Humane Endpoints for Genetically Engineered Animal Models
Melvin B. Dennis, Jr.
Introduction
I
n the early 1980s, the introduction of transgenic mice revolutionized the production of genetically altered animals.
Before then, the methods for modifying the genome of
animals were spontaneous and induced mutagenesis, hybridization, and selective breeding. These tools were used in
agriculture to improve quality and yield of products and in
biomedical research to produce models for the study of physiologic and pathologic processes. Mutagenesis remains a commonly used instrument and is used to generate large numbers
of experimental animals. In addition, transgenic technology
expanded the ability to manipulate the genome and dramatically increased the number of studies involving use of genetically engineered animals. It has facilitated efforts to understand the role of specific genes and study ways to replace
defective ones. Proponents of the use of transgenic animals
assert that the models produced are more specific and reliable than spontaneous disease models. They maintain that
their use will increase the yield of medical research while
eventually reducing the numbers of animals used. Opponents
believe the technology is wasteful because large numbers of
donor, recipient, and breeder mice, as well as a large percentage of nontransgenic offspring, are euthanized and do not
produce usable data. Opponents also charge that some of
these animals may experience significant pain and distress as
a result of the genetic alternatives.
Genetic engineering involves insertion, deletion, or alteration of a segment(s) of DNA followed by observation of
the effects in the animal and/or offspring. Methodologies
include manipulation of the genome by (1) pronucleus microinjection to produce overexpression of a gene(s); (2) targeted mutagenesis in embryonic stem cells to knock out or
inactivate genes; and (3) introduction of new genetic material, including promoter and regulatory sequences via various vectors. Descriptions of the procedures used are available (Pinkert 1994; Silver 1995).
New fields of study, including xenotransplantation and
gene therapy, have been fostered by the increased ability to
manipulate and modify the genome. Gene therapy involves
incorporating a normal gene sequence into a viral or nonviral
vector that can carry it into target cells to replace a defective
sequence. The replacement gene(s) and any necessary regulatory sequences to be inserted are spliced into the vector,
such as replication-deficient retroviruses, adenoviruses, or
herpes viruses (Makrides 1999). Other vector work uses replication-competent viruses (e.g., herpes simplex and vaccinia
virus), which are administered at a low dose to produce subclinical infection while achieving gene transfer. Nonviral
vectors such as cationic liposomes are also being developed
to accomplish gene transfer (Liu et al. 1995).
Determination of endpoints for mutant animals is not
appreciably different than for animals in other studies. The
objective of the experiments is often to study the effect of
overexpression or deletion of a gene of interest on the presence, absence, or alteration of a protein, enzyme, or cytokine;
to study a human disease; or to develop a treatment. A robust
phenotype for the mutant gene that can be readily observed is
helpful. Death is not usually intended in genetic engineering
studies, but lethality or animals with severe health problems
are commonly encountered. However, whereas the goal of
agricultural research is to produce a normal animal with superior qualities, the goal of biomedical research is often to
study the animals with induced abnormalities. Genetically
engineered animals with decreased ability to resist disease,
with increased tumor production, or that have compromised
basic bodily functions such as eating or breathing are not
unusual. Establishing humane endpoints in such studies can
present challenges, depending on the particular phenotype of
the genetic line. The situation is complicated by the unpredictability of genetic manipulation and the variety of phenotypic expressions possible. It is aided by the consistency
of phenotypic expression within a line and the ability to manipulate expression of problem genes. These and other factors that should be considered when establishing humane
endpoints for genetically engineered animal models are discussed below in the context of oversight by the attending
veterinarian and the institutional animal care and use committee (IACUC1).
Melvin B. Dennis, Jr., DVM, is Professor and Chairman of the Department
of Comparative Medicine, School of Medicine, University of Washington,
Seattle, Washington,
IACUC Review of Genetic Engineering
Studies
'Abbreviations used in this article: IACUC, institutional animal care and
use committee; PI, principal investigator.
In the United States, the IACUC has oversight of animal use
in a framework of regulations, policies, and guidelines. The
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ILAR Journal
Department of Agriculture has published regulations (CFR
1998) that currently apply only to studies involving species
other than rats, mice, and birds. Provisions in the Guide for
the Care and Use of Laboratory Animals (NRC 1996) apply
to all vertebrate animals. I have discussed regulation of animal research in the United States (Dennis and Van Hoosier
1994) and of general IACUC review of genetic engineering
protocols (Dennis 1999). Guidelines for Research Involving
Recombinant DNA (CFR 1998; Federal Register 1994) provides direction for oversight of genetic engineering experiments. Institutions with animal studies involving introduction of recombinant DNA into the germline of animals are
required to have an institutional biosafety committee and an
animal containment specialist. Most studies in which the
animal's genome is altered by the stable introduction of recombinant DNA into the germline require approval by both
the institutional biosafety committee and the IACUC.
Genetic engineering protocols present the IACUC with a
unique set of problems with regard to humane endpoints.
IACUC members have traditionally asked the principal investigator (PI1) to list anticipated experimental outcomes and
to describe how situations involving pain or distress will be
addressed. Investigators may be able to provide this information in situations where animals with a well-characterized
genome are used, but they may not when new lines are to be
generated. Manipulations of the genome can involve a shift
away from the traditional hypothesis-driven methodology to
one of discovery. Often the hypothesis may propose that the
genetic alteration will cause significant effects that cannot be
predicted accurately. For instance, a gene of interest is overexpressed, knocked out, or inactivated, and the offspring are
carefully observed to learn the effects of the manipulation.
Unanticipated Outcomes
A significant problem in establishing humane endpoints for
genetic engineering studies is the occurrence of unanticipated adverse outcomes. Incorporation of variable numbers
of strands of heterologous DNA into a variable number of
insertion sites can produce innumerable outcomes, many of
which are unpredictable. An example of this incorporation
occurred in a study involving the pronuclear injection of the
gene for human growth hormone into mice. One would predict that the offspring overexpressing human growth hormone would be larger and heavier than the parent mice. In
addition, however, some of the transgenic lines also had increased liver failure, kidney dysfunction, tumor development, infant and juvenile mortality, shortened lifespan, reduced fertility, and structural changes in the heart and spleen
(Wolf and Wanke 1995). This case illustrates that for the PI
and the IACUC to make an informed decision regarding the
humane aspects of such studies, there must be ongoing monitoring of the progeny. If only the anticipated outcomes are
considered, animals with severe problems could be produced
for which there is no adequate plan for addressing pain and
distress issues.
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2000
There is also a need for ongoing monitoring of succeeding generations of genetically altered progeny as was illustrated in a line of transgenic pigs with high levels of bovine
growth hormone. The first two generations had improved
feed conversion efficiency and low levels of subcutaneous
fat; however, subsequent generations were characterized by
ulcers, arthritis, nephritis, cardiomegaly, and infertility
(Pursel et al. 1989). In some lines, problems may not become
apparent until the gene of interest is homozygous. This occurred in mice transfected with a drosophila heat shock gene
(hsp70) and a herpesvirus thymidine kinase gene. First generation heterozygotes appeared phenotypically normal, but
F 2 homozygotes had loss of hind limbs, malformed forelimbs, facial clefts, and olfactory lobe defects (McNeish et
al. 1988).
Even after producing several lines using a particular gene
construct, the phenotype of subsequent lines cannot always
be predicted accurately. One group made multiple lines of
lck-IL-4 transgenic mice without the occurrence of observable problems. In a subsequent line, however, it was observed that animals 3 to 6 mo of age became progressively
humpbacked. Further study revealed that the animals had
osteoporosis (Lewis et al. 1993). This effect could have resulted from incorporation of the gene construct in different
numbers or into different sites than had previously occurred.
In this case, an unanticipated outcome produced a useful
model. The case also illustrates that the search for models of
human and animal diseases may require the continued breeding of compromised animals until they are fully characterized. However, in principle IX of the US Government Principles for the Utilization and Care of Vertebrate Animals
Used in Testing, Research, and Training, it is stated that such
decisions for continued breeding should not rest with the
investigators but instead with a review group such as the
IACUC. However, it is incumbent on the investigator to describe how new conditions involving pain and distress will
be handled, including establishment of appropriate and humane endpoints.
Protocol Oversight
To adequately meet its responsibilities for oversight of genetic engineering studies, it is advisable for an institution to
have a system that will identify animal welfare problems so
they may be dealt with in an effective and timely fashion.
The IACUC review and approval processes should be followed by surveillance of the animals involved in the study
and periodic reviews of the welfare of the animals being
produced.
Initial IACUC Review
If a proposed study involves use of a strain that has been
characterized, the PI should be expected to predict outcomes
and provide clearly defined endpoints for animals found to
95
be in pain or distress. The welfare issues can be readily anticipated in well-defined transgenic and knockout animals,
many of which are available commercially. However, there
are other situations with a need for in-house breeding of lines
that have not been well characterized. Additionally, phenotype information will not be available for newly developed
lines. In these cases, the PI may be asked to predict outcomes
based on what is known about the gene(s) of interest or related genes. In most situations, the PI will be the best person
to predict outcomes because he or she is most knowledgeable about the gene.
Surveillance
Because outcomes are often unpredictable, the IACUC can
use surveillance or monitoring of ongoing studies to ensure
adequate oversight of welfare considerations. Such surveillance can be accomplished by using a morbidity and mortality reporting program with feedback to the IACUC proceeding through the attending veterinarian. In my institution, the
program resulted in identification of transgenic and knockout lines with many unexpected adverse outcomes, examples
of which are listed in Table 1. As problems are identified,
IACUC and veterinary personnel meet with the PI to discuss
the situation and devise strategies to improve welfare or establish endpoints.
Continuing Review
Rereview of animal use protocols is required annually by the
US Department of Agriculture and every 3 yr by US Public
Health Service policy. Approval to continue breeding a particular line beyond a minimal number required for maintenance and basic phenotyping could be accomplished during
these required reviews. Due to the high frequency of occurrence of unexpected adverse outcomes, it seems prudent to
require Pis to return to the IACUC for approval of continued
breeding of each genetically altered line as soon as the phe-
Table 1 Examples of adverse outcomes identified
in genetic engineering experiments
Allergic encephalomyelitis anasarca
Arterial wall calcification
Diabetes
Dopamine deficiency
Epilepsy
Hydrocephalus
Increased tumor incidence
Malocclusion
Osteoporosis
96
notype is known. IACUC members should ask questions such
as, what morbidity or mortality is associated with the phenotype of this line, what endpoints will be used when animals
develop painful or distressful conditions, or is there evidence
of health problems in this line of animals. The results of
phenotyping could be submitted to the IACUC, which would
review the data and grant or withhold approval for the continued breeding of the line. Additional data of potential relevance to the review (i.e., immune competency or disease
model data) could also be submitted.
The task of the IACUC is to weigh animal welfare considerations and the potential utility of a line to decide whether
to allow continued breeding. The dilemma for an IACUC is
that founder and early generation individuals with debilitating phenotypes can represent a precious resource. Breeding
of sufficient numbers of these lines to assess phenotype and
determine its utility is warranted. However, continued maintenance of lines that are not actively studied is difficult to
justify. Whether to allow continued breeding of a line with
well-being problems may be more important than establishing humane endpoints for individual animals.
Strategies for Increasing Animal Welfare
In addition to approving or withholding approval to continue
breeding a particular line with health problems, other methods to preserve a particularly problematic gene or DNA sequence may be considered. In some cases, embryo or embryonic stem cell cryopreservation may be a useful alternative
to continued breeding of animals with well-being problems.
The IACUC can work with a PI to devise ways of altering the
phenotypic expression of a particular problematic gene to
enable its study and characterization. It is possible to experiment with strategies such as changing the background strain
or treatment interventions. In addition, changing the construct using promoter and regulatory sequences can enable
the PI to turn effects on and off.
The consistency of phenotypic expression within a particular line can be helpful in establishing endpoints based on
the age of the animals. For example, C57BL/6 x 129/Sv mice
rarely have tumors before 1 yr of age. When the tumor suppressor gene p53 is knocked out, mice have high tumor rates
and die by 10 mo of age (Roths et al. 1999). Endpoints for
studies with these animals may be constructed so that the
study is terminated before the age when tumors become a
welfare problem.
Treatment of a condition that compromises the welfare
of the animals can be a useful solution, as was done in dopamine-deficient mice created by inactivating the tyrosine hydroxylase gene in dopaminergic neurons. It was initially
found that inactivation of both alleles results in midgestational lethality. Administration of L-DOPA to pregnant females resulted in complete rescue of mutant mice in utero
(Zhou et al. 1995). Without additional treatment, however,
the DA-/- mice were adipsic and aphagic. They became
hypoactive and runted at 2 to 3 wk of age and died shortly
ILAR Journal
thereafter. After daily administration of L-DOPA, it was possible to maintain and study them (Zhou and Palmiter 1995).
Not all treatments were successful, however, as was revealed
in additional studies using gene therapy with two recombinant adeno-associated viruses expressing the human tyrosine
hydroxylase gene and guanosine 5c-triphosphatecyclohydrolase 1 with these animals. Feeding behavior was restored for
several months; however, locomotion and coordination improved only partially (Szczypka et al. 1999).
Another strategy to potentially improve welfare is to
maintain the line by breeding phenotypically normal heterozygotes. For example, transforming growth factor beta 1deficient null-mutation mice die by 3 wk of age. Heterozygote mothers provide sufficient transforming growth factor
beta 1 to the fetus for normal development, but the homozygous offspring cannot produce their own transforming
growth factor beta-1 and so die after birth (Boivin et al.
1995). The number of mice with health problems can be
reduced by breeding heterozygotes to maintain the line. However, this system may require a larger number of animals,
depending on the number of breeders necessary to perpetuate
the line and whether the wild type offspring produced could
be used. Approximately 25% of the offspring would be homozygous and useful for study, 50% would be heterozygotes
usable for maintaining the line, and 25% of the offspring
produced would be wild type.
Background strain is an important consideration in phenotypic expression of a gene, as seen in the diabetic (db)
mutation in mice. On a C57BL/6J background, db/db mice
are obese and have insulin resistance; however, they do not
develop diabetes (Roths et al. 1999). On the closely related
C57BL/KsJ background, the mice have a severe diabetes
syndrome. Such background differences could be used to
maintain a particularly valuable mutant allele without producing the severely debilitating phenotype.
The age of mice at the time a gene is expressed is another
factor that can vary according to strain background. Homozygous beige (bg/bg) mice differ from littermates only in coat
color until they are 12 to 13 mo of age when on a C3H/He
background. Then, they develop tremors, ataxia, lethargy,
and die. The same syndrome is not seen until 20 to 24 mo of
age, when the same mutation is on a C57BL/6J background
(Murphy and Roths 1978).
Inducible promoters can be used to regulate expression
of genes, enabling limitation of the effects of a gene to a
particular period of time and allowing control over severity
of expression of the induced phenotype. Many promoters
have been used, including tetracycline and its derivative
doxycycline (Shockett and Schatz 1996). When a tetracycline promoter is used, the gene is inactive when tetracycline
is administered and is activated when tetracycline is removed.
Incorporation of a doxycycline control switch into a gene
construct allows animals not fed doxycycline to live without
phenotypic expression of the altered genome. When the animals are fed doxycycline, the effects of the construct will be
elicited, whereas removal of doxycycline turns off the gene
and allows the animal to return to normal.
Volume 41, Number 2
2000
Phenotyping Protocols
Each line of animals with a different number or sequence of
genes has a specific, unique genotype with a potentially novel
and useful phenotype. When a new genetic line is created, its
phenotype must be documented to assess its possible utility
(van der Meer and van Zutphen 1995). The initial phenotyping should include basic data to provide a general picture of
the major characteristics of the line. Such basic data might
include parameters listed in Table 2. Basic data can be
supplemented by results of specialized tests for identifying
more unusual traits or specific interests of the PI. For ex-
Table 2 Sample phenotyping protocol3
1. Morbidity and mortality
A. Fetal death
B. Life span
2. Fertility
A. Litter size at birth
B. Litter size at weaning
3. Development
A. Birth weight
B. Growth rate
C. Hair growth
D. Development of neonatal reflexes
E. Age at incisor eruption
F. Age eyes and ears open
G. Age at standing and walking
4. Clinical parameters
A. Physical examination for malformations
B. Coat condition
C. Nasal or ocular discharge
D. Hemogram
E. Serum chemistry profile
F. Tumor development
5. Simple behavioral parameters
A. Posture, climbing, and locomotion
B. Eating and drinking
C. Grooming
D. Activity level, exploration
E. Alertness
F. Aggression
G. Twitches, tremors
H. Stereotypic behaviors
I. Righting
J. Auditory startle
K. Seizures
L. Reflexes
6. Necropsy and histology
7. Specialized testing
A. T and B cell function
B. Cytokine profile
C. Pathogen susceptibility
D. Complex behavioral testing
E. Learning testing
a
Items can be evaluated and data submitted for each line for which
continued breeding is requested.
97
ample, there is much interest in behavior phenotyping and
protocols for specialized testing in this area have been proposed (Costa 1996; Crawley and Paylor 1997).
As mentioned above, IACUC members can request or
require submission of phenotype data to conduct its review
of a particular line. Table 2 is a basic list of suggested parameters Pis can submit to an IACUC to obtain approval for
continued breeding of lines. The PI may also wish to submit
the results of specialized testing to establish the importance
of a particular line.
Finally, there are some unique safety issues the IACUC
should consider when reviewing protocols involving breeding and housing animals with altered germlines, some of
which may also have an impact on animal welfare. It is important to ensure containment to preclude inadvertent sexual
contact with other animals and to prevent escape of genetically altered animals into the environment. Even donation of
excess animals to zoos and shelters for other animals' food
should not include genetically altered animals. In addition to
control of sexual contact, when replication-deficient viruses
are used for a vector, there is a theoretical danger of horizontal transfer of viruses and genetic material to other animals.
For instance, if an injected animal carried a helper virus, it
might enable the vector virus to replicate and produce clinical disease, produce a recombination creating a new infectious agent, or activate an oncogene (Chong et al. 1998). To
help prevent this, vectors should be tested to ensure that
there is no replication-competent virus before administration
to animals. It is prudent for animals to be contained under
Biosafety Level 2 conditions after gene transfer with replication-deficient virus vectors until it is established that indeed,
there is no virus shedding. There is also a potential danger to
humans from accidental needle sticks.
Summary
Genetic engineering studies are increasing and they offer an
exciting tool for the study of disease processes. Due to the
discovery nature of many of these studies, it is difficult to
predict the outcomes of the genetic manipulations planned.
The result can be creation of new lines of animals with debilitating phenotypes. It is crucial for institutions to supervise and continually review the studies to identify problems
as they occur and to ensure that appropriate, humane endpoints are established. When identified, there are some novel
strategies that can be attempted to resolve or minimize the
impact of problems on compromised animals. Successful
resolution of welfare concerns may be achieved when the PI,
attending veterinarian, and IACUC members cooperate.
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