Humane Endpoints for Genetically Engineered Animal Models

Melvin B. Dennis, Jr.

Introduction manipulate and modify the genome. Gene therapy involves

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incorporating a normal gene sequence into a viral or nonviral

I
n the early 1980s, the introduction of transgenic mice revo- vector that can carry it into target cells to replace a defective
lutionized the production of genetically altered animals. sequence. The replacement gene(s) and any necessary regu-
Before then, the methods for modifying the genome of latory sequences to be inserted are spliced into the vector,
animals were spontaneous and induced mutagenesis, hybrid- such as replication-deficient retroviruses, adenoviruses, or
ization, and selective breeding. These tools were used in herpes viruses (Makrides 1999). Other vector work uses rep-
agriculture to improve quality and yield of products and in lication-competent viruses (e.g., herpes simplex and vaccinia
biomedical research to produce models for the study of physi- virus), which are administered at a low dose to produce sub-
ologic and pathologic processes. Mutagenesis remains a com- clinical infection while achieving gene transfer. Nonviral
monly used instrument and is used to generate large numbers vectors such as cationic liposomes are also being developed
of experimental animals. In addition, transgenic technology to accomplish gene transfer (Liu et al. 1995).
expanded the ability to manipulate the genome and dramati- Determination of endpoints for mutant animals is not
cally increased the number of studies involving use of ge- appreciably different than for animals in other studies. The
netically engineered animals. It has facilitated efforts to un- objective of the experiments is often to study the effect of
derstand the role of specific genes and study ways to replace overexpression or deletion of a gene of interest on the pres-
defective ones. Proponents of the use of transgenic animals ence, absence, or alteration of a protein, enzyme, or cytokine;
assert that the models produced are more specific and reli- to study a human disease; or to develop a treatment. A robust
able than spontaneous disease models. They maintain that phenotype for the mutant gene that can be readily observed is
their use will increase the yield of medical research while helpful. Death is not usually intended in genetic engineering
eventually reducing the numbers of animals used. Opponents studies, but lethality or animals with severe health problems
believe the technology is wasteful because large numbers of are commonly encountered. However, whereas the goal of
donor, recipient, and breeder mice, as well as a large percent- agricultural research is to produce a normal animal with su-
age of nontransgenic offspring, are euthanized and do not perior qualities, the goal of biomedical research is often to
produce usable data. Opponents also charge that some of study the animals with induced abnormalities. Genetically
these animals may experience significant pain and distress as engineered animals with decreased ability to resist disease,
a result of the genetic alternatives. with increased tumor production, or that have compromised
Genetic engineering involves insertion, deletion, or al- basic bodily functions such as eating or breathing are not
teration of a segment(s) of DNA followed by observation of unusual. Establishing humane endpoints in such studies can
the effects in the animal and/or offspring. Methodologies present challenges, depending on the particular phenotype of
include manipulation of the genome by (1) pronucleus mi- the genetic line. The situation is complicated by the un-
croinjection to produce overexpression of a gene(s); (2) tar- predictability of genetic manipulation and the variety of phe-
geted mutagenesis in embryonic stem cells to knock out or notypic expressions possible. It is aided by the consistency
inactivate genes; and (3) introduction of new genetic mate- of phenotypic expression within a line and the ability to ma-
rial, including promoter and regulatory sequences via vari- nipulate expression of problem genes. These and other fac-
ous vectors. Descriptions of the procedures used are avail- tors that should be considered when establishing humane
able (Pinkert 1994; Silver 1995). endpoints for genetically engineered animal models are dis-
New fields of study, including xenotransplantation and cussed below in the context of oversight by the attending
gene therapy, have been fostered by the increased ability to veterinarian and the institutional animal care and use com-
mittee (IACUC1).

Melvin B. Dennis, Jr., DVM, is Professor and Chairman of the Department

of Comparative Medicine, School of Medicine, University of Washington, IACUC Review of Genetic Engineering
Seattle, Washington, Studies
'Abbreviations used in this article: IACUC, institutional animal care and In the United States, the IACUC has oversight of animal use
use committee; PI, principal investigator. in a framework of regulations, policies, and guidelines. The

94 ILAR Journal
Department of Agriculture has published regulations (CFR There is also a need for ongoing monitoring of succeed-
1998) that currently apply only to studies involving species ing generations of genetically altered progeny as was illus-
other than rats, mice, and birds. Provisions in the Guide for trated in a line of transgenic pigs with high levels of bovine
the Care and Use of Laboratory Animals (NRC 1996) apply growth hormone. The first two generations had improved
to all vertebrate animals. I have discussed regulation of ani- feed conversion efficiency and low levels of subcutaneous
mal research in the United States (Dennis and Van Hoosier fat; however, subsequent generations were characterized by
1994) and of general IACUC review of genetic engineering ulcers, arthritis, nephritis, cardiomegaly, and infertility
protocols (Dennis 1999). Guidelines for Research Involving (Pursel et al. 1989). In some lines, problems may not become
Recombinant DNA (CFR 1998; Federal Register 1994) pro- apparent until the gene of interest is homozygous. This oc-
vides direction for oversight of genetic engineering experi- curred in mice transfected with a drosophila heat shock gene
ments. Institutions with animal studies involving introduc- (hsp70) and a herpesvirus thymidine kinase gene. First gen-
tion of recombinant DNA into the germline of animals are eration heterozygotes appeared phenotypically normal, but

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required to have an institutional biosafety committee and an F 2 homozygotes had loss of hind limbs, malformed fore-
animal containment specialist. Most studies in which the limbs, facial clefts, and olfactory lobe defects (McNeish et
animal's genome is altered by the stable introduction of re- al. 1988).
combinant DNA into the germline require approval by both Even after producing several lines using a particular gene
the institutional biosafety committee and the IACUC. construct, the phenotype of subsequent lines cannot always
Genetic engineering protocols present the IACUC with a be predicted accurately. One group made multiple lines of
unique set of problems with regard to humane endpoints. lck-IL-4 transgenic mice without the occurrence of observ-
IACUC members have traditionally asked the principal in- able problems. In a subsequent line, however, it was ob-
vestigator (PI1) to list anticipated experimental outcomes and served that animals 3 to 6 mo of age became progressively
to describe how situations involving pain or distress will be humpbacked. Further study revealed that the animals had
addressed. Investigators may be able to provide this informa- osteoporosis (Lewis et al. 1993). This effect could have re-
tion in situations where animals with a well-characterized sulted from incorporation of the gene construct in different
genome are used, but they may not when new lines are to be numbers or into different sites than had previously occurred.
generated. Manipulations of the genome can involve a shift In this case, an unanticipated outcome produced a useful
away from the traditional hypothesis-driven methodology to model. The case also illustrates that the search for models of
one of discovery. Often the hypothesis may propose that the human and animal diseases may require the continued breed-
genetic alteration will cause significant effects that cannot be ing of compromised animals until they are fully character-
predicted accurately. For instance, a gene of interest is over- ized. However, in principle IX of the US Government Prin-
expressed, knocked out, or inactivated, and the offspring are ciples for the Utilization and Care of Vertebrate Animals
carefully observed to learn the effects of the manipulation. 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
Unanticipated Outcomes IACUC. However, it is incumbent on the investigator to de-
scribe how new conditions involving pain and distress will
A significant problem in establishing humane endpoints for be handled, including establishment of appropriate and hu-
genetic engineering studies is the occurrence of unantici- mane endpoints.
pated adverse outcomes. Incorporation of variable numbers
of strands of heterologous DNA into a variable number of
insertion sites can produce innumerable outcomes, many of Protocol Oversight
which are unpredictable. An example of this incorporation
occurred in a study involving the pronuclear injection of the To adequately meet its responsibilities for oversight of ge-
gene for human growth hormone into mice. One would pre- netic engineering studies, it is advisable for an institution to
dict that the offspring overexpressing human growth hor- have a system that will identify animal welfare problems so
mone would be larger and heavier than the parent mice. In they may be dealt with in an effective and timely fashion.
addition, however, some of the transgenic lines also had in- The IACUC review and approval processes should be fol-
creased liver failure, kidney dysfunction, tumor develop- lowed by surveillance of the animals involved in the study
ment, infant and juvenile mortality, shortened lifespan, re- and periodic reviews of the welfare of the animals being
duced fertility, and structural changes in the heart and spleen produced.
(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 moni- Initial IACUC Review
toring of the progeny. If only the anticipated outcomes are
considered, animals with severe problems could be produced If a proposed study involves use of a strain that has been
for which there is no adequate plan for addressing pain and characterized, the PI should be expected to predict outcomes
distress issues. and provide clearly defined endpoints for animals found to

Volume 4 1 , Number 2 2000 95

be in pain or distress. The welfare issues can be readily an- notype is known. IACUC members should ask questions such
ticipated in well-defined transgenic and knockout animals, as, what morbidity or mortality is associated with the pheno-
many of which are available commercially. However, there type of this line, what endpoints will be used when animals
are other situations with a need for in-house breeding of lines develop painful or distressful conditions, or is there evidence
that have not been well characterized. Additionally, pheno- of health problems in this line of animals. The results of
type information will not be available for newly developed phenotyping could be submitted to the IACUC, which would
lines. In these cases, the PI may be asked to predict outcomes review the data and grant or withhold approval for the con-
based on what is known about the gene(s) of interest or re- tinued breeding of the line. Additional data of potential rel-
lated genes. In most situations, the PI will be the best person evance to the review (i.e., immune competency or disease
to predict outcomes because he or she is most knowledge- model data) could also be submitted.
able about the gene. The task of the IACUC is to weigh animal welfare con-
siderations and the potential utility of a line to decide whether

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to allow continued breeding. The dilemma for an IACUC is
Surveillance that founder and early generation individuals with debilitat-
ing phenotypes can represent a precious resource. Breeding
Because outcomes are often unpredictable, the IACUC can of sufficient numbers of these lines to assess phenotype and
use surveillance or monitoring of ongoing studies to ensure determine its utility is warranted. However, continued main-
adequate oversight of welfare considerations. Such surveil- tenance of lines that are not actively studied is difficult to
lance can be accomplished by using a morbidity and mortal- justify. Whether to allow continued breeding of a line with
ity reporting program with feedback to the IACUC proceed- well-being problems may be more important than establish-
ing through the attending veterinarian. In my institution, the ing humane endpoints for individual animals.
program resulted in identification of transgenic and knock-
out lines with many unexpected adverse outcomes, examples
of which are listed in Table 1. As problems are identified, Strategies for Increasing Animal Welfare
IACUC and veterinary personnel meet with the PI to discuss
the situation and devise strategies to improve welfare or es- In addition to approving or withholding approval to continue
tablish endpoints. breeding a particular line with health problems, other meth-
ods to preserve a particularly problematic gene or DNA se-
quence may be considered. In some cases, embryo or embry-
Continuing Review onic stem cell cryopreservation may be a useful alternative
to continued breeding of animals with well-being problems.
Rereview of animal use protocols is required annually by the The IACUC can work with a PI to devise ways of altering the
US Department of Agriculture and every 3 yr by US Public phenotypic expression of a particular problematic gene to
Health Service policy. Approval to continue breeding a par- enable its study and characterization. It is possible to experi-
ticular line beyond a minimal number required for mainte- ment with strategies such as changing the background strain
nance and basic phenotyping could be accomplished during or treatment interventions. In addition, changing the con-
these required reviews. Due to the high frequency of occur- struct using promoter and regulatory sequences can enable
rence of unexpected adverse outcomes, it seems prudent to the PI to turn effects on and off.
require Pis to return to the IACUC for approval of continued The consistency of phenotypic expression within a par-
breeding of each genetically altered line as soon as the phe- ticular 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 sup-
pressor 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
Table 1 Examples of adverse outcomes identified study is terminated before the age when tumors become a
in genetic engineering experiments welfare problem.
Treatment of a condition that compromises the welfare
of the animals can be a useful solution, as was done in dopam-
Allergic encephalomyelitis anasarca
Arterial wall calcification ine-deficient mice created by inactivating the tyrosine hy-
Diabetes droxylase gene in dopaminergic neurons. It was initially
Dopamine deficiency found that inactivation of both alleles results in midgesta-
Epilepsy tional lethality. Administration of L-DOPA to pregnant fe-
Hydrocephalus males resulted in complete rescue of mutant mice in utero
Increased tumor incidence (Zhou et al. 1995). Without additional treatment, however,
Malocclusion the DA-/- mice were adipsic and aphagic. They became
Osteoporosis hypoactive and runted at 2 to 3 wk of age and died shortly

96 ILAR Journal
thereafter. After daily administration of L-DOPA, it was pos- Phenotyping Protocols
sible to maintain and study them (Zhou and Palmiter 1995).
Not all treatments were successful, however, as was revealed Each line of animals with a different number or sequence of
in additional studies using gene therapy with two recombi- genes has a specific, unique genotype with a potentially novel
nant adeno-associated viruses expressing the human tyrosine and useful phenotype. When a new genetic line is created, its
hydroxylase gene and guanosine 5c-triphosphatecyclohydro- phenotype must be documented to assess its possible utility
lase 1 with these animals. Feeding behavior was restored for (van der Meer and van Zutphen 1995). The initial phenotyp-
several months; however, locomotion and coordination im- ing should include basic data to provide a general picture of
proved only partially (Szczypka et al. 1999). the major characteristics of the line. Such basic data might
Another strategy to potentially improve welfare is to include parameters listed in Table 2. Basic data can be
maintain the line by breeding phenotypically normal het- supplemented by results of specialized tests for identifying
erozygotes. For example, transforming growth factor beta 1- more unusual traits or specific interests of the PI. For ex-

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deficient null-mutation mice die by 3 wk of age. Heterozy-
gote mothers provide sufficient transforming growth factor
beta 1 to the fetus for normal development, but the homozy-
gous offspring cannot produce their own transforming Table 2 Sample phenotyping protocol3
growth factor beta-1 and so die after birth (Boivin et al.
1995). The number of mice with health problems can be
1. Morbidity and mortality
reduced by breeding heterozygotes to maintain the line. How-
A. Fetal death
ever, this system may require a larger number of animals,
B. Life span
depending on the number of breeders necessary to perpetuate 2. Fertility
the line and whether the wild type offspring produced could A. Litter size at birth
be used. Approximately 25% of the offspring would be ho- B. Litter size at weaning
mozygous and useful for study, 50% would be heterozygotes 3. Development
usable for maintaining the line, and 25% of the offspring A. Birth weight
produced would be wild type. B. Growth rate
Background strain is an important consideration in phe- C. Hair growth
notypic expression of a gene, as seen in the diabetic (db) D. Development of neonatal reflexes
E. Age at incisor eruption
mutation in mice. On a C57BL/6J background, db/db mice
F. Age eyes and ears open
are obese and have insulin resistance; however, they do not G. Age at standing and walking
develop diabetes (Roths et al. 1999). On the closely related 4. Clinical parameters
C57BL/KsJ background, the mice have a severe diabetes A. Physical examination for malformations
syndrome. Such background differences could be used to B. Coat condition
maintain a particularly valuable mutant allele without pro- C. Nasal or ocular discharge
ducing the severely debilitating phenotype. D. Hemogram
The age of mice at the time a gene is expressed is another E. Serum chemistry profile
factor that can vary according to strain background. Homozy- F. Tumor development
gous beige (bg/bg) mice differ from littermates only in coat 5. Simple behavioral parameters
color until they are 12 to 13 mo of age when on a C3H/He A. Posture, climbing, and locomotion
B. Eating and drinking
background. Then, they develop tremors, ataxia, lethargy,
C. Grooming
and die. The same syndrome is not seen until 20 to 24 mo of D. Activity level, exploration
age, when the same mutation is on a C57BL/6J background E. Alertness
(Murphy and Roths 1978). F. Aggression
Inducible promoters can be used to regulate expression G. Twitches, tremors
of genes, enabling limitation of the effects of a gene to a H. Stereotypic behaviors
particular period of time and allowing control over severity I. Righting
of expression of the induced phenotype. Many promoters J. Auditory startle
have been used, including tetracycline and its derivative K. Seizures
doxycycline (Shockett and Schatz 1996). When a tetracy- L. Reflexes
cline promoter is used, the gene is inactive when tetracycline 6. Necropsy and histology
7. Specialized testing
is administered and is activated when tetracycline is removed.
A. T and B cell function
Incorporation of a doxycycline control switch into a gene B. Cytokine profile
construct allows animals not fed doxycycline to live without C. Pathogen susceptibility
phenotypic expression of the altered genome. When the ani- D. Complex behavioral testing
mals are fed doxycycline, the effects of the construct will be E. Learning testing
a
elicited, whereas removal of doxycycline turns off the gene Items can be evaluated and data submitted for each line for which
and allows the animal to return to normal. continued breeding is requested.

Volume 41, Number 2 2000 97

ample, there is much interest in behavior phenotyping and CFR [Code of Federal Regulations]. 1998. Title 9 (Animals and Animal
protocols for specialized testing in this area have been pro- Products), Subchapter A (Animal Welfare) (9 CFR 1-4).
Chong H, Starkey W, Vile RG. 1998. A replication-competent retrovirus
posed (Costa 1996; Crawley and Paylor 1997).
arising from a split-function packaging cell line was generated by re-
As mentioned above, IACUC members can request or combination events between the vector, one of the packaging constructs,
require submission of phenotype data to conduct its review and endogenous retroviral sequences. J Virol 72:2663-2670.
of a particular line. Table 2 is a basic list of suggested param- Costa P. 1996. Neuro-behavioural tests in welfare assessment of transgenic
eters Pis can submit to an IACUC to obtain approval for animals. In: O'Donoghue PN, ed. Sixth FELASA Symposium: Har-
monization of Laboratory Animal Husbandry. London. Royal Society of
continued breeding of lines. The PI may also wish to submit Medicine Press, p 51-53.
the results of specialized testing to establish the importance Crawley JN, Paylor R. 1997. A proposed test battery and constellations of
of a particular line. specific behavioral paradigms to investigate the behavioral phenotypes
Finally, there are some unique safety issues the IACUC of transgenic and knockout mice. Horm Behav 31:197-211.
should consider when reviewing protocols involving breed- Dennis MB Jr. 1999. Institutional animal care and use committee review of

Downloaded from [Link] by guest on 09 December 2018

genetic engineering. In: Gonder JC, Prentice ED, Russow LM, editors.
ing and housing animals with altered germlines, some of Genetic Engineering and Animal Welfare: Preparing for the 21st Cen-
which may also have an impact on animal welfare. It is im- tury. Greenbelt MD: SCAW. p 20-31.
portant to ensure containment to preclude inadvertent sexual Dennis MB Jr, Van Hoosier GL Jr. 1994. North American legislation and
contact with other animals and to prevent escape of geneti- regulation of the use of live animals in scientific research. In: Svendsen
cally altered animals into the environment. Even donation of P, Hau J, editors. Selection and Handling of Animals in Biomedical
Research. Vol 1 of Handbook of Laboratory Animal Science. CRC Press,
excess animals to zoos and shelters for other animals' food p 23-36.
should not include genetically altered animals. In addition to Federal Register. 1994. Guidelines for Research Involving Recombinant
control of sexual contact, when replication-deficient viruses DNA. Vol 59, July 5, Separate Part IV.
are used for a vector, there is a theoretical danger of horizon- Lewis DB, Liggitt HD, Effmann EL, Motley ST, Teitelbaum SL, Jepsen KJ,
tal transfer of viruses and genetic material to other animals. Goldstein SA, Bonadio J, Carpenter J, Perlmutter RM. 1993. Osteoporo-
sis induced in mice by overproduction of interleukin 4. Proc Natl Acad
For instance, if an injected animal carried a helper virus, it
SciUS A 90:11618-11622.
might enable the vector virus to replicate and produce clini- Liu Y, Liggitt D, Zhong W, Tu G, Gaensler K, Debs R. 1995. Cationic
cal disease, produce a recombination creating a new infec- liposome-mediated intravenous gene delivery. J Biol Chem 270:24864-
tious agent, or activate an oncogene (Chong et al. 1998). To 24870.
help prevent this, vectors should be tested to ensure that Makrides SC. 1999. Components of vectors for gene transfer and expression
in mammalian cells. Protein Exp Pur 17:183-202.
there is no replication-competent virus before administration
McNeish JD, Scott WJ, Potter SS. 1988. Legless, a novel mutation found in
to animals. It is prudent for animals to be contained under PHT1-1 transgenic mice. Science 241:837-839.
Biosafety Level 2 conditions after gene transfer with replica- Murphy ED, Roths JB. 1978. Purkinje cell degeneration, a late effect of
tion-deficient virus vectors until it is established that indeed, beige mutations in mice. Annu Rep Jackson Lab 49:108-109.
there is no virus shedding. There is also a potential danger to NRC [National Research Council]. 1996. Guide for the Care and Use of
Laboratory Animals. Washington DC: National Academy Press.
humans from accidental needle sticks.
Pinkert CA. Editor. 1994. Transgenic Animal Technology: A Laboratory
Handbook. Orlando: Academic Press.
Pursel VG, Pinkert CA, Miller KF, Bolt DJ, Campbell RG, Palmiter RD,
Summary Brinster RL, Hamer RE. 1989. Genetic engineering of livestock. Sci-
ence 244:1281-1288.
Roths JB, Foxworth WB, McArthur MJ, Montgomery CA, Kier AB. 1999.
Genetic engineering studies are increasing and they offer an Spontaneous and engineered mutant mice as models for experimental
exciting tool for the study of disease processes. Due to the and comparative pathology: History, comparison, and developmental
discovery nature of many of these studies, it is difficult to technology. Lab Anim Sci 49:12-34.
predict the outcomes of the genetic manipulations planned. Shockett PE, Schatz DG. 1996. Diverse strategies for tetracycline-regulated
The result can be creation of new lines of animals with de- inducible gene expression. Proc Natl Acad Sci U S A 93:5173-5176.
Silver LM. 1995. Mouse Genetics: Concepts and Applications, New York:
bilitating phenotypes. It is crucial for institutions to super- Oxford University Press.
vise and continually review the studies to identify problems Szczypka MS, Mandel RJ, Donahue BA, Snyder RO, Leff SE, Palmiter RD.
as they occur and to ensure that appropriate, humane end- 1999. Viral gene delivery selectively restores feeding and prevents le-
points are established. When identified, there are some novel thality of dopamine-deficient mice. Neuron 22:167-178.
strategies that can be attempted to resolve or minimize the van der Meer M, van Zutphen LFM. 1995. Use of transgenic animals and
welfare considerations. In: van Zutphen LFM, van der Meer M, editors.
impact of problems on compromised animals. Successful Welfare Aspects of Transgenic Animals. Berlin: Springer-Verlag. p 78-
resolution of welfare concerns may be achieved when the PI, 89.
attending veterinarian, and IACUC members cooperate. Wolf E, Wanke R. 1995. Growth hormone overproduction in transgenic
mice: Phenotypic alterations and deduced animal models. In: van
Zutphen LFM, van der Meer M, eds. Welfare Aspects of Transgenic
Animals. Berlin: Springer-Verlag. p 26-47.
References Zhou QY, Palmiter RD. 1995. Dopamine-deficient mice are severely
hypoactive, adipsic, and aphagic. Cell 83:1197-1209.
Boivin GP, O'Toole BA, Ormsby IE, Diebold RJ, Eis MJ, Doetschman T, Zhou QY, Quaife CJ, Palmiter RD. 1995. Targeted disruption of the tyrosine
Kier AB. 1995. Onset and progression of pathological lesions in trans- hydroxylase gene reveals that catecholamines are required for mouse
forming growth factor-beta 1-deficient mice. Am J Pathol 146:276-288. fetal development. Nature 13:640-643.

98 ILAR Journal