thanks to analysis within the Undiagnosed Disease Project, the doctors found two more children, a boy and a girl, with similar symptoms, who carried an identical point mutation in the Early B-Cell Factor 3 (EBF3)-coding gene. The EBF3 gene is responsible for the regulation of various other genes’ expression, therefore it is highly possible that its alterations can widely influence nervous and muscle systems. Therefore, the researchers decided to check the consequences of EBF3 gene mutation in the fruit fly model. The experimental outcomes lead to the conclusion that EBF3 is indeed responsible for disease symptoms observed in small patients. The additional 20 patients all around the world have been found to carry the mutation(s) in this gene, leading to the conclusion that it can be responsible for several cognitive or speech disorders, and hypotonia of unknown origin. The knowledge regarding the disease’s origin allows for easier diagnosis in the future saving families from the “diagnosis odyssey”: a long, uncertain and difficult journey through a variety of specialists and medical tests. Additionally, animal models can be used to test potentially therapeutic compounds leading to the discovery of efficient treatment (Dunlap, n.d.).
7 Animal Models Limitations
Animal models have great importance in the life sciences, however, these models are not devoid of disadvantages. There is no such animal model, which could mimic all features of human disease, simply because we are non-identical. The differences between species cause differences in phenotype, morphology, molecular or physiological pathways, etc. Therefore, it is important to
8 Ethical Issues
Experimentation on animals and ethical issues connected with such activity has been a topic of wide-world debate for decades. The necessity of conducting such experiments, which saves human lives, is undoubtful. However, the price is high because it causes animal distress. The discussion focusing on ethical issues leads scientists to establish rules, which allow them to avoid unnecessary experiments, and protect animals from suffering. In 1959 William Russell and Rex Burch described ethical guidelines, called the three R’s (3Rs). The “R” letters relate to the terms Replacement, Reduction, and Refinement – three main, important practices, which should be applied during planning and conducting experiments involving animals (Tannenbaum & Bennett, 2015).
The first “R” says that, if it is possible the researcher should replace the experiment on animals with another technique. For example, one can use cell cultures
The second “R” obliged the investigator to reduce the number of animals used in the experiment to a minimum. This approach can be achieved by careful planning, also by using computational tools to calculate the minimal number of individuals in tested groups. Another example of following the “reduction” rule, is obtaining as much data from tested animals as possible. Let’s imagine that we treat fish with a potentially therapeutic substance to observe overall phenotype. We can assess the changes using a variety of tests, for example, record a movie showing the swimming speed and pattern, make a photo of live fish to show its length and shape, and finally use the animal muscles for biochemical tests. In this way, we save at least two or three experimental groups. Additionally, we can use animal tissues, which were not used in experiments, for didactic purposes or other kinds of investigations and research.
The third “R” regards animal experiment refinement to avoid or ameliorate the animal suffering, distress and pain. This is an especially subtle issue because it is really hard to assess if any human intervention in animal natural life rhythms is harmless. Even tests taken as harmless, such as microscopic observations of live animal behaviour, introduce into their lives some additional stimuli such as different light, additional sounds or physical vibrations. All procedures, which could be painful, are carried out on appropriately anaesthetized animals (anaesthetic agents cause loss of feeling in all or part of the body, with or without loss of consciousness). But there are also several other ways to ameliorate the distress, that is. by maintaining stable, proper conditions in the breeding facility. It is also worth noting here, that the animals for research purposes are bred and maintained in proper, strictly controlled conditions. The animal facilities are obliged to keep rigid conditions such as, in the case of zebrafish, a certain quality of water and food, stable temperature, adequate aquatic systems, enough space and take into account many other important factors.
The 3Rs were stated to serve as a base for the development of future alternatives to the use of animals in research. Indeed, nowadays there are expanding range of options to replace experimentations on animals such as 3D cell cultures, computer modelling and bioinformatic tools (Andersen & Winter, 2019; Balls, 2020; Díaz et al., 2020; Petetta & Ciccocioppo, 2021; Robinson et al., 2019).
9 Conclusions
Simple organisms such as bacteria, yeasts, worms, and fruit flies are indispensable to gaining knowledge regarding human organisms and health. The model means something simpler than the original, which we used as an example. The fast development of alternative experimental systems will lead us to use in the future other than animal disease models, at least in some cases (Benam et al., 2015). Nevertheless, the use of animals as a model of the human body is of utmost importance in drug testing and medicine, simply because we, as mankind, are not skilled enough to imitate nature in a sufficient way. The simplest organism created by nature is far too complex to replace it fully with any artificial creation.
Abbreviations
ZNF s | zinc finger nucleases |
TALEN s | transcription activator-like effector nucleases |
CRISPR | clustered regularly interspaced short palindromic repeats |
Cas9 | caspase 9 |
Notes
https://marrvel.org/; retrieved October 6, 2021
https://flybase.org/; accessed June 24, 2024
https://zfin.org/; accessed June 24, 2024
References
Andersen, M. L., & Winter, L. M. F. (2019). Animal models in biological and biomedical research – experimental and ethical concerns. Anais Da Academia Brasileira de Ciencias, 91(suppl 1), e20170238.
Balls, M. (2020). It’s time to reconsider the principles of humane experimental technique. Alternatives to Laboratory Animals: ATLA, 48(1), 40–46.
Benam, K. H., Dauth, S., Hassell, B., Herland, A., Jain, A., Jang, K.-J., Karalis, K., Kim, H. J., MacQueen, L., Mahmoodian, R., Musah, S., Torisawa, Y.-S., van der Meer, A. D., Villenave, R., Yadid, M., Parker, K. K., & Ingber, D. E. (2015). Engineered in vitro disease models. Annual Review of Pathology, 10, 195–262.
Burggren, W. W., & Warburton, S. (2007). Amphibians as animal models for laboratory research in physiology. ILAR Journal/National Research Council, Institute of Laboratory Animal Resources, 48(3), 260–269.
Chenouard, V., Remy, S., Tesson, L., Ménoret, S., Ouisse, L.-H., Cherifi, Y., & Anegon, I. (2021). Advances in genome editing and application to the generation of genetically modified rat models. Frontiers in Genetics, 12, 615491.
Cordier, A. C., & Haumont, S. M. (1980). Development of thymus, parathyroids, and ultimo-branchial bodies in NMRI and nude mice. The American Journal of Anatomy, 157(3), 227–263.
Díaz, L., Zambrano, E., Flores, M. E., Contreras, M., Crispín, J. C., Alemán, G., Bravo, C., Armenta, A., Valdés, V. J., Tovar, A., Gamba, G., Barrios-Payán, J., & Bobadilla, N. A. (2020). Ethical considerations in animal research: The principle of 3R’s. Revista de Investigacion Clinica; Organo Del Hospital de Enfermedades de La Nutricion, 73(4), 199–209.
Dickinson, M. E., Flenniken, A. M., Ji, X., Teboul, L., Wong, M. D., White, J. K., Meehan, T. F., Weninger, W. J., Westerberg, H., Adissu, H., Baker, C. N., Bower, L., Brown, J. M., Caddle, L. B., Chiani, F., Clary, D., Cleak, J., Daly, M. J., Denegre, J. M., … Murray, S. A. (2016). High-throughput discovery of novel developmental phenotypes. Nature, 537(7621), 508–514.
Dubińska-Magiera, M., Daczewska, M., Lewicka, A., Migocka-Patrzałek, M., Niedbalska-Tarnowska, J., & Jagla, K. (2016). Zebrafish: A model for the study of toxicants affecting muscle development and function. International Journal of Molecular Sciences, 17(11). https://doi.org/10.3390/ijms17111941
Dubińska-Magiera, M., Migocka-Patrzałek, M., & Cegłowska, A. (2022). Danio adventure. Developmental biology of the zebrafish in science popularisation. Journal of Biological Education, 56(3), 245–255.
Dubińska-Magiera, M., Migocka-Patrzałek, M., Lewandowski, D., Daczewska, M., & Jagla, K. (2021). Zebrafish as a model for the study of lipid-lowering drug-induced myopathies. International Journal of Molecular Sciences, 22(11).
Dunlap, E. (n.d.). How fruit flies helped to find a diagnosis for my daughter. Texas Children’s Hospital. https://nri.texaschildrens.org/blog/how-fruit-flies-helped-find-diagnosis-my-daughter
Ericsson, A. C., Crim, M. J., & Franklin, C. L. (2013). A brief history of animal modeling. Missouri Medicine, 110(3), 201–205.
Gondo, Y., Fukumura, R., Murata, T., & Makino, S. (2009). Next-generation gene targeting in the mouse for functional genomics. BMB Reports, 42(6), 315–323.
Groza, T., Gomez, F. L., Mashhadi, H. H., Muñoz-Fuentes, V., Gunes, O., Wilson, R., Cacheiro, P., Frost, A., Keskivali-Bond, P., Vardal, B., McCoy, A., Cheng, T. K., Santos, L., Wells, S., Smedley, D., Mallon, A.-M., & Parkinson, H. (2023). The International Mouse Phenotyping Consortium: comprehensive knockout phenotyping underpinning the study of human disease. Nucleic Acids Research, 51(D1), D1038–D1045.
Hartung, T. (2008). Thoughts on limitations of animal models. Parkinsonism & Related Disorders, 14(Suppl 2), S81–S83.
Hau, J. (2008). Animal models for human diseases. In P. M. Conn (Ed.), Sourcebook of models for biomedical research (pp. 3–8). Humana Press.
Kiełbwna, L., & Migocka-Patrzałek, M. (2017). Models of amphibian myogenesis – the case of Bombina variegata. The International Journal of Developmental Biology, 61(1–2), 17–27.
Migocka-Patrzałek, M., & Elias, M. (2021). Muscle glycogen phosphorylase and its functional partners in health and disease. Cells, 10(4). https://doi.org/10.3390/cells10040883
Muñoz-Fuentes, V., Cacheiro, P., Meehan, T. F., Aguilar-Pimentel, J. A., Brown, S. D. M., Flenniken, A. M., Flicek, P., Galli, A., Mashhadi, H. H., Hrabě de Angelis, M., Kim, J. K., Lloyd, K. C. K., McKerlie, C., Morgan, H., Murray, S. A., Nutter, L. M. J., Reilly, P. T., Seavitt, J. R., Seong, J. K., … The IMPC Consortium. (2018). The International Mouse Phenotyping Consortium (IMPC): A functional catalogue of the mammalian genome that informs conservation. Conservation Genetics, 19(4), 995–1005.
Onaciu, A., Munteanu, R., Munteanu, V. C., Gulei, D., Raduly, L., Feder, R.-I., Pirlog, R., Atanasov, A. G., Korban, S. S., Irimie, A., & Berindan-Neagoe, I. (2020). Spontaneous and induced animal models for cancer research. Diagnostics (Basel, Switzerland), 10(9). https://doi.org/10.3390/diagnostics10090660
Petetta, F., & Ciccocioppo, R. (2021). Public perception of laboratory animal testing: Historical, philosophical, and ethical view. Addiction Biology, 26(6), e12991.
Plantié, E., Migocka-Patrzałek, M., Daczewska, M., & Jagla, K. (2015). Model organisms in the fight against muscular dystrophy: Lessons from drosophila and Zebrafish. Molecules, 20(4), 6237–6253.
Robinson, N. B., Krieger, K., Khan, F. M., Huffman, W., Chang, M., Naik, A., Yongle, R., Hameed, I., Krieger, K., Girardi, L. N., & Gaudino, M. (2019). The current state of animal models in research: A review. International Journal of Surgery, 72, 9–13.
Rosenthal, N., & Brown, S. (2007). The mouse ascending: perspectives for human-disease models. Nature Cell Biology, 9(9), 993–999.
Szadvari, I., Krizanova, O., & Babula, P. (2016). Athymic nude mice as an experimental model for cancer treatment. Physiological Research / Academia Scientiarum Bohemoslovaca, 65(Suppl 4), S441–S453.
Tannenbaum, J., & Bennett, B. T. (2015). Russell and Burch’s 3Rs then and now: The need for clarity in definition and purpose. Journal of the American Association for Laboratory Animal Science: JAALAS, 54(2), 120–132.