Comparative Medicine
Copyright 2000
by the American Association for Laboratory Animal Science
Vol 50, No 1
February 2000
Overview
Phenotype Assessment:
Are You Missing Something?
Philip A. Wood
Background and Purpose: Phenotype assessment of genetically modified rodents is an essential component of animal model development and their eventual use. Described here is a paradigm to consider as we collectively pursue
phenotype assessment of the constantly increasing number of genetically modified rodent models, as well as those
with spontaneously occurring mutations.
Methods: Review of past experiences and the literature provides useful examples to illustrate the principles described.
Conclusion: A practical approach to phenotype assessment can be divided into a primary level of assessment to
find abnormalities, and a secondary level of more specialized assessment to quantify and evaluate the abnormalities detected. There are many subtle, but important phenotypic characteristics that can be markedly affected by
the background genetics and environment of the animal being assessed.
The phenotype of an animal is determined by a complex interaction of genetics and environment. It is the evaluation of phenotype that allows us to determine the usefulness of a mutant
strain as a model for biomedical research. In the present era of
designing genetically altered animals, particularly mice, we are
faced with the daunting task of determining the phenotype.
This requirement for phenotype assessment also applies to the
wealth of spontaneous mutant animals. This assessment requires sorting out genetic effects from environmental ones. A
specific phenotype is usually expected from genetically altered
mice whether they are transgenic over-expression models or
gene knockout models where a particular gene function has
been modified or ablated altogether. Thus, for any given genetic
alteration, we often try to predict what the phenotype will be.
Many times we find the predicted phenotypes and more. It is,
however, common to hear that surprisingly a given model has
“no phenotype” (1). Perhaps an abnormality was missed because
a very exclusive phenotype was all that was looked for. There
have been other recent overviews of rodent models, the techniques involved in their generation and analysis, and specific
phenotypes found and characterized (1, 2, 3). The purpose of
this paper is to describe a general scheme of approaching the
overall phenotype assessment of the large number of genetically
altered or spontaneous mutant mice, as well as other rodent
models currently being developed.
As national mutant mouse resources are being developed,
there is a growing interest in phenotype assessment of large
numbers of mice. An initial “reality check” tells us that we cannot possibly evaluate every mutant mouse line for every possible abnormality. The limitations include the availability of
adequate assessment procedures with high through-put capability, the numbers of mutant and control animals available to
Department of Comparative Medicine, Schools of Medicine and Dentistry,
University of Alabama at Birmingham, Birmingham, Alabama 35294-0019
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obtain a meaningful result, and the surprising costs involved in
determining the overall phenotype. Therefore, we must think in
terms of a paradigm to follow (4). Such programs should be useful during development of regional or national phenotype assessment resource centers. Therefore, a practical approach is
presented to consider for assessment of the many mutant rodent lines being developed. Highlighted here also are several
example phenotypes that were not obvious or presented as
unique environmental situations that markedly affected the
phenotypes being evaluated.
The overall process of phenotype assessment can be divided
into two major components. The first component is a primary
level of assessment where the goal is to simply find abnormalities. This is in effect a screening process. This component can
likely be achieved at most institutions. The second component is
the secondary level assessment where the goal is to quantify
and evaluate the abnormalities found in the primary level of assessment. This will consist of more specialized analyses and will
be much more limited in availability. This component lends itself to development of regional or national resource centers.
The primary level assessment is a fairly straightforward
clinical/pathological assessment that should be available at
most institutions.
General Paradigm for Phenotype Assessment — Adapted
from (4) Primary Level Assessment: Find Abnormalities
1. Clinical Assessment: Transgenic or mutant mice or rats are
frequently found on a mixed genetic background. Many
knockout mice are initially on C57BL/6 x 129/Sv mixed genetic background, therefore controls should be wild-type, gender-matched littermates with a similar mixed background.
a. Evaluate litter size: record number born/weaned, sex and
genotype distribution.
b. Visual observation particularly during the dark cycle
when rodents are most active. Observe for aggressive,
Phenotype Assessment
hyperactive, hypoactive behaviors etc.
c. Examine for any changes in pelage, skeletal or other
body conformational changes, excessive size or failure to
thrive.
2. Pathologic examination: Recommend evaluating weanlings,
retired breeders, and any that are obviously sick.
a. General necropsy to evaluate for any visible lesions and
histopathology of all organs by an experienced rodent pathologist.
b. Test mice or rats for any background infectious disease
that may confuse the phenotype resulting from a genetic
alteration using microbiologic/serologic/parasite evaluation to detect pathogens.
c. Clinical pathology measures such as blood cell counts,
simple urine analysis for protein and glucose.
d. Determine lifespan of the mice or rat line.
e. Re-evaluate phenotypes in old age.
Secondary Level Assessment: Quantify and Evaluate Abnormalities
1. Embryologic evaluation.
a. If abnormal litter size and genotype distribution is observed, these animals should be evaluated for gestational
loss versus neonatal loss. This often requires timed
matings with careful embryologic evaluation to detect
the specific gestational stages when the animals die.
2. Specialized pathologic evaluation.
a. Specialized stains for lesions detected by standard histopathology.
b. Electron microscopy for cellular lesions that are not
discernable at the light microscope level.
c. Further evaluation of any blood cell count abnormalities
with fluorescent-activated cell sorting (FACS) analysis of
leukocytes and other more specific immunologic investigations.
d. Specialized organ assessment, e.g., pathologic evaluation
of heart changes, eye changes, bone changes, analyses of
neuron and neurotransmitter distribution, etc.
3. Specialized biochemical and genetic analyses.
a. Metabolite analyses on blood, urine, tissue extracts for
specific metabolites such as amino acids, lipids, carbohydrates in deficient or excessive concentrations. These assays may require very specialized equipment and
expertise with small sample size.
b. Enzyme or other specific protein analyses. This would include not only assays that demonstrate the presence or
absence of a protein, but also functional assays that may
be essential for substantiating a specific enzyme deficiency indicated by abnormal metabolite assays or blood
cell abnormalities.
c. Hormone analyses. This can be particularly important in
diabetic animals, as well as those with failure to thrive,
small body size, infertility, skeletal abnormalities, behavior abnormalities or skin disease.
d. Genetic analyses. It is often useful to analyze the biochemical changes identified for inheritance patterns to look for
autosomal recessive inheritance patterns as is common
with enzyme deficiencies. The inheritance pattern of phenotype may not follow the expected pattern from the known
mutation involved, e.g., genomic imprinting or other factors
may influence the inheritance pattern.
4. Physiologic assessment.
a. Pathologic evaluation may indicate organ dysfunction
such as hyperplastic or hypertrophic enlargement, atrophy or absence. Technologies are being developed to more
thoroughly assess physiologic function such as miniaturized devices that can transmit data via telemetry for
these valuable physiologic measures in the awake, unrestrained animal. Miniaturized instrumentation for procedures such as ultrasonography, magnetic resonance
imaging, dual-energy X-ray absorptiometer (DEXA)
analyses, indirect calorimetry studies and other such devices are becoming increasingly available for these specialized measures in rodents including those procedures
that are especially difficult in mice.
5. Behavioral assessment.
a. There already are many behavioral differences observed
among the inbred strains of rodents. There are many mutant or transgenic mice and rats that also have abnormal
behavior that need evaluation. These models will allow
pursuit of genotype/phenotype correlations that will be
crucial in elucidating the genetic components of drug
abuse and mental illness. This will require not only the
current behavior assessment paradigms, but also specialized physiologic assessment with location specific
brain implants and EEG type measures.
6. Pathologic effects:
a. Rodents have a wide range of susceptibilities to common
laboratory pathogens, many of which alter biologic responses. This has been well documented for several infectious agents. This points out at least two issues to
consider. One point is that to fully evaluate the phenotype of a new model, it should be documented free of
pathogens that may induce unwanted phenotypes.
b. Secondly, for genetic manipulations involving immune
functions and related effects, the animal’s susceptibility
to even opportunistic pathogens may markedly influence
the phenotype. Thus, when evaluating the phenotype
with a specific intended effect on these systems, this
must be carefully controlled for and assessed.
The secondary level of assessment is much more specialized.
Several common characteristics are listed that frequently require a more in-depth analysis. Physiologic assessment, especially of mice, is currently evolving rapidly primarily due to the
miniaturization of instrumentation. Behavioral assessment is
another area that is very important and is likewise evolving.
On a cautionary note, there are two aspects that are frequently proposed or discussed for widespread mouse phenotype
screening. This includes behavioral testing, which is very important but needs further refinement for use on mice on a massive
screening scale. A standardized set of meaningful tests are
needed that can be done reproducibly in many laboratories. Another major concern with some of the behavioral testing paradigms available is the requirement for high throughput. As an
example, if the behavioral test takes one hr/mouse and the laboratory can only analyze four mice at a time, this will not be a
practical test for a primary level screening procedure. Some of
the current testing methods will work well for specific behavioral traits, but this will only be practical as a secondary level
specialized test.
Another mass screening procedure frequently discussed is a
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Vol 50, No 1
Comparative Medicine
February 2000
gene expression profile on mutant mice on the primary level.
This is both impractical and useless since you may be mostly
looking at a huge number of secondary gene expression effects.
That is, e.g., a gene knockout mutant has a gene targeted for a
heart function abnormality. Taking the approach of blind
screening with a gene chip screen of 30,000 genes to determine
the level of expression from the liver of this mouse you find elevated expression of 200 genes and abnormally low expression
of 100 genes compared to the control. We have certainly obtained a “gene expression phenotype” from this mutant, but
most likely what we have found are a large number of “red herrings” as far as different gene expression signals that will really
be useful. We should consider several points. What are you looking for with this screening approach? What age of animal do you
evaluate? How many tissues and which ones? How many animals are needed to have a significant result? This technology is
currently very expensive and should be reserved for well
planned gene expression experiments, and not be used as a
screening procedure for phenotype assessment. Using the heart
gene mutant mouse example above, let us consider examining
hearts from the male mutants at a given age when they develop
hypertrophy. We are interested specifically to evaluate different
levels of expression of fatty acid oxidation genes as compared
with the glucose oxidation genes. We possibly also find abnormal expression of myosin genes in the hypertrophied hearts.
Thus when we plan the experiment with a large enough number of mice, mutants and controls, with a specific end-result in
mind, this now becomes a worthwhile endeavor.
Environmental influences.
Dietary changes due to unsuspected ingredients or deficiencies may play important roles in expression of phenotypes. Although many rodent diets appear fairly similar, subtle changes
in constituents can have significant effects on the animals.
Some examples include the possible roles that phytoestrogens,
found in soybean and alfalfa-derived protein, may have in
masking gender specific phenotypes. Likewise, studies involving blood pressure evaluation may be significantly affected by
simply changing vendors who supply different quantities of salt
in their diets. Significant alterations in reproductive phenotype
can result with major changes in animal room temperature,
humidity, pheromone effects, and noise.
To follow are examples to demonstrate aspects of the general
principles outlined.
Genetic Background Effects
A mutant mouse model was developed with a CD18 gene
knockout resulting in a deficiency of the ␤-subunit of ␤-2
integrin on the PLJ background (5). This mouse strain has severe psoriasis, even when germ-free. When this mutant allele is
on the C57BL/6 background there are no observable skin abnormalities (5). Another common genetic and environmental effect
to recognize is that genetic background determines the wide
range of susceptibility or resistance to common infectious disease agents (6). As an example there is a wide range and severity of lesions found in different inbred strains of mice when
experimentally infected with Mycoplasma pulmonis. C57BL/6
mice are very resistant to lung disease, while DBA and C3H
mice are extremely sensitive to this experimental infection (7).
This is an important issue for two reasons. One reason is the
possible effects on phenotype from indigenous infection altering
the phenotype in susceptible strains more so than resistant
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strains. Secondly, experimental infection would result in a
markedly different phenotype depending on the background
strain used. Thus, background strain can have profound effects
as to whether or not we see an abnormal phenotype.
Inconspicuous But Important Phenotypes
A few years ago we evaluated amino acid metabolism in the
Dahl/Rapp rat models of hypertension (8). We investigated
three different but genetically related rat strains(the wild-type
Sprague-Dawley strain, the salt resistant and salt sensitive
Dahl/Rapp rat models of hypertension.) These rats were maintained on either a low or high salt diet that affected their blood
pressures. Sanders and colleagues had discovered previously (9)
that the blood arginine concentrations were significantly different among these strains. The blood arginine variation correlated with changes in blood pressure, i.e., the resistant rat
strain maintained a normal blood pressure with a concomitant
increase in blood arginine, while the sensitive strain did not.
Serum amino acid analysis is a fairly specialized evaluation and
at the time not a blatantly obvious one to consider in this disease. This turned out to be a nitric oxide-mediated mechanism
of blood pressure control and the arginine played a pivotal role.
Further specialized enzymatic studies of mechanism have not
fully uncovered the metabolic basis of increasing blood arginine
(8). This also emphasizes that rat models like these can be sensitive to unknown changes in salt concentrations, e.g., when rodent food vendors are changed.
What is the Target Phenotype for Developing New Models?
Sometimes previously established models can bias our expectations of phenotype for developing new models. As an example
there is a lot of interest in screening mouse mutants for finding
new models of obesity. What are we looking for? That is, we
should consider the common obesity characteristics found in
humans and use those characteristics to guide what we are
looking for in new mouse models. We often get a mindset that
we should only be looking for obese mutants like the famous
leptin deficient obese mutant mouse model (10). We find that at
about any age, obese mutants have approximately three times
the body weight of wild-type controls. Obesity found in people,
however, is not usually this excessive. This would be modeling
human obesity in situations where the patient would weigh
450-500 pounds. These are extremely rare obesity situations in
human medicine. Therefore, more subtle obesity mouse models
may be very important, e.g., obesity phenotypes in the range of
25-50% overweight.
More Subtle and Important Phenotypes in Models of
Fatty Acid Oxidation Deficiency
The next series of examples concerns our research in the area
of mitochondrial ␤-oxidation of fatty acids (11). The emphasis
here is the enzymatic deficiency of the first step of mitochondrial ␤-oxidation of fatty acids, the acyl-CoA dehydrogenase deficiency diseases. The acyl-CoA dehydrogenase step is catalyzed
by four chain-length specific acyl-CoA dehydrogenase enzymes.
Here two of the four enzymes will be discussed, namely short
chain acyl-CoA dehydrogenase (SCAD) and long-chain acyl-CoA
dehydrogenase (LCAD). The first mouse model we identified in
this pathway was the SCAD deficient mouse (12). It has all of
the biochemical and molecular features confirming the enzyme
deficiency and related metabolic abnormalities (12, 13). This
spontaneously occurring deficiency was found in the BALB/c
subline, BALB/cByJ. This is a standard inbred strain sold by the
Phenotype Assessment
Jackson Laboratory. There are two points to this example. All
BALB/cByJ mice currently obtained from the Jackson Laboratory have the inborn error of fatty acid oxidation of SCAD deficiency. So many investigators believe they are ordering a
“normal” BALB/c mouse, but it has at least this one significant
metabolic abnormality. These mice also are behaviorally very
different than the BALB/cJ mice. BALB/cByJ mice are very docile, while BALB/cJ mice are much more hyperactive, and the
males are especially aggressive. This is of significant practical
importance. The Jackson Laboratory catalog shows that one
cannot order BALB/cJ males after 4 weeks of age because of
their severely aggressive behavior. Therefore, BALB/c mice can
be very different, including inherent traits that can profoundly
affect the net phenotype.
Finally, the LCAD deficient mouse model provides interesting
examples of phenotype assessment (14). This mouse was developed by gene knockout procedures and resulted in a mouse
model with no detectable LCAD activity (14). One of the first
abnormalities we found in these mutants was abnormally small
litter sizes along with an abnormal genotype distribution of the
LCAD mutant allele. By crossing parents heterozygous for
LCAD deficiency, we found small litter sizes of 5.8 pups/litter as
compared to over 8 pups/litter in our control matings (14). We
genotyped 479 of the pups and found a significantly abnormal
distribution of only 60% of the expected number of homozygous
pups and surprisingly, only 45% of the expected heterozygous
pups (14). The mechanisms for this gestational loss are currently unknown.
Another example of an unexpected phenotype that has profound effects was discovered while studying mitochondrial uncoupling protein-1 (UCP-1). Through a series of studies done by
Kozak and colleagues, they discovered that the BALB/cByJ
mice described earlier were profoundly cold intolerant like mice
with UCP-1 deficiency (15). They mapped the cold intolerance
trait to a genetic region on chromosome 5 that included the
SCAD gene (Acads) (16). Since we also had LCAD deficient
mice, together we decided to test the hypothesis that mitochondrial fatty acid oxidation provides the energy required for
nonshivering thermogenesis by brown fat. This would further
support the role SCAD deficiency had in their original observation of cold intolerance. These experiments demonstrated that
LCAD deficiency also caused a severe cold intolerance, and thus
showed that complete acyl-CoA dehydrogenation and presumably fully intact fatty acid oxidation is essential for
nonshivering thermogenesis (16).
Conclusion
In summary, several issues related to determining the phenotypes of genetically altered rodent models have been reviewed.
A paradigm was suggested that included a primary level of assessment be used to detect abnormalities, followed by a secondary level of more specialized assessment. This will be required
to quantify and evaluate the abnormalities found in the initial
screening. Most biomedical research institutions will have the
expertise needed to do the primary level assessment. In contrast, the secondary level assessment is more specialized and
could be organized in regional or national centers to provide the
expertise required to gain the full benefits of genetically modified rodent models. Several examples were provided to illus-
trate the important, but sometimes overlooked influences of genetic background and environment on phenotype. Some phenotypic characteristics are not always obvious to look for, such as
abnormal amino acid metabolism in hypertension or cold intolerance in a standard inbred mouse strain (BALB/cByJ). Finally,
environmental factors can profoundly influence the net phenotype
such as common influences like animal room conditions to more
cryptic situations such as indigenous pathogen infection, or dietary
characteristics such as salt or phytoestrogen concentrations.
Acknowledgements
I thank my many colleagues who contributed to the studies reviewed
here. Many of the studies described as examples were supported in
part by NIH grants RR02599 and RR00463.
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