Online Issues

Journal Vol 47(2)

Phenotyping of Genetically Engineered Mice: Humane, Ethical, Environmental, and Husbandry Issues

Marilyn J. Brown and Kathleen A. Murray

Marilyn J. Brown, D.V.M., M.S., DACLAM, DECLAM, is Executive Director of Animal Welfare and Training and President of Charles River Laboratories Foundation, Wilmington, Massachusetts. Kathleen A. Murray, D.V.M., M.S., DACLAM, is Senior Director of Animal Program Management at Charles River Laboratories.

Abstract

The growing use of genetically engineered (GE) mice in scientific research has raised many concerns about the animal welfare of such mice. The types of welfare concerns may differ within the three stages that comprise the establishment of GE animal models: development, production, and research use. The role and impact of the members of the research team on these concerns may also vary with each stage. To make both scientific and animal welfare decisions at each stage, it is necessary to have a thorough knowledge of the animal model—in this case, the phenotypic expression of the GE animal. Phenotype screening is the analysis of visible or measurable characteristics of an animal that result from the genotype and its interaction with the environment. Phenotypes expressed that are relevant to the research program are usually carefully investigated; however, those that may have an impact on the animal's welfare but have little or no impact on the disease process under study are often less carefully studied. Thorough analysis and documentation of the animal welfare aspects of phenotype provide the research team with the information they need to control the environment to minimize negative animal welfare effects. Such information is also essential to allow members of the institutional animal care and use committee to perform necessary cost:benefit ethical review of proposed GE animal studies. Investigators who submit information about models for publication should document all aspects of a phenotype, including the area of scientific interest as well as those areas that affect animal welfare, for clarity and for subsequent research with the respective models.

Key Words: animal welfare; ethics; genetically engineered mice; IACUC; phenotype

Introduction

The growing use of genetically engineered (GE¹) mice in scientific research has raised many concerns about the animal welfare of such mice (Balls 1999; Buehr et al. 2003; Glenn 2003; Moore and Mepham 1995). However, some have suggested that the use of GE mice may not actually increase welfare concerns. Frank Lowe, in a Guest Editorial in the British Veterinary Journal, asked, “Is there a morally relevant difference between conventional mice given viruses or chemicals to induce cancer and transgenic mice expressing a tumour gene?” He goes on to recommend performing a case by case ethical analysis of genetic manipulation and to urge investigators not to neglect potential ethical issues (Loew 1994).

Clearly the ethical issues and animal welfare concerns that must be considered whenever animals are used in research must also be considered in the use of genetically modified (GM^1,2) animals. The ethical issues are similarly influenced by the animal welfare concerns raised in a particular study, and especially the cost:benefit analysis that focuses on the justification for animal use, the justification of species and numbers to be used, and the necessity of balancing relevance and research design with the cost to the animal. The welfare concerns (e.g., potential for pain and distress) due to the characteristics of the disease state under study may not be significantly different between the naturally occurring or induced animal model for a disease and the model created through genetic manipulation. The GM animal is usually “designed” to manifest the disease, but actual clinical expression can range from the full symptomatology of the disease to much milder effects. The GM animal may have only one gene in a polygenetic condition, in which case serious clinical signs in the animal are not evident, yet the condition still facilitates study of the disease mechanism. In fact, targeting a disease or specific aspects of a disease using genetically modified animals often permits the use of fewer animals in the experimental process, thus leading to one of Russell and Burch's (1959) three principles (3Rs¹) of humane experimental technique—reduction. For example, the ability to harvest tissue that expresses a specific gene may allow for studies to be conducted in vitro on those tissues rather than using large number of animals for in vivo studies. However, in some models, in addition to the expected clinical expression based on the disease under study, additional phenotypic impacts may exist due to the side effects of the genetic manipulation itself (Mertens 2000). In developing a new GM mouse, phenotypic expression that could result in conditions that would have animal welfare implications might occur any time during the pre- and postnatal development period or at any subsequent age. Moreover, these conditions could involve alterations in physiological or behavioral functions as well as other parameters. For this reason, the severity of the alternations, hence the impact, on animal welfare could vary considerably.

It should be noted that not all genetic manipulations are performed to model disease and not all genetically modified animals express clinical disease. In many cases, no clinical signs or disease may be expressed, but changes exist in metabolic pathways or physiological processes that may have no visible or substantial effect on animals. The issues addressed in this article involve that subset of animals in which disease develops, potentially leading to animal welfare issues. In addition to clinical conditions that may be predicted based on model design, the expression of unexpected phenotypes that may have an impact on animal welfare can present additional challenges to the scientist, laboratory animal veterinarian, technician, and members of both the institutional animal care and use committee (IACUC¹) and the ethics committee (EC¹).

Animal welfare is a concept that may mean different things to different constituents. It is a phrase often used but rarely precisely defined. For the purpose of this discussion, we define animal welfare as follows:

“Animal welfare is a human responsibility that encompasses all aspects of animal well being, including appropriate housing, clinical and behavioral management, nutrition, disease prevention and treatment, responsible care and use, humane handling, and, when necessary, euthanasia. Responsible care and use, in the context of laboratory animal welfare and the use of animals for scientific purposes, includes a commitment to avoid unnecessary pain, distress, and discomfort” (Kathryn Bayne, The American Society of Laboratory Animal Practitioners [ASLAP] Animal Welfare Committee, personal communication, 2005).

The types of welfare concerns may differ within the three stages that comprise the establishment of GE animal models: development, production, and research use. The roles and impact of the members of the research team (scientist, veterinarian, technician, and IACUC or EC) on these concerns may also vary with each stage. To make both scientific and animal welfare decisions at each stage, it is necessary to have a thorough knowledge of the animal model—in this case, phenotypic expression of the GM animal.

Phenotype analysis or screening looks at visible or measurable characteristics of an animal that result from the genotype and its interaction with the environment. The environment, in this case, includes such variables as husbandry practices, diet, light cycle, and microbiological status. Phenotypes expressed that are relevant to the research program are usually carefully investigated; however, those that may have an impact on the animal's welfare but have little or no impact on the disease process under study are often less carefully studied, if at all. Thorough analysis and documentation of the animal welfare aspects of phenotype provide scientists, veterinarians, and technicians with the information they need to control the environment to minimize negative animal welfare impacts. Such information is also essential to allow IACUC or EC members to perform necessary cost:benefit ethical review of proposed GM studies.

Development

Genetic modification techniques enable one to manipulate the genome to create unique animal models for studies in various disciplines as well as to establish causal relationships between genotypes and phenotypes (Polites and Pinkert 2002). These technologies, which are well described in textbooks, include pronuclear microinjection, homologous recombination in embryonic stem cells, viral-mediated gene transfer, nuclear transfer, RNAi, and chemical mutagenesis (Nagy et al. 2003; Plagge et al. 2000; Polites and Pinkert 2002). In general, one inserts, deletes, or alters a segment of DNA resulting in overexpression, reduced expression, or inactivation of the gene of interest. One then characterizes or phenotypes the GE animals to delineate the effects of the genetic manipulation (Dennis 2000).

When designing a transgenic mouse model, there are many factors to consider. One important design decision that will directly influence the phenotype of the GE mouse is the background strain one selects to receive the genetic manipulation (Linder 2001). Most common inbred strains of mice are very well characterized phenotypically. Taking the time to think about the expected outcome of the genetic manipulation and how gene expression might be modified by different background strains, particularly in the context of the experimental goals, may be critical to the overall success of the research program. For example, if one is creating a model that will be used to study learning and memory and plans on utilizing several mazes that depend on the animal's ability to see visual cues, it would be important for the selected background strain to have normal vision.

During the animal model design and development phase, it is important to predict outcomes with respect to animal welfare and adverse phenotypes. For example, if one expects that the genetic alteration will result in death or an extremely debilitating phenotype when expressed in all tissues, then one should consider the use of either spatial or temporal deletion strategies to avoid such phenotypes. Recombination systems such as Cre-loxP and FLP-FRT can be used to limit gene expression to certain tissues (Nagy et al. 2003; Polites and Pinkert 2002). The use of an inducible promoter such as tetracycline-based systems allows one to control expression in a time-dependent fashion, turning the gene on or off based on the presence or absence of tetracycline (Plagge et al. 2000; Polites and Pinkert 2002).

Although it is difficult to predict outcomes with respect to animal welfare and adverse phenotypes, an important reason for attempting to list potential detrimental outcomes of a genetic manipulation is to aid in the development of monitoring protocols and endpoints. It is the responsibility of the investigator to provide this specific type of information to the IACUC. Careful consideration should be given to anticipated life span issues, adequacy of immune system function, reproductive abilities, altered anatomical and physiological functions, impaired sensory abilities, and development and severity of clinical disease. It is possible for the researcher and veterinary team to work together to develop a customized monitoring plan to ensure that unacceptable deterioration of animal welfare does not occur due to the development of an adverse phenotype. Nevertheless, we acknowledge that in creating GM animals, the outcomes are often unknown and unpredictable. For this reason, the type of phenotype that will be expressed may not be available for the IACUC members' deliberations and thus, there may be no basis to set specific monitoring protocols or endpoints before model creation. Although it is certainly the responsibility of the investigator to report abnormal findings that might have animal welfare considerations to the IACUC once they occur, and the IACUC should make clear the types of findings that would be of concern and need to be reported, the lack of phenotypic information to set such parameters and standards before model creation would not necessarily prevent a protocol from being approved.

Acknowledging that predictions may be inaccurate and that the unexpected happens even with the best planning, numerous investigators have suggested developing a list of parameters for monitoring and tracking all newly created lines: Mertens and Rulicke (1999, 2000), Crawley (1999, 2000), van der Meer and colleagues (2001), and Dennis (2002). In this context, the main purpose of collecting the phenotypic data is to ensure adequate review of animal welfare considerations (Dennis 2002). These same data, however, are valuable from a scientific perspective because they further characterize the model.

Dennis (2002) proposes the use of a system with two interdependent components—clinical surveillance and assessment of phenotype—as an effective means to address potential animal welfare issues in newly created lines. The clinical surveillance component is directed by the animal care and veterinary staff, and the phenotype assessment program is managed by the research team. These data can be used to refine appropriate monitoring and endpoints for a given line. In addition, the information may be reported back to the IACUC and used by members in their cost:benefit ethical review to determine whether to approve further breeding and use of this line.

During the phenotype assessment phase of animal model development, the program designed by the research team should be sufficiently broad to allow detection of both expected and unexpected phenotypes. In addition to characterizing the line, these data are needed to determine the model's suitability for subsequent analyses. We suggest including the following tests that will serve as a standard baseline for all models in a minimum general phenotyping screen: clinical chemistries, complete blood count and differential, urinalysis, gross and histopathology of major organs, abnormal gross tissues and expected target organs, and a physical examination similar to that described by Crawley with components from the Irwin Observational Battery and the SHIRPA test (Crawley 2000). This general background screen is meant to be non-defect and non-model-specific screening. The tests are relatively simple to perform and do not require large investments in capital or labor. The use of standardized baseline testing enables the evaluation of the general health of each line, as well as the respective sensory abilities and basic anatomical, physiological, behavioral, and motor function.

Part of the program of providing adequate veterinary care includes frequent monitoring of all animals on a regular basis. To facilitate data collection, we recommend that all personnel working with GE animals (including veterinary technicians, research technicians, animal care staff, and investigators) receive clinical observation training that covers normal rodent behavior, reproductive biology, and frequently observed clinical conditions (e.g., malocclusion, hydrocephaly, dermatitis, bite wounds, barbering). Personnel should be trained to report anything that appears abnormal and to use standardized and precise descriptive terms (i.e., not only “morbidity” and “mortality”).

A well-defined process for reporting observations, including the use of standardized forms, as well as data review and the development of an action plan to address issues should be part of the model development process. It is the responsibility of all who use or care for GE animals to report clinical abnormalities using this process. After the veterinary staff members review the clinical observation data, they should work with the investigative team to develop an action plan for further monitoring, should apply treatment or intervention as appropriate, and should refine the endpoints associated with the clinical disease development or debilitation associated with these animals.

During phenotypic assessment by research staff, findings may be observed (e.g., abnormalities in serum chemistries or early gross or histopathological lesions) that are not causing overt clinical signs at that time. Veterinary and animal care staff should be alerted to these abnormalities and should adjust plans accordingly.

Production

Following initial development and phenotypic characterization, the next stage in the establishment of a GE animal model is the production phase. The phenotypic data, collected during the development phase of the model, including morbidity and mortality statistics, should be used to help determine the optimal breeding strategy for maintenance of the line. The goal is to minimize welfare issues associated with the breeding colony, yet still produce the animals needed for experimental studies. Key points to consider in developing a production plan include any known phenotype or life span issues that may affect reproduction, any expression or phenotype differences based on zygosity, differences in fertility based on sex or zygosity, as well as the goal or goals of the breeding colony.

Location of the mutation on the X or Y chromosome and infertility related to zygosity, sex, or the development of an adverse phenotype that interferes with the reproductive ability of the mutant animal will limit choices for breeding schemes (Murray and Parker 2005). For example, if only the homozygous animals have adverse phenotypes, one might consider maintaining the colony by breeding heterozygous X wild-type so that only heterozygous and wild-type mice are routinely maintained. When experimental animals are needed, a special mating of heterozygous X heterozygous animals can be set up. Homozygous animals as well as any heterozygous and wild-type littermates can then be used on study. It should be noted that maintaining a colony as described above with the goal of minimizing animal welfare concerns will produce more animals over time than a colony of either heterozygous X heterozygous or homozygous X homozygous matings, which may be in conflict with the 3Rs. IACUC members will need to weigh the numbers of animals produced and not utilized against the animal welfare concerns of those animals expressing a detrimental phenotype. In addition, using a mating scheme as described above will require advance planning and coordination with those responsible for initiating breeding to ensure that matings are established at the appropriate time to deliver the required number of experimental animals.

It is often possible to obtain well-established models from a variety of sources, including commercial breeders, mutant mouse repositories, and other institutions. Before obtaining an established line, it is important to gather as much information as possible regarding the phenotype of the model to determine whether any special requirements are necessary to maintain the health and well-being of the animals. Sources of this information include the commercial supplier, repository or institution of origin, and the laboratory that initially created the model, as well as publications or publicly available databases. A useful starting point for a web-based search is the Mouse Genome Informatics resource (www.informatics.jax.org), which provides integrated access to data on genetics, genomics, and biology of laboratory mouse strains including a section on phenotypes and alleles.

The authors' institution receives large numbers of mutant lines from facilities around the world. The institution provides a model information form for the investigator's completion before receipt of the animals. Pertinent information includes reproductive characteristics, age of onset for various phenotypic parameters, and the possible existence of differences in expression based on sex or zygosity, life span issues, and any special care needed to maintain these animals. Institutional staff members review the information, which may trigger a request for additional information, including copies of relevant publications. Based on the available information, a plan is developed to accommodate the special needs of each model, including a monitoring program and endpoints. These forms are then reviewed with the investigator and agreed upon before permission is given to ship the animals to our facility. Examples of the types of special needs frequently identified during this process are listed in Table 1.

Jegstrup and coworkers (2003) recently suggested performing a thorough animal welfare evaluation as part of a general phenotypic characterization. The goal of this evaluation would be to reveal any special needs or problems with a strain. This information would then be compiled as an instruction document that would accompany the strain when it is sold or provided to another investigator. The practice is similar to the readily accepted practice of providing health status information before shipping animals, as Mertens and Rulicke (2000) have suggested. We are pleased to report significant progress toward the goal of investigators having adequate and high-quality phenotypic information upon receipt of animals at our institution over the last 2 to 3 yr.

Sadly, even with established lines, we have noted discrepancies between the phenotypic data shared with us before the mice are shipped and the clinical observations that we make once we are maintaining the animals in our facility. For this reason and for all lines, we maintain a clinical surveillance program similar to that described in the Development section above. Morbidity and mortality data are reviewed weekly. Unexpected observations are reported to the research team. The production management and animal care teams share the responsibility for developing a customized monitoring program and action plan to define potential production or animal welfare problems further. Based on analysis of the findings, modifications to the breeding strategy, a monitoring program, and attention to special needs requirements may be instituted as needed.

It is important to note that not all detected discrepancies are detrimental. In many cases, the incidence of morbidity and/or mortality that is observed may be less than the incidence the investigator reports. It is important to remember that an animal's phenotype is the result of the interaction between its genotype and the environment. It is likely that different husbandry practices and environmental conditions are responsible for at least some of these discrepancies. It is well established that a multitude of factors, including husbandry practices and environmental conditions under which production and research are conducted, may influence the results of animal research (Lipman and Perkins 2002). For this reason, it is important to document husbandry practices and environmental conditions and to include any altered husbandry practices that have been instituted in response to adverse phenotypes (see examples in Table 1).

Throughout the production process, the research team and the animal care staff overseeing the breeding colony should communicate regularly. In addition to providing morbidity and mortality updates and sharing all clinical observations with the investigative staff, the two groups should communicate regarding production goals and experimental animal needs. Regular communication between the two groups is also critical to balance the supply and demand of animals successfully.

In addition, at several points—early in the characterization phase, after the model is developed and established, and finally when the need for the line diminishes—the investigator should strongly consider cryopreserving the line as embryos, sperm, and/or ovaries. During the early and mid-stage GM model development process, cryopreservation provides an important insurance policy, should the unexpected happen. The technique has value toward the end of the animal model's use in the institution's research program both from the perspective of animal welfare and for cost savings. Cryopreservation is as an effective means to “store” the line for potential future access by either the investigator or some future collaborator, rather than maintaining the line as live animals for the same purposes.

Use

Most of the special considerations involving GE mice occur during the development and production stages of model establishment. As with any proposed animal study, the IACUC or EC must conduct an ethical review of animal use. Investigators should consider and be prepared to describe the following aspects of the proposed study: justification for the animal model proposed and the animal numbers to be used, the types of clinical effects that can be anticipated in the animals, how pain and distress will be minimized, the endpoints that will be used, any special husbandry that will be necessary, the qualifications and training of personnel involved, the appropriateness of the procedures proposed, and details of animal monitoring. With respect to a well- established and well-characterized line of GM mice, greater knowledge of the phenotype will make it possible to answer these questions with greater certainty. However, with animals for which all of the details of the phenotype are not known, and if the potential is high for animal welfare problems due to the nature of the gene modification being created, it may be appropriate to handle the protocol as one would handle a pilot study. In these studies, approval typically occurs in a step-wise fashion, with small numbers of animals and more frequent feedback to the committee as the phenotype is characterized.

As mentioned above, the use of GE animals may offer some unique opportunities to enhance animal welfare and the 3Rs. Through careful phenotyping, early indicators of disease may be used that minimize the development of more serious clinical signs. Tissues expressing the gene of interest may be able to replace some in vivo studies. To foster animal welfare and the 3Rs, IACUCs and ECs should expect as much information as possible about the phenotype of the proposed GE animal and ensure that all members of the research team, including the animal care and veterinary staff, are prepared to meet any special needs of such animals. As with any protocol, unexpected complications of any model should be promptly brought back to the IACUC or EC for review.

Summary

The use of GE mice offers many opportunities to continue to expand our knowledge of biological systems and disease. The development of such models must involve all members of the research team, scientists, technicians, veterinarians, animal care staff, and the IACUC and/or the EC. Careful evaluation for how genetic modifications affect animal welfare and for planning studies and animal care to minimize adverse outcomes is essential. Sound professional judgment plays a key role in helping to achieve this goal, given the large numbers of GM lines of mice and the often limited amount of phenotypic information that has been generated for any given line. It is important to share with the scientific community the documentation of all aspects of a phenotype, including both the area of scientific interest as well as areas that affect animal welfare and husbandry considerations. In scientific articles related to the use of GE mice, this shared information should include descriptions of special care needs as well as the environmental parameters and husbandry practices.

¹Abbreviations used in this article: 3Rs, refinement, reduction, replacement; EC, ethics committee; IACUC, institutional animal care and use committee; GE, genetically engineered; GM, genetically modified.

²In the context of this article, the terms genetically engineered, genetically modified, and genetically manipulated are used synonymously.

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