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Genetically Modified Mice

Introduction

Rudolph Jaenisch bred the first genetically mod-ified (GM), or transgenic, mouse in 1974, and their use has exploded in the past few decades. In

Great Britain, GM animals are used in 53 percent of all experiments, with mice representing 91.4 percent of all GM animals used.1,2 While it’s impossible to count the total number of animals used in research in the U.S. or the number of GM mice bred or used each year estimates are in the hundreds of millions of animals and growing. This dramatic increase in the use of GM animals requires an examination of the scientific and ethical consequences of using them in experiments. There are two ways to create a genetically modified mouse: (A) Scientists can inject a DNA sequence con-taining the gene of interest into the fertilized egg of a pregnant mouse, a process called pronuclear injection. They use this method when they want to add additional genes to the mouse’s genome. (B) Scientists can mod-ify mouse embryonic stem cells using human DNA, and then inject them into the blastocyst (pre-embryo) of a pregnant mouse. This is a common method used to remove or “knock out” a single gene in the mouse’s genome.

GM Mice in Research

GM mice are used in large numbers to study human disease. Scientists use knockout mice to study human conditions where a single gene is missing or altered in the genome, such as sickle-cell anemia, muscular dys-trophy, diabetes, and Parkinson’s disease. Or, scientists study a specific gene that’s been inserted in the mouse and make estimates on the effects in humans. Tu-mor-promoting genes called oncogenes can be inserted into the mouse’s genome, causing it to develop tumors throughout his body. These “oncomice” are commonly used to test the effectiveness of new cancer drugs. GM mice are also used extensively in the study of Alzhei-mer’s disease and other forms of dementia. In addition to disease, GM mice are often used for studying drug- or chemical-induced immunotoxicity, genotoxicity, carcinogenicity, and metabolic processes.Problems with GM Mice

There are several scientific problems with the use of GM mice to study human diseases, and their daily lives can be of poor quality. Therefore the continued large-scale generation of GM mice raises compelling ethical issues that cannot be ignored.

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Mice engineered to express a green fluorescent protein flank a normal mouse under ultraviolet light.

NATIONAL INSTITUTES OF HEALTH

BMC CANCER / WIKIPEDIA CREATIVE COMMONS

A laboratory mouse in which a gene affecting hair growth has been knocked out (left) is shown next to a normal lab mouse.

Scientific Drawbacks:

• Many of the transgenic and knockout experiments fail to produce the desired result. Even if everything goes right, only 1/8 to 1/4 of the offspring in each lit-ter will have the desired gene mutation. Introducing a foreign gene (or knocking one out) can disrupt normal gene expression in the adjacent genes, known as ‘leak-iness’.3 It is also difficult to control the precise location of insertion into the genome. As a result, many of the offspring that result from modification of a mouse’s genome are not viable as test subjects. Even worse, the modification doesn’t express itself in the offspring, which happens in 85 percent of mice who are manip-ulated.4 These animals are simply considered “waste” and killed. • Genetic-modification is a resource intensive process. As discussed above, genetic modification is error-prone. It can also negatively impact the fertili-ty and survival rates of its subjects, so those that do have the desired trait are less likely to reproduce and pass that trait along to offspring.5 Because of this, large numbers of mice are used to obtain a few subjects that are healthy enough to survive the course of the experi-ment and have the desired modification. The mice that lack the modification or are not healthy enough are killed. This process wastes research money. • Disease models based off of animal data often don’t reflect human biology accurately. One of the drawbacks of using GM mice to study disease is that mouse and human biology doesn’t always overlap. The size difference and dissimilar physiology between the two species makes comparison difficult, and expres-sion of the same gene can be vastly different.6 For ex-ample, humans and mice share the [γc] gene. A muta-tion in this gene results in a loss of T and NK cells in both humans and mice, causing severe immunological defects. However, mice also lose B cells, while humans do not.7 This distinction, which seems subtle, has enormous consequences for drug development. • Interpreting studies done in GM mice is difficult and error-prone. The differences between human and animal biology have direct implications for how results are interpreted. Promising results in studies involving GM mice have failed miserably when moved to human trials. GM mice have been widely used to test treat-ments for Alzheimer’s disease, with promising results. However, when treatments began clinical (human) tri-als they failed miserably and often had “unforeseen ad-verse events or negative therapeutic outcomes.”8 One

treatment caused inflammation of the meninges and brain that “ultimately turns out to be worse than the original disease.”8 Relying on animal studies, geneti-callymodified or otherwise, always carries a risk (see PCRM’s factsheet “Dangerous Medicine: Examples of animal-based safety tests gone wrong” for more infor-mation).

Welfare Concerns• Many procedures are painful and invasive. A sur-vey from the Canadian Council on Animal Care found that most procedures that GM mice undergo cause moderate to severe physical discomfort to the mouse.9 Typical procedures include: inducing superovulation in females, surgically placing embryos, vasectomy, and ear and tail biopsy.4 After the study has been conduct-ed, mice are euthanized, usually via CO₂ suffocation or manual dislocation of their neck. Many of these proce-dures are performed without pain relief.• Unexpected side-effects can have a negative impact on welfare. In addition to the disease or condition that is induced in GM mice, transgenesis can result in any number of unanticipated side-effects, such as lameness, susceptibility to disease, stress, reduced fertility, reduced adult body weight, and immune impairment.5,10 A loss of gene function, called an insertional mutation, can also occur. Some side effects of insertional mutations include: loss of limbs (the legless mutation), craniofa-cial and visceral malformations, defects in the olfacto-ry lobes and cerebrum of the brain, deafness, physical deformities, and alteration of reproductive function.11-14 Estimates of insertional mutation frequency in GMM range from 7-20 percent.10

• Even when nothing goes wrong, GM mice have a rough time. GM mice are subjected to a number of dai-

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HUMAN GENOME PROJECT/U.S. DEPARTMENT OF ENERGY

The inability to produce leptin leads to extreme obesity in a genetically modified mouse.

ly stressors in addition to those directly related to the experiment. Routine lab procedures such as handling, blood collection, drug dosing, and separation from oth-er mice can cause marked and prolonged psychological and physiological stress.15 More than 50 percent of mice in laboratories exhibit behaviors that are indicative of distress, which can accumulate over time and result in severe mental trauma which parallels that seen in hu-mans kept in similar conditions.16,17

• Questionable Relevance. Mice have been modified to express green fluorescence protein so that they glow in the dark, grow a structure similar to a human ear on their back, and to become cognitively deficient “Forest Gump” mice, who can run twice as far as regular mice but have little working and spatial memories.18-20 GM mice are seen as scientific “playtoys” rather than sen-tient beings.

Alternatives to Using GM Mice in Research

Human-relevant methods of studying disease ad-dress the lack of overlap between mouse and human biology, the true contributions genetic defects have to human disease, the cost-intensive nature of using an-imals as subjects, and serious welfare concerns. Some alternatives include: epidemiological studies, bioinfor-matics, systems biology, tissue engineering, microfluid-ics, in vitro (human cell and tissue cultures) research, in silico (computer-based) techniques, stem cell methods, and safe human-based studies. More information about alternative methods of studying disease can be found in PCRM’s factsheet “Problems Associated with Animal Experimentation.”

References 1. Home Office of Great Britain. Annual Statistics of Scientific Procedures on living animals:

2012. Stationary Office Limited, 2013:7.

2. Ormandy EH, Schuppli CA, Weary DM. Worldwide trends in the use of animals in re-search: the contribution of genetically-modified animal models. Altern Lab Anim. 2009;37:63-68.

3. Matthaei KI. Genetically manipulated mice: a powerful tool with unsuspected caveats. J Physiol. 2007;582(Pt 2):481-488.

4. Robinson V, Jennings M; Working Group. Refinement and reduction in production of ge-netically modified mice: sixth report of the BVAAWF/FRAME/RSPCA/UFAW Joint Work-ing Group on Refinement. Altern Lab Anim. 2004;32(Suppl 1A):373-375.

5. Laible G. Enhancing livestock through genetic engineering: recent advances and future prospects. Comp Immunol Microbiol Infect Dis. 2009;32:123-137.

6. Fougerousse F, Bullen P, Herasse M, et al. Human-mouse differences in embryonic ex-pression patterns of developmental control genes and disease genes. Human Molecular Genetics. 2000;9:165-173.

7. Mestas J, Hughes CW. Of mice and not men: differences between mouse and human immunology. J Immunol. 2004;172:2731-2738.

8. Kokjohn TA, Roher A. E. Amyloid precursor protein transgenic mouse models and Alz-heimer’s disease: understanding paradigms, limitations, and contributions. Alzheimers Dement. 2009;5:340-347.

9. Canadian Council on Animal Care. 2009 CCAC survey of animal use. Web: 8 July 2013. http://www.ccac.ca/Documents/Publications/Statistics/Survey_2009.pdf

10. Van Reenen CG, Meuwissen TH, Hopster H, Oldenbroek K, Kruip TH, Blokhuis HJ. Trans-genesis may affect farm animal welfare: a case for systematic risk assessment. J Anim Sci. 2001;79:1763-1779.

11. McNeish JD, Thayer J, Walling K, Sulik KK, Potter SS, Scott WJ. Phenotypic characteriza-tion of the transgenic mouse insertional mutation, legless. J Exp Zool. 1990;253:151-162.

12. Alagramam KN, Kwon HY, Cacheiro NL, et al. A new mouse insertional mutation that causes sensorineural deafness and vestibular defects. Genetics. 1999;152:1691-1699.

13. Woychik RP, Stewart TA, Davis LG, D’Eustachio P, Leder P. An inherited limb deformity created by insertional mutagenesis in a transgenic mouse. Nature. 1985;318:36-40.

14. Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach KS, Smithies O. Alteration of repro-ductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene. Proc Natl Acad Sci U S A. 1993;90:11162-11166.

15. Balcombe JP, Barnard ND, Sandusky C. Laboratory routines cause animal stress. Contem-porary Topics. 2004;43:42-51.

16. Mason GJ, Latham NR. Can’t stop, won’t stop: is stereotypy a reliable animal welfare in-dicator? Animal Welfare. 2004;13:57-69.

17. Ferdowsian H, Merskin D. Parallels in sources of trauma, pain, distress, and suffering in humans and nonhuman animals. J Trauma Dissociation. 2012;13:448-468.

18. Liu H, Kishi T, Roseberry AG, et al. Transgenic mice expressing green fluorescent protein under the control of the melanocortin-4 receptor promoter. J Neurosci. 2008;23:7143-7154.

19. Cao Y, Vacanti JP, Paige KT, Upton J, Vacanti CA. Transplantation of chondrocytes utiliz-ing a polymer-cell construct to produce tissue-engineered cartilage in the shape of a human ear. Plast Reconstr Surg. 1997;100:297-302.

20. Kolisnyk B, Guzman MS, Raulic S, et al. ChAT-ChR2-EYFP mice have enhanced motor endurance but show deficits in attention and several additional cognitive domains. J Neurosci. 2013;33:10427-10438.

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