MEDG520
Block 7
Behavioural Genetics
Concepts:
 What is behaviour?
 Explain and give examples of how genetic and environmental factors can interact to
cause disease
o Gene-Environment Interaction
o Cancer as a model for gene/environment interaction
o Carcinogen metabolizing enzymes
 Explain how a multifactorial trait is recognized
o What is multifactorial inheritance?
o multifactorial traits
o How to determine how many genes are involved in a trait
 Describe X-linked inheritance
o X-linked Dominant
o X- linked Recessive
 Describe how genetic heterogeneity and phenocopies can confound the
interpretation of familial patterns in common conditions
 Describe the importance of defining a clinical phenotype in determining the genetic
etiology of common conditions
 Regression analysis
 Founder Mutation
 Mouse Technology
 ES cells and chimeric mice
 Transgenic mice
o What is Transgenesis and how does the term apply to mouse technology
o How are transgenic mice used?
o More Transgenic Mouse info.
 Two methods of producing transgenic mice are widely used:
o The Embryonic Stem Cell Method (Method "1")
o The Pronucleus Method (Method "2")
 Gene targeting
 Knockins
 Knockouts
 Description of random insertion transgenic mice
 Description of targeted insertion transgenic mice
 Random vs. Targeted Gene Insertion
 Explanation of the conditional targeted mutations
 Describe quantitative trait loci and their mapping
o A Quick Guide to QTLs
 What is the difference between QTLs and Mendelian mutants?
 What is QTL mapping?
 What statistical issues must be considered?
o QTLs in Biology: What they are and why they are worth mapping
o Mapping Quantitative Trait Loci (QTL)
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o Types of QTL mapping:
o Statistical methods
Describe how studies in laboratory animals can be used to identify genes involved in
complex diseases
There are 2 classic animal model methods that are especially useful
o Inbred stains
o Selective inbreeding
Animal Models and Genetics of Alcoholism
o Inbred, Selectively Bred, and Recombinant Inbred Strains for Alcoholism
Quantitative Trait Loci (QTL) for alcoholism
o Knockout Animals for Alcoholism
o Contribution of other animal models to genetics of alcoholism…
o Summary
Compare and contrast utility of different animal models available.
1.) Caenorhabditis elegans
2.) Zebra Fish
3.) Mouse
4.) Drosophila
5.) The cat as an animal model for human hereditary disease
6.) Fugu Rubripes (Pufferfish)
How closely related are mice and humans? How many genes are the same?
What are the comparative genome sizes of humans and other organisms being
studied?
Describe what is meant by a “candidate gene approach”
o Strategies for the Candidate Gene Approach
o Advantages of the Candidate Gene Approach
o Disadvantages of the Candidate Gene Approach
Discuss why genetic research into areas like behaviour or intelligence often evokes
public concern
Genetic research into these areas evokes public concern not so much because of
what the research will find, but more, the use of that information
Assigned Papers
o Cases et al., 1995
o Brunner et al. 1993
o Caspi et al. 2002.
o Ikemoto et al. 1997.
o Young et al. 2002.
o Abrahams et al. 2002.
Useful links
Definitions
What is behaviour?
 What we and other species do. Or, more strictly, what we see others doing.
 Thus, asthma and diabetes could be considered behavioural traits. We usually limit study
to behaviours that are more directly the result of brian processes.
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Even this can be too broad. For example, motor skills would fall under the category of a
brain process but we typically don’t consider unconsciousness in response to drugs a
behaviour.
Once further distinction to make is to require that the behavior be the result of a
psychological process.
This also is difficult to define and thus, most studies are forced to make some simplifying
definitions to assess the behaviour of interest. Eg. Define behavior of interest such as
aggression by record of violent crimes, etc.
Explain and give examples of how genetic and environmental factors can interact to cause
disease
Gene-Environment Interaction
Defined as a change in the direction or magnitude of the effect of a genetic variant when the
environment changes.
There is growing evidence that susceptibility genes for common diseases do not have a primary
etiological role in predisposition to disease, but rather act as response modifiers to exogeneous
factors such as stress, environment, disease and drug intake.
Examples:
In the U.S., African-American people have more vascular disease and dementia than white
people, but both conditions are relatively infrequent in West Africa. It is assumed that the
African population has more genetic variants that harbour a latent susceptibility to the diseases
that are only fully expressed in the US environment.
Some of the authors from the Caspi group (Paper 2 from Nov.18) studied the same cohort of
1037 children and found a functional polymorphism in the promoter region of the serotonin
transporter gene that moderates the influence of stressful life events on depression. They found
two alleles, the short and the long. People homozygous for the short allele displayed more
depressive symptoms than homozygotes of the long allele.
Cancer as a model for gene/environment interaction
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Originally thought to be mostly environmental causes because cancer rarely follows
Mendelian inheritance. Discovery of oncogenes, tumour suppressor genes, DNA repair
genes, have also led to claims that cancer is purely genetic.
Now known that cancer is usually caused by a combination of environmental and genetic
effects. Environment can act through the genetic mechanism of mutation, while genetic
mechanism can modify the effect of environment.
Few highly penetrant ‘cancer causing’ genes have been found (eg. BRCA)
Research has turned to more common genetic alterations that by themselves do not cause
cancer but act in concert with environmental exposures to cause cancer
Carcinogen metabolizing enzymes
 primary candidates for gene-environment interaction studies
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Variants of these genes exist which may affect the exposure of an individual to the
carcinogen they metabolize.
Polymorphisms in these genes can result in enzymes that are less efficient at
metabolizing carcinogens, thus giving the carcinogen more time and concentration to
cause damaging mutations.
Eg. N-acetyltransferase enzymes acetylates heterocyclic amines found in cooked meat
and tobacco. Can be both beneficial or detrimental depending on lifestyle.
Cytochrome P450 superfamily of enzymes – eg. CYP1A1 catabolizes polyaromatic
hydrocarbons including benzo(a)pyrene and other constituents of smoke.
Explain how a multifactorial trait is recognized
What is multifactorial inheritance?
Multifactorial inheritance means that many factors (multifactorial) are involved in causing a birth
defect. The factors are usually both genetic and environmental, where a combination of genes
from both parents, in addition to unknown environmental factors, produce the trait or condition.
Often one gender (either males or females) is affected more frequently than the other in
multifactorial traits. There appears to be a different threshold of expression, which means that
one gender is more likely to show the problem over the other gender. For example, hip dysplasia
is nine times more common in females than males.
Multifactorial traits do recur in families, because they are partly caused by genes. The chance for
a multifactorial trait or condition to happen again depends upon how closely the family member
with the trait is related to you.
multifactorial traits--determined by one or more genes AND by environment. In these cases,
each gene is being inherited in a Mendelian fashion, but the combination of the genes produces a
variable expression of a certain trait. Thus, overall, you do not see the typical ratios of single
gene inheritance.
Height and skin color are both polygenic and multifactorial. If one is deprived of proper
nourishment, one might be short even though one’s genetic makeup predicts tallness.
Environment thus has an effect on realizing the genetic potential. Usually, traits that are due to
multiple genes also are affected by environment.
How to determine how many genes are involved in a trait
When geneticists analyze the frequency with which certain traits appear in the population, they
can determine whether the trait is polygenic or if it is produced by a single gene. They do this by
plotting the frequency with which a trait is seen against the different phenotypes of the trait. For
example, height . Height in pea plants falls into one of two categories--dwarf or tall (called
discontinuous variation), while height in humans shows a bell-shaped distribution (continuous
variation) (fig 7.3). This continuous variation, or bell-shaped curve, is typical of multifactorial
traits.
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The greater the number of genes involved in a particular trait, the wider the bell curve because
there are more possible combinations. Remember, in contrast, in a dihybrid cross for two genes
controlling two distinct phenotyes, there are only 4 distinguishable phenotypic classes (9:3:3:1).
When two or more genes contribute to one trait, one calculates the possible phenotypes based on
the total number of dominant alleles in the genotype (the two genes are contributing to the same
phenotype). Thus, two genes yields 5 possible phenotypes, based only on the number of
dominant alleles. Take eye color as an example. In the 16 genotype grid of a dihybrid cross, 1
genotype will have 4 dominant alleles (AABB) and 1 will have 0 dominant alleles (aabb). 4 will
have 3 dominant alleles, 6 will have 2 dominant alleles, and 4 will have 1 dominant allele (fig
7.4). Again, this produces 5 possible phenotypes.
With three loci, the possibilities expand to 7 combinations, with four, 9. Thus, the more genes
involved in a trait, the greater number of possible phenotypes produced, and the more continuous
the variation.
As more loci are included, this binomial distribution quickly approaches the Gaussian
distribution, or the bell-shaped normal curve, observed with human quantitative traits. Three loci,
each with three alleles, are enough to produce population frequencies indistinguishable from a
normal curve. The multifactorial model is then:
1. Several, but not an unlimited number, loci are involved in the expression of the
trait.
2. There is no dominance or recessivity at each of these loci.
3. The loci act in concert in an additive fashion, each adding or detracting a small
amount from the phenotype.
4. The environment interacts with the genotype to produce the final phenotype.
As an example of 4. above, women are, on average, three inches shorter than men with the same
genome. Environmental factors (hormones) affect the final phenotype.
Not all human traits that show a continuous distribution in the population are multifactorial traits.
Any bimodal distribution is not controlled by multifactorial expression. It is more likely to be
under the control of a single dominant/recessive gene with modifying environmental factors.
Multifactorial traits all show a unimodal bell-shaped distribution.
In summary, the hallmarks for multifactorial inheritance are:
1. Most affected children have normal parents. This is true of diseases and
quantitative traits. Most geniuses come from normal parents, most mentally
challenged come from normal parents.
2. Recurrence risk increases with the number of affected children in a family.
3. Recurrence risk increases with severity of the defect. A more severely affected
parent is more likely to produce an affected child.
4. Consanguinity slightly increases the risk for an affected child.
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5. Risk of affected relatives falls off very quickly with the degree of relationship.
Contrast this with autosomal dominant inheritance with invomplete penetrance,
where the recurrence risk falls off proportionately with the degree of relationship.
6. If the two sexes have a different probability of being affected, the least likely sex, if
affected, is the most likely sex to produce an affected offspring.
Multifactorial inheritance typically results in continuous variation. The more genes involved, the
more classes of phenotypes will result. The environment affects each of these classes and
provides even more diversity of phenotypes--the result is that if we examine a large population,
we see “continuous variation”, rather than a few distinct phenotypes.
Describe X-linked inheritance
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Definition: A Mendelian disease that is encoded by a mutant gene on the X-chromosome.
X-linked diseases are fully expressed in males who have inherited the mutant X.
In females expression is variable due to random X inactivation.
There are 2 forms of X-linked inheritance: Dominant and Recessive
X-linked Dominant
Individuals possessing ONE copy of the mutant gene will be affected. Therefore, it affects both
males and females.
Modes of inheritance:
 A male or female child of an affected mom have a 50% chance of inheriting the mutation.
 All female children of an affected father will be affected.
 NO male children of an affected father will be affected.
X- linked Recessive
Affects individuals with NO normal copy of the gene.
Also characterized by rare clinical expression in heterozygous females.
Modes of inheritance:
 Heterozygous females are CARRIERS and generally don’t display clinical symptoms.
 All males possessing an X-linked recessive mutation are clinically affected.
 All offsprings of a carrier female have 50% chance of inheriting the mutation.
 All female children of affected fathers will be carriers
 No male children of an affected father will be affected.
There is NO male-male transmission in X-linked inheritance. This hallmark feature of Xlinked inheritance is true for both the dominant and recessive forms.
Examples:
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Duchenne Muscular Distrophy (DMD)
 Males are affected
 Disease is transmitted by healthy females who are heterozygous carriers.
 In females, Duchenne is the result of a rare chromosome rearrangement involving X and
an autosomal chromosome.
 Females may show x-linked recessive traits for other reasons such as having only one X
chromosom. Such is the case in Turner Syndrome.
Describe how genetic heterogeneity and phenocopies can confound the interpretation of
familial patterns in common conditions
 Genetic heterogeneity and phenocopies (definitions in Mendelian section) can increase
the complexity of distinguishing familial patterns:
 Linkage studies allow for the mapping of disease genes on the basis of phenotype. It
follows then, that if mutations in more than one gene (ie. Gene 1 and gene 2) can create
the same phenotype (genetic heterogeneity), linkage between disease phenotype and any
given marker in close proximity to gene 1 will be weakened by the fact that several of the
families in your study may be expressing the disease due to a mutation in gene 2. When
one condition can be attributed to a mutation in more than one gene (loci), it is referred to
as locus heterogeneity.
 If genetic heterogeneity occurs as allelic heterogeneity (more than one mutation at the
same locus causes the same condition), the condition may occur at varying degrees of
severity. This may confuse classification of individuals as affected or unaffected.
 Some individuals may not be “symptomatic” and therefore not produce a noticeable
phenotype. You may not distinctly visualize inheritance of a disease, particularily on
first glance and thus may incorrectly score the individual as “unaffected”.
 Multiple degrees of expression of a condition (due to allelic heterogeneity) may exist in a
family, and thus again, individuals may be wrongly labelled “unaffected”, or expression
of the same disease in different individuals may be labelled “unrelated”. Diseases in
which this often occurs are myotonic dystrophy and mental illnesses.
 Inclusion of phenotypic traits not associated with the genetic condition (ie. Phenocopies)
will skew analysis, making the information found only suitable for the particular family
studied.
 A good example of a condition displaying both allelic and locus heterogeneity is
Osteogenesis Imperfecta.
Definitions
 A phenocopy is a trait not caused by inheritance of a gene but mimics the phenotype of a
specific genotype.
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Genetic heterogeneity refers to different mutations that can cause a similar phenotype.
Both of these concepts are well demonstrated by the following example.
Huntington’s Disease (HD) is an autosomal dominant neurodegenerative disease that results in
motor abnormalities and dementia. The HD (CAG)n repeat expansion is a sensitive and specific
marker for HD. However there are a significant number of HD cases where the repeat expansion
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is absent, suggesting that mutations in other genes may be provoking HD-like disorders. These
phenocopies are the result of a mutation in the prion protein (prP) gene (PRNP). Affected
individuals are heterozygous for a 192-nucleotide insertion within the prP coding region. This
HD phenocopy is in fact a familial prion disease with an autosomal dominant pattern of
inheritance and a remarkable range of clinical features many of which overlap with those of HD.
A slightly different situation can also cause a phenocopy in which the phenotype is the result of
an environmental condition. For example, Thalidomide disrupts normal embryonic development
in a manner that mimics the effects of a rare mutation known as phocomelia. Babies with this
genetic disorder are born with one or more abnormal ‘flipper-like’ limbs. Thalidomide causes
similar birth defects in babies with a normal genotype.
Describe the importance of defining a clinical phenotype in determining the genetic etiology
of common conditions
 Contamination of the groups of “affected” and “unaffected” persons may make findings
insignificant (not linked) even if truly significant (linked) or vice versa.
 If a phenotype is included in the criteria for grouping individuals affected which is not
related to the disease (for example, it is a phenocopy), individuals possessing only this
trait will wrongly be classified as affected.
 If a phenotype is not included in the criteria for grouping individuals as affected,
individuals with milder forms of the condition may not be classified as affected and
wrongly classified as unaffected.
 In short, contamination of unaffected and affected groups will skew linkage analysis.
 A good example to use would be Myotonic Dystrophy.
Regression analysis
Regression analysis is a very useful statistical process for forecasting. Its purpose is to take a
series of independent variables and determine whether a particular dependent variable is related
to the independent variables. It can, therefore, be described as the statistical method of finding
the “best“ mathematical model to describe one variable as a function of another.
A regression line displays how well independent and dependent variables fit together. Points are
plotted on a graph, and after regression calculation, a line is drawn that is the "best fit" to the
points on the graph. The closer the line is to each point, the stronger the relationship is between
the independent and dependent variables. Any future forecast on the dependent variable can be
found on the regression line.
In linear regression the relationship is constrained to be a straight line and least-squares analysis
is used to determine the best fit. In logistic regression the dependent variable is qualitative rather
than continuously variable and likelihood functions are used to find the best relationship. In
multiple regression the dependent variable is considered to depend on more than a single
independent variable.
Founder Mutation
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A founder mutation is a mutation that arose in a founder population. As population
expansion occurred, the mutation in the founder population became prevalent in the
larger population.
Founder mutations can be identified because:
1) "Identical mutations in apparently unrelated different families are nonetheless located
on the same genetic background, so that we can assume that these families have had a
common ancestor in the distant past. These mutations are called "founder-mutations",
because they are the "founders" of a "colony" of descendants.
2) The haplotypes in the genomic region containing the mutated gene will be the same
across individuals with the same common ancestor.
3) Identifying founder mutations is valuable in calculating disease risk for the members
of a specific population.
For example: 1300 mutation variations has been identified in the BRCA1 and BRCA2
genes (predisposition to breast cancer genes) of which 700 are causal. A number of these
mutations have been identified repeatedly across populations. For example, the 5382insC
mutation on BRCA1 has been reported in individuals of Jewish, Dutch, Germanic,
Lithuanian, Russian, Hungarian, French, Italian, French Canadian and British ancestries.
This is a classic example of a founder mutation that was spread through human
migration.
Mouse Technology
ES cells and chimeric mice
Embryonic stem (ES) cells are pluripotent cell lines with the capacity of self-renewal and a broad
differentiation plasticity. They are derived from pre-implantation embryos and can be propagated
as a homogeneous, uncommitted cell population for an almost unlimited period of time without
losing their pluripotency and their stable karyotype. Even after extensive genetic manipulation,
mouse ES cells are able to reintegrate fully into viable embryos when injected into a host
blastocyst or aggregated with a host morula. After these pre-implantation embryos are implanted
into a surrogate mother, they develop into mosaic offspring known as chimeras. The tissues of
chimeric mice are comprised of a mixture of cells that originated from both the host embryo and
the ES cells. The contribution of each originating cell population is seen most visibly in the fur,
which is generally striped black (from host cells) and brown (from ES cells). Healthy ES cells
can give rise to descendants in all cell types, including functional gametes to produce more and
more mice containing the desired genetic modification (Thompson et al., 1989). If the
proportion of ES cell descendents in the coat of the animal is high, the probability that ES cells
are represented in gametes is also high, since ES cells mix thoroughly with host cells early in
embryogenesis. ES cells give rise to brown coat color because they are Aw/Aw (dominant
White-bellied Agouti), and the host cells give rise to black coat color because they are a/a
(recessive non-agouti). The ES cells used most commonly are from the 129 strain of mice, while
the host embryos are from the C57BL6 strain of mice. If the chimeras are bred to a/a non-agouti
mice (for example C57BL6 or Black Swiss), then any brown offspring (Aw/a) must have arisen
from ES cell-derived gametes, and 50% of the brown offspring are expected to carry the genetic
modification.
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Transgenic mice
What is Transgenesis and how does the term apply to mouse technology
 By definition, transgenesis is the introduction of DNA from one species into the genome
of another species.
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Many of the first transgenic mice fit this description well as they were generated
to study the overexpression of a human protein, often an oncogene. Currently,
the phrase "transgenic mouse" generally refers to any mouse whose genome
contains an inserted piece of DNA, originating from the mouse genome or from
the genome of another species, and the term includes the standard transgenic
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mouse as well as a knockin or knockout mouse.
A transgenic animal is one that carries a foreign gene that has been deliberately inserted
into its genome. The foreign gene is constructed using recombinant DNA methodology.
In addition to a structural gene, the DNA usually includes other sequences to enable it
o to be incorporated into the DNA of the host.
o to be expressed correctly by the cells of the host.
Transgenic sheep and goats have been produced that express foreign proteins in their
milk.
Transgenic chickens are now able to synthesize human proteins in the "white" of the
eggs.
How are transgenic mice used?
One of the simplest ways to study gene function in a mouse is exogenous expression of a
protein in some or all tissues. For this type of genetic modification, a new piece of DNA is
introduced into the mouse genome. This piece of DNA includes the structural gene of interest, a
strong mouse gene promoter and enhancer to allow the gene to be expressed and vector DNA to
enable the transgene to be inserted into the mouse genome. Successful integration of this DNA
results in the expression of the transgene addition to the wild type, basal protein levels in the
mouse. Depending on the goal of the experiment, the transgenic mouse will exhibit overexpression of a non-mutated protein, expression of a dominant-negative form of a protein, or
expression of a fluorescent-tagged protein.
To generate a standard transgenic mouse, a bacterial or viral vector containing the
transgene and any desired markers are injected into a fertilized mouse egg. The DNA usually
integrates into one or more loci during the first few cell divisions of preimplantation
development. The number of copies of the transgenic fragment can vary from one to several
hundred, arranged primarily in head-to-tail arrays, and the transgenic founder mice are mosaic
for the presence of the transgene. Founders are very likely to have germ cells with the integrated
transgene, and therefore will be able to vertically transmit the integrated gene, and all cells of the
progeny transgenic mouse contain the transgene. This method is relatively quick, but includes
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the risk that the DNA may insert itself into a critical locus, causing an unexpected, detrimental
genetic mutation. Alternatively, the transgene may insert into a locus that is subject to gene
silencing. If the protein being expressed from the transgene causes toxicity, excessive
overexpression from multiple insertions can be lethal to some tissues or even to the entire mouse.
For these reasons, several independent lines mice containing the same transgene must be created
and studied to ensure that any resulting phenotype is not due to toxic gene-dosing or to the
mutations created at the site of transgene insertion.
Transgenic Mouse info from http://darwin.bio.uci.edu/~tjf/tmf_tgms.html
A transgenic mouse (or any other species) is simply an organism that has had DNA
introduced into one or more of its cells artificially. This is commonly done in one of two ways.
DNA can be integrated in a random fashion by injecting it into the pronucleus of a fertilized
ovum. In this case, the DNA can integrate anywhere in the genome, and multiple copies often
integrate in a head-to-tail fashion. There is no need for homology between the injected DNA and
the host genome.
Targeted insertion, the other common method of producing transgenic animals, is accomplished
by introducing the DNA into embryonic stem (ES) cells and selecting for cells in which the DNA
has undergone homologous recombination with matching genomic sequences. For this to occur,
there must be several kilobases of homology between the exogenous and genomic DNA, and
positive selectable markers (e.g., antibiotic resistance genes) must be included. In addition,
negative selectable markers (e.g., "toxic" genes) are often used to select against cells that have
incorporated DNA by non-homologous recombination (i.e., random insertion).
Pronuclear injection of DNA is often used to characterize the ability of a promoter to direct
tissue-specific gene expression. For example, promoter/enhancer constructs may be used to drive
expression of a reporter gene, such as LacZ, whose protein product, beta-galactosidase, is
detected histochemically. Another major use for transgenic mice produced by pronuclear
injection of DNA is to examine the effects of overexpressing and misexpressing endogenous or
foreign genes at specific times and locations in the animal.
Many factors influence whether a promoter/transgene construct will express (produce the
appropriate mRNA and protein) in transgenic mice. The promoters that are used must be known
to function appropriately in vivo (in vitro function does not always guarantee this). Transgene
constructs may have accumulated mutations during cloning (especially if PCR was involved).
Perhaps the most important consideration has to do with the trangene's insertion site in the mouse
genome. At many chromosomal locations, transgenes will be transcriptionally silent. At others
they may express, but with a tissue- and temporal specificity that is not identical to what has
previously been seen with the same promoter construct. The intrinsic ability of a promoter
construct to drive transgene expression reliably and with faithful tissue specificity also varies
from promoter to promoter, for reasons that are not well understood. For these reasons, the TMF
can guarantee to produce mice that have integrated the injected DNA, but we cannot make
guarantees about transgene expression.
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One of the most common uses of targeted insertion of DNA is to make "knockout mice".
Typically, homologous recombination is used to insert a selectable gene (e.g., neo, which confers
G418 (neomycin) resistance) driven by a constitutive promoter (e.g., PGK) into an essential exon
of the gene one wishes to disrupt (often the first coding exon). To accomplish this, the neo gene
(or other DNA) is flanked by large stretches of DNA (on the order of 2-7 kb) that exactly match
the genomic sequences surrounding the desired insertion point. Once this construct is
electroporated into ES cells, the cells' own machinery performs the homologous recombination.
To make it possible to select against ES cells that incorporate DNA by non-homologous
recombination, it is common for targeting constructs to include a negatively selectable gene
outside the region intended to undergo recombination (typically the gene is cloned adjacent to
the shorter of the two regions of genomic homology). Because DNA lying outside the regions of
genomic homology is lost during homologous recombination, cells undergoing homologous
recombination cannot be selected against, whereas cells undergoing random integration of DNA
often can. A commonly used gene for negative selection is the herpes virus thymidine kinase
gene, which confers sensitivity to the drug gancyclovir.
Following positive selection (e.g. G418, to select for neo) and negative selection if desired, ES
cell clones need to be screened for incorporation of the targeting construct into the correct
genomic locus. Typically, one designs a targeting construct so that a band normally seen on a
Southern blot or following PCR amplification becomes replaced by a band of a predicted size if
and only if homologous recombination occurs. Since ES cells are diploid, only one allele is
usually altered by the recombination event so, when appropriate targeting has occurred, one
usually sees bands representing both wild type and targeted alleles.
The embryonic stem (ES) cells that are used for targeted insertion are derived from the inner cell
masses of blastocysts (early mouse embryos). These cells are pluripotent, meaning they can
develop into any type of tissue. ES cells must be maintained on a layer of feeder cells, typically
mouse embryo fibroblasts that have been irradiated to prevent them from dividing. ES cells must
be passaged every 2-3 days to keep them from differentiating (and losing pluripotency).
It is important that the genomic DNA used in making a targeting construct is derived from the
same strain of mouse as the ES cells one intends to use. Even small gaps in homology due to
sequence polymorphisms between mouse strains can dramatically reduce the efficiency of
homologous recombination. Since the vast majority of ES cell lines are derived from the 129
mouse strain, the TMF has purchased a set of "dot-blot" membranes representing arrayed
genomic clones of 129 DNA, with 5-fold coverage of the entire genome. A screening service
will be offered to investigators to assist them in finding appropriate genomic clones from this
library.
Once positive ES clones have been grown up and frozen, the production of transgenic animals
can begin. Donor females are mated, blastocysts are harvested, and 10-15 ES cells are injected
into each blastocyst. Eight to ten blastocysts are then implanted into a uterine horn of each
pseudopregnant recipient. By choosing the appropriate donor strain, the detection of chimeric
offspring (i.e., those in which some fraction of tissue is derived from the transgenic ES cells) can
be as simple as observing hair and/or eye color. If the transgenic ES cells do not contribute to the
germline (sperm or eggs), the transgene cannot be passed on to offspring. The TMF cannot
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guarantee that ES cells will "go germline", but, if handled properly (e.g. not allowed to become
differentiated or aneuploid in vitro), those ES lines that are in common use generally contribute
to the germline an acceptable percentage of the time.
It is important to note (and perhaps not obvious) that production of transgenic mice by
pronuclear injection can also occasionally result in a mosaic animal. This happens when
integration of the transgene is delayed until after the first cell division. There can also be
multiple insertion events at different genomic loci and at different times. Thus, a single founder
can be mosaic for one insertion site but not the other.
Transgenic animals are a powerful tool for studying gene function and testing drugs. Many
human genetic diseases can be modeled by introducing the same mutation into a mouse or other
animal. Although similar genetic manipulations can be performed in tissue culture, the
interaction of transgenes with proteins, hormones, neurotransmitters, and other components of an
intact organism provides a much more complete and physiologically relevant picture of the
transgene's function than could be achieved in any other way. With the development of
transgenic livestock and plants, new uses for this technology have become apparent, some of
potentially great economic and medical value. These include the ability to produce medicallyuseful recombinant proteins and antibodies on an industrial scale, as well as disease-resistant
crops.
Two methods of producing transgenic mice are widely used:
 Transforming embryonic stem cells (ES cells) growing in tissue culture with the desired
DNA (Method “1”);
 injecting the desired gene into the pronucleus of a fertilized mouse egg (Method “2”);
The Embryonic Stem Cell Method (Method "1")
Embryonic stem cells (ES cells) are harvested from the inner cell mass (ICM) of mouse
blastocysts. They can be grown in culture and retain their full potential to produce all the cells of
the mature animal, including its gametes.
1. Make your DNA
Using recombinant DNA methods, build molecules of DNA containing



the structural gene you desire (e.g., the insulin gene)
vector DNA to enable the molecules to be inserted into host DNA molecules
promoter and enhancer sequences to enable the gene to be expressed by host cells
2. Transform ES cells in culture

Expose the cultured cells to the DNA so that some will incorporate it.
3. Select for successfully transformed cells.
13
4. Inject these cells into the inner cell mass (ICM) of mouse blastocysts.
5. Embryo transfer



Prepare a pseudopregnant mouse (by mating a female mouse with a vasectomized male).
The stimulus of mating elicits the hormonal changes needed to make her uterus receptive.
Transfer the embryos into her uterus.
Hope that they implant successfully and develop into healthy pups (no more than onethird will).
6. Test her offspring

Remove a small piece of tissue from the tail and examine its DNA for the desired gene.
No more than 10-20% will have it, and they will be heterozygous for the gene.
7. Establish a transgenic strain


Mate two heterozygous mice and screen their offspring for the 1:4 that will be
homozygous for the transgene.
Mating these will found the transgenic strain.
The Pronucleus Method (Method "2")
1. Prepare your DNA as in Method 1
2. Transform fertilized eggs



Harvest freshly fertilized eggs before the sperm head has become a pronucleus.
Inject the male pronucleus with your DNA.
When the pronuclei have fused to form the diploid zygote nucleus, allow the zygote to
divide by mitosis to form a 2-cell embryo.
3. Implant the embryos in a pseudopregnant foster mother and proceed as in Method 1.
14
Gene targeting
Homologous recombination in embryonic stem cells is now a routine method for modifying the
mouse genome at a specific locus. The technique was first developed for site-directed
mutagenesis in yeast, and has been successfully adapted for mammalian cells (Smithies et al.,
1985). In theory, any deletion, point mutation, inversion or translocation can now be modeled in
mice. The DNA construct to be introduced into the genome of the ES cells should contain
several kilobases of DNA that are homologous to the mouse genome to provide the best odds of
recombination. The vector also contains the modifications to be introduced as well as genes
conferring drug resistance or sensitivity so researchers can select the rare recombination events
from a large population of ES cells. In yeast, true homologous recombination at the correct locus
occurs at a very high rate, so that random integration of the vector into the yeast genome is rare.
In mammalian genomes, however, the majority of recombination events do not occur at the
desired locus, so ES cells showing resistance to the selective agent must also be screened by
Southern blot or by PCR to discover which clones have been correctly targeted.
15
Knockins - To avoid the problems of a standard transgenic, many researchers now rely on
knockin mice to study the exogenous expression of a protein. A knockin mouse is generated by
targeted insertion of the transgene at a selected locus. The insert is flanked by DNA from a noncritical locus, and homologous recombination allows the transgene to be targeted to that specific,
non-critical integration site. In this way, a researcher has complete control of the genetic
environment surrounding the overexpression cassette and it is likely that the DNA did not
16
incorporate itself into multiple locations. Site-specific knockins result in a more consistent level
of expression of the transgene from generation to generation because it is known that the
overexpression cassette is present as a single copy. Also, because a targeted transgene is not
interfering with a critical locus, the researcher can be more certain that any resulting phenotype
is due to the exogenous expression of the protein. Although the generation of a knockin mouse
does avoid many of the problems of a traditional transgenic mouse, this procedure requires more
time to assemble the vector and to identify ES cells that have undergone homologous
recombination.
Knockouts - While traditional transgenic and knockin mice are generated to express a protein,
much information can be learned from the elimination of a gene or the deletion of a functional
domain of the protein. This can be achieved through random mutation using chemical
mutagenesis or a gene trap approach, or through gene targeting to generate a knockout mouse.
Homologous recombination allows a researcher to completely remove one or more exons from a
gene, (see Figure 1 below) which results in the production of a mutated or truncated protein or,
more often, no protein at all. The process and time line for making a knockout mouse with the
Cell Migration Consortium Transgenic & Knockout Mouse Initiative has been outlined in this
Time line document. The phenotypes of knockout mice can be very complex because all tissues
of the mouse are affected, though it is not uncommon for a knockout mouse to display
embryonic lethality or to show no phenotype at all.
Description of random insertion transgenic mice
 Involves injection of raw DNA construct (usually circularized, or in a vector?) into the
male pronuclei of an embryo, before fusion of the male and female pro-nuclei. Use male
pro-nuclei because it is bigger.
 The DNA construct might consist of a gene of interest and some promoter.
 Once the pro-nuclei fuse, the embryo is implanted into another mouse.
 The DNA is randomly incorporated into the genome by the rolling circle mechanism.
 Insertion can disrupt genes.
 There can be multiple insertions, but you try to minimize this. Multiple insertions tend to
segregate and are lost after a few generations.
 The transgene will be hemizygous.
 Insertion can be validated by southern hybridization to check for DNA inserted and
northern hybridization to see if gene is being transcribed into mRNA.
 Because insertion is random, position effects can be a problem.
 Much faster and cheaper than a targeted insertion approach.
Description of targeted insertion transgenic mice
The early vectors used for gene insertion could, and did, place the gene (from one to 200 copies
of it) anywhere in the genome. However, if you know some of the DNA sequence flanking a
particular gene, it is possible to design vectors that replace that gene. The replacement gene can
be one that

restores function in a mutant animal or
17

knocks out the function of a particular locus.
In either case, targeted gene insertion requires
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

the desired gene
neor, a gene that encodes an enzyme that inactivates the antibiotic neomycin and its
relatives, like the drug G418, which is lethal to mammalian cells;
tk, a gene that encodes thymidine kinase, an enzyme that phosphorylates the nucleoside
analog gancyclovir. DNA polymerase fails to discriminate against the resulting
nucleotide and inserts this nonfunctional nucleotide into freshly-replicating DNA. So
ganciclovir kills cells that contain the tk gene.
Step 1
Treat culture of ES cells with preparation of vector DNA.
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
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Most cells fail to take up the vector; these cells will be killed if exposed to G418.
In a few cells: the vector is inserted randomly in the genome. In random insertion, the
entire vector, including the tk gene, is inserted into host DNA. These cells are resistant to
G418 but killed by gancyclovir.
In still fewer cells: homologous recombination occurs. Stretches of DNA sequence in the
vector find the homologous sequences in the host genome and the region between these
homologous sequences replaces the equivalent region in the host DNA.
Step 2
Culture the mixture of cells in medium containing both G418 and ganciclovir.
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
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The cells (the majority) that failed to take up the vector are killed by G418.
The cells in which the vector was inserted randomly are killed by gancyclovir (because
they contain the tk gene).
This leaves a population of cells transformed by homologous recombination (enriched
several thousand fold).
Step 3
Inject these into the inner cell mass of mouse blastocysts.
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Random vs. Targeted Gene Insertion
Random Insertion
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Inject transgene into the male
pronucleus of embryo before
fusion.
Gives rise to mosaic mice(one
fertilization event) if transgene
incorporated after first division.
Insertion is random
An increased chance of multiple
insertions.
Insertions occur at various sites.
Greater chance of insertion
mutagenesis.
No sequence homology
necessary.
Easier to make and takes less
time (3 months average)
Targeted insertion
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
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
Homologous recombination in
Emryonic Stem Cells.
Gives rise to chimeras (two
fertilization events).
Insertion is targeted.
Less likelihood of multiple
insertions.
Known region of insertion.
Lesser chance of insertion
mutagenesis.
Specific sequence homology to
the mouse genome is necessary.
Very difficult and takes about
1.5 years to get the desired
mouse.
Explanation of the conditional targeted mutations
19
Many genes that participate in interesting genetic pathways are essential for either mouse
development, viability or fertility. Therefore, a traditional knockout of the gene can never lead to
the establishment of a knockout mouse strain for analysis. Conditional gene modification using
Cre-lox technology allows the gene of interest to be knocked-out in only a subset of tissues or
only at a particular time, circumventing lethality. Because gene targeting can be controlled both
spatially and temporally, the function of a given gene can be studied in the desired cell types and
at a specific time point. This genetic dissection allows researchers to define gene function in
development, physiology or behavior.
Cre recombinase, a site-specific integrase isolated from the P1 bacteriophage, catalyzes
recombination between two of its consensus DNA recognition sites (Sauer & Henderson 1988).
These loxP sites are 34 base pairs in length, consisting of two 13bp palendromic sequences that
flank a central sequence of 8bp which determines the directionality of the loxP site. Two loxP
sites are most often placed in a trans orientation on either side of an essential, functional part of a
gene so that recombination removes that functionality and knocks-out the gene. (See Figure 2)
LoxP sites can also be placed in a cis orientation to invert the intervening sequence. LoxP sites
placed on different chromosomes can be used to generate targeted translocations, though this
recombination event occurs at a relatively low frequency compared to the highly-efficient intragene recombination.
20
21
LoxP sites do not recombine in the absence of Cre recombinase, so regulation of the expression
of Cre recombinase also regulates the DNA recombination and the initiation of the genomic
alteration. An extensive collection of mice have been generated, each line expressing Cre from a
promoter that is either tissue specific, cell specific, developmentally specific or responsive to an
exogenous agent like tetracycline. With such a collection available, several promoter-specific
mouse models can be studied in parallel. Additionally, researchers have generated an extensive
collection of vectors that express Cre recombinase from a reliable promoter, and transient
expression of Cre results in high rates of recombination in cultured cells. Thus, recombination
can be triggered in ES cells to generate a more traditional knockout mouse in addition to the
tissue-specific knockout. Alternatively, the mouse can be bred and grown as a pseudo- wild type
with out any recombination, and then a population of cells cultured from this mouse can be
transfected with a Cre-expression vector to generate recombined cells.
Recently, Flp recombinase (and its Frt DNA sites) have also proven useful in mouse transgenics
(Vooijs et al., (1998; Dymecki & Tomasiewicz 1998). Although few lines of mice have been
generated to express Flp in vivo, this system is very useful for the removal of the selection gene
from the targeted gene at the ES cell stage. The presence of a Neomyosin resistance cassette in
an intron can result in an alteration of gene function and therefore produce an unwanted or even
lethal phenotype (Scacheri et al., 2001). This problem can be avoided if the investigator utilizes
both the Cre and Flp recombination systems. A targeting vector containing both a Flp-flanked
neoR marker and a loxP-flanked exon can be introduced into ES cells. After selection, the
Neomyocin resistance cassette can be removed with Flp recombinase before the ES cells are
injected into host blastocysts to make mice. (See Figure 3) With this system, the chimeric
offspring contain only a minimal genetic modification (the addition of two loxP sites and one Frt
site) in the gene of interest, limiting the likelihood of a complicating phenotype. As with a loxPonly targeting, the regulated expression of Cre results in the regulated alteration of this gene.
22
23
Describe quantitative trait loci (QTLs) and their mapping
A Quick Guide to QTL
What is the difference between Quantitative Trait Loci (QTLs) and Mendelian mutants?
 Many Mendelian mutants can be considered QTLs, but not all QTLs are Mendelian
mutants.
 Mendelian mutations tend to refer to large-effect changes in the DNA sequence that
destroy or alter a genes function.
 Some QTLs may have only small effects by subtly altering gene expression levels for
example.
 Mainly a difference in effect size
What is QTL mapping?
 The identification of molecular variants that contribute to phenotypic variation
 QTL detection often uses recombinant inbred strains or crosses between two inbred
strains.
 Phenotypes are compared to genotypes to look for correlations. Can not only identify
genetic effects but can also identify the type of genetic action. Eg. if aa and ab are
equally present and more commonly found than bb in individuals with a certain
phenotype, then the a allele is likely responsible for the phenotype and dominant to b.
 QTL is not a linkage study. It is a test of association between QTL and marker.
What statistical issues must be considered?
 A significant QTL is one in which the linkage (association?) has a p-value that would
occur only once in 20 whole genome scans by chance.
 If more than one phenotype is considered, more stringent LOD-scores are required for
significance. Why? More likely to observe association by chance? Must meet demands
imposed by multiple testing.
 Can use permutation tests to overcome this problem. (1) Randomly assign phenotypes to
genotypes. (2) Apply test of relationship between phenotype and genotype to permuted
data set. (3) Repeat (1) and (2) thousands of times to determine distribution of statistical
test. Then, found out how often result with unpermuted set can be expected by chance
and establish threshold for significance.
 Should consider power of test to assess extent of false negatives and false positives. Can
determine number of animals needed for specified level of power or conversely, the level
of power provided by the number of animals. Eg. A recombinant inbred strain set with
26 strains has %90 power to detect a QTL which explains %50 of phenotypic variance
(this is optimistic, most QTL will explain a smaller fraction of phenotypic variance).
Reference:
Doerge, R. Mapping and analysis of quantitative trait loci in experimental populations (2002).
Nature Reviews Genetics 3: 43-52.
QTLs in Biology: What they are and why they are worth mapping
24
Quantitative trait loci are normal genes in every sense of the word. The somewhat unwieldy term
quantitative trait loci highlights the fact that variant forms—or alleles—of QTLs have relatively
subtle quantitative effects on phenotypes. QTLs identified in human populations are often
referred to as susceptibility genes because humans carrying certain alleles are at greater risk of
developing particular diseases. QTLs are often contrasted with Mendelian loci that have
pronounced and usually dichotomous effects on phenotypes, but the demarcation between
quantitative and qualitative traits is often blurred. QTLs that have particularly large effects—
often verging on producing Mendelian segregation patterns (e.g., 1:2:1)—are referred to as
major-factor or major-effect QTLs. Their large effects make them the easiest QTLs to map.
If large numbers of genes collectively control variation in a CNS phenotype (neuron number, cell
ratios, volume of a region, etc.), then allelic differences at any one locus will be associated with
small, possibly undetectable differences in phenotype. This phenomenon is referred to as the
infinitesimal model of polygenic action. In contrast, if only a small number of genes control
much of the variation and if their alleles exert relatively large effects on the phenotypes, then it
will often be possible to map single QTLs. Quantitative genetic analysis of variation in bristle
number in Drosophila provides a superb example of what can be achieved by "disassembling" a
polygene into its constituent QTLs. Normal allelic variants at the achaete-scute complex and at
the scabrous locus each account for 5–10% of the variance in numbers of these sensory organs.
In this system, the QTLs controlling natural variation are also known to be vitally important
developmental genes that had been identified previously based on mutant phenotypes associated
with null alleles.
The ability to map QTLs in the CNS depends critically on the amount of variance explained by
allelic differences at single QTLs and the technical precision with which traits can be measured
(a signal-to-noise problem). The more accurate and reliable the method of phenotyping, the
higher the heritability, and the greater the number of resolvable QTLs.
Mapping Quantitative Trait Loci (QTL)
The aim of QTL mapping is to identify chromosomal regions, which contain genes that affect
quantitative traits (traits affected by many genes and environmental factors). In order to do so,
genetic markers with known positions are identified and associated with phenotypic records.
The first step in any QTL-mapping experiment is usually to construct populations that originate
from homozygous, inbred parental lines. The resulting F1 lines will tend to be heterozygous at
all markers and QTL. From the F1 population, crosses are made (for example, backcross, F2
interchross and crosses to generate recombinant inbred lines), and the segregation of markers and
QTL are statistically modeled. In general, experimenters assume that markers are segregating
randomly, but if, in fact, markers are subject to segregation distortion, it is not possible to
anticipate how the resulting estimates of recombination will be affected, as well as any potential
QTL locations. Once the data are collected on each individual, statistical association between
the markers and quantitative trait are established through basic statistical approaches that range
from simple techniques, such as analysis of variance, to models that include multiple markers
and interactions. The simpler statistical approaches tend to be methods of QTL detection that
assess differences in the phenotypic means for single-marker genotypic classes. The actual
25
location of QTL involves an estimated genetic map with known distances between markers, and
evaluation if a likelihood function that is maximized over the established parameter space.
Types of QTL mapping:



Single-marker tests – assesses the segregation of a phenotype with respect to a marker
genotype, indicating which markers are associated with the quantitative trait of interest.
o The null hypothesis tested is that the mean of the trait value is independent of the
genotype at a particular marker
o The null hypothesis is rejected when the test statistic is larger than a crucial value
and the implication is that a QTL is linked to the marker under investigation
o A large sample of individuals provides the opportunity to observe recombinant
events and to estimate parameters with greater accuracy and therefore a greater
ability to detect QTL through a single-marker test
Genetic Maps – recombination is first estimated for all markers that are segregating as
expected, and then any marker that is linked to any other marker is placed in the same
linkage group
o the linear arrangement of markers into linkage groups, or chromosomes, provides
the genetic map for locating QTL that are relative to intervals of markers
o the estimated genetic map benefits the estimation of missing marker information
by using the surrounding marker genotypes to infer knowledge of the missing
marker genotypes
o QTL can be mapped more easily in an interval of defined genetic distance
o The methods for linearly ordering the molecular markers rely on minimizing the
recombination between pairs of markers
o As the estimated genetic distance between markers is a function of the average
number of observed recombination events between them, minimizing these values
best represents the frequency of recombination
o The accuracy of locating QTL is limited by the information, particularly by the
number of recombinants, that is gained from observing the genotypic states of the
markers
o Therefore, scoring a larger number of individuals is more effective in accurately
mapping QTL
Interval Mapping – uses an estimated genetic map as the framework for the location of
QTL
o The intervals that are defined by ordered pairs of markers are searched in
increments and statistical methods are used to test whether a QTL is likely to be
present at the location within the interval or not
o This method statistically tests for a single QTL at each increment across the
ordered markers in the genome
o The results of the tests are expressed as LOD scores which compare the
evaluation of the likelihood function under the null hypothesis (no QTL) with the
alternative hypothesis (QTL at the testing position) for the purpose of locating
probably QTL.
26
Statistical methods
Least squares methods are the simplest methods used in QTL detection. The model assumes
QTL genotype effects to be fixed, and regresses probability of the possible genotypes given
marker information on the phenotypic data.
In the Maximum Likelihood method variance component models assume random QTL effects.
The covariance structure between the QTL effects are modeled using an identity by descent
(IBD) matrix, containing probabilities that each combination of alleles are identical by descent
given the observed marker genotypes. Polygenic variation not explained by the QTL effects can
be modeled as random effects. This model is appropriate for simple and complex designs.
Bayesian linkage analysis is very flexible in terms of the complexity of the QTL model, number
of QTL, interaction between QTL, polygenic variation, robust distributions for error variance,
and is appropriate for simple or complex designs.
Describe how studies in laboratory animals can be used to identify genes involved in
complex diseases
Researchers are increasingly using animal models to study the genetic basis of complex human
behaviours. The most commonly used animal species are rodents, but other species, such as
nonhuman primates, fruit flies, and zebrafish, can also provide important information. In
behavioural studies, mouse models are especially common because the developmental and
anatomical features of the mouse and human brains are very similar. Further, the human and
mouse genomes are remarkably similar and a mouse ortholog exists for almost every human
gene and vice versa. Since genetic studies of behaviour cannot be done on human subjects, the
mouse provides an ideal model to identify and characterize genes that influence brain function
and behaviour in relation to psychiatric disorders.
There are 2 classic animal model methods that are especially useful:
1) Inbred stains in which all allelic diversity has vanished and each same sex member of the
inbred strain is essentially a monozygotic twin of all other members. There are over 100 strains
available. If environmental conditions are maintained as constant and uniform as possible,
differences observed between strains will be attributed to specific allele differences at each gene
that were ‘trapped’ in each strain during its formation.
2) Selective inbreeding by which the experimenter selects for those traits that they wish to study
by mating together those animals that best represent the trait of interest for several generations.
A variety of approaches are employed in identifying complex genetic traits; particularly
knockout and transgenic mice as well as specially bred animal lines that can be used for various
genetic analyses, including quantitative trait locus mapping. Other strategies applied in genetic
studies in animal models include random mutagenesis, virus-mediated gene transfer, and gene
expression profiling.
Animal Models and Genetics of Alcoholism
(taken from AlcoholCME.com)
27
Alcoholism is a complex genetic trait and susceptibility is influenced by multiple genes of small
effect. There are 4 distinguishing features that define alcoholism clinically;
1)
2)
3)
4)
Alcohol’s rewarding effects
The development of tolerance
The pathological consequences to brain systems
Dependence on alcohol inferred from the presence of withdrawal symptoms when the
drug is removed.
Inbred, Selectively Bred, and Recombinant Inbred Strains for alcoholism
Early studies identified differences in alcohol preference between standard inbred strains of mice
(McClearn and Rodgers, 1959). Later, inbred mouse and rat strains were shown to differ in
alcohol and drug tolerance, severity of withdrawal, and operant responding (self-administration)
(Crabbe et al., 1994). Alcohol-related phenotypic differences standard between inbred strains
may be associated with a particular polymorphism by chance, or because of parallel differences
at another, functionally significant locus. Genetic differences between strains selectively bred to
differ in a particular trait, however, are likely to be either functionally related to the behavior
variation or closely linked to such functional polymorphism.
The development of recombinant inbred (RI) strains has allowed mapping of the genes
responsible for the variation in drug responses. RI strains are reinbred from the F2 generation
(second offspring generation) of the cross between two inbred progenitor strains (Bailey, 1971).
The result is that each of the progenitor's chromosomes in every RI strain has recombined several
times. A correlation between a strain distribution pattern (SDP) for a trait (represented by
intrastrain mean phenotypic values) and a gene marker indicates linkage. This linkage is
detectable for major gene effects (non-overlapping SDPs) and for continuous (quantitative) traits
(Klein, 1978; Gelman et al., 1988; Plomin and McClearn, 1993). A quantitative trait locus (QTL)
can then be confirmed using the F2 offspring of the cross between the progenitor strains,
congenic strains (strains differing in one small chromosomal region containing a locus of
interest), or other approaches. A number of provisional markers and candidate genes are
identified in the QTL research as associated with various alcohol-related behaviors and reactions
to psychoactive drugs.
Quantitative Trait Loci (QTL) for alcoholism
For a QTL marker identified for an alcohol-use-related process in mice to be useful as a
candidate gene marker in humans, - unless the marker itself has functional significance - the
regions in mice and humans should be syntenic, i.e., in the same order in the chromosomes. As
much as 80% of the mouse genome is syntenic with the human genome (Copeland et al., 1993).
This suggests that the drug-related QTL information obtained in mice may be applicable in
humans (Phillips et al., 1994). Indeed, a significant proportion of the murine alcohol-related QTL
have human homologs and, moreover, are known to be involved in the determination of central
nervous system functioning and even drug reaction.
For example, a QTL for acute alcohol withdrawal severity was detected (lod score 4.1) for a
marker located on murine (mouse) chromosome 11 (Buck et al., 1997). The region covered
included the potentially relevant loci of GABA-A receptor subunits which have homologs on
28
human chromosome 5. In the same study, loci on chromosomes 1 and 4 were found to have even
higher lod scores for liability to physiological dependence (5.6) and each explained 26% of
genetic variance and 6% of the total phenotypic variance in this trait.
Sex-specific QTL for alcohol preference in mice were found on chromosome 2 for males (a
region including the cluster of sodium channel alpha-subunit genes), and on chromosome 11 for
females (a region including the serotonin transporter locus) (Melo et al., 1996). A mutation at the
gene encoding the transcription factor, CREB (cAMP-responsive element-binding protein) in
mice prevents physical symptoms of morphine withdrawal (Maldonado et al., 1996); the human
homolog of this gene is located on chromosome 2. A genome-wide QTL mapping study (Buck et
al., 2002) yielded significant evidence for QTLs on chromosomes 19 and distal 1 that account for
45% of the genetic variance in ethanol withdrawal severity.
Knockout Animals for Alcoholism
By completely obliterating the function of a specific gene in selectively bred mice, scientists can
create knockout mice - these animals provide an excellent means to isolate the function of
particular genes. For instance, male mice lacking monoamine oxidase A function have been
shown to manifest increased aggressiveness with parallel dramatic increases in serotonin brain
concentrations in the pups (normal levels in adults) as well as elevations in dopamine and
norepinephrine (Cases et al., 1995); these neurotransmitters are known to be involved in the
central nervous system (CNS) mechanisms of alcohol response. The DRD4 knockout mice
display elevated locomotor sensitivity to ethanol, cocaine, and methamphetamine (Rubinstein et
al., 1997). It should be noted, however, that since the knocked-out genes are silenced throughout
the animal's lifespan, the behavioral manifestations observed in them may be indicative of
disruption in processes normally unrelated to variation in alcohol response.
Summary
Animal studies of the contribution of specific genes to the liability to alcoholism have
illuminated several important aspects of liabilty to alcoholism. These studies have significant
implications for explaining human heritability of this trait. Animal studies allow a more careful
examination of the effects of specific genes given the ability of scientists to genetically engineer
mice.
Utilizing animal strains exhibiting differential distribution patterns of
alcohol-related traits for a gene marker, laboratory studies allow for the
detection of multiple polymorphic sites potentially contributing to liability
variation.
Studies of inbred mice suggest the similar origin of severity of withdrawal
from multiple drugs, including alcohol.
Utilizing recombinant inbred strains of mice in the analysis of quantitative
trait loci allows for detecting genes linked to alcohol-related behaviours.
QTL identification from mice are relevant to human genetics if the genes are
syntenic or QTL have functionla significance; some 80% of the mouse
29
genome is syntenic with the human genome.
Use of engineered knockout animals allow for an isolation of the influence
of specific genes on a particular behavior of interest.
Contribution of other animal models to genetics of alcoholism…
In the fruit fly Drosophila melanogaster, genes that influence sensitivity or tolerance to alcohol
have been extensively studied. One Drosophila mutant with markedly increased alcohol
sensitivity has been named “cheapdate”. The mutation in this strain affects the cAMP signalling
pathway, which is important for many regulatory processes in the cell. The cAMP signaling
pathway also has been implicated in determining alcohol consumption and sensitivity to alcohol
in studies of a mouse knockout model and of human alcoholics. These observations suggest that
Drosophila will provide an important model system for at least some aspects of alcohol’s effects.
Further, non-alcohol related research in Drosophila has led to the isolation of a mouse gene that
corresponds to a Drosophila gene called neuralized, which subsequently was found to alter
sensitivity to alcohol.
Compare and contrast utility of different animal models available.
1.) Caenorhabditis elegans


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nearly transparent roundworm has intestines, muscles and a complex nervous system that
are startlingly similar to humans in chemical structure.
40 to 45% of C. elegans genes have homologs in humans, and many more are conserved
in organisms like yeast and drosophila
generation time is about 3.5 days, and can be manipulated by growing the worms at
higher or lower temperatures
important strains can be frozen in liquid nitrogen and stored, then used much later
since the cuticle is transparent, cell division can be observed in real time
the genome contains fewer repetitive sequences than humans
one worm produces 300 progeny on average, so large progeny numbers make genetic
results more statistically significant
The worm's DNA sequences are known in full and are, moreover, very much shorter than
those of humans (100 million bases for the worm, compared to 3 billion in the human),
which greatly simplifies the search for their functions in the genome.
In nature, 99.9 percent of worms are self-fertilizing hermaphrodites (which makes them
easy to keep in the lab), but males also occur rarely. The frequency of males can be
increased in the lab by heat shocking the worms (this increases the frequency of X nondisjunction), then male strains can be maintained by mating several males to one
hermaphrodite. Males are useful in genetic crosses because if found in the F1 progeny,
they are a marker for the success of the cross.
30

The worm has been studied as a model organism since the early 1970’s, so there is much
information on the genetics of the organism, gene structure and function, tools for gene
and molecular analysis, genome organization, etc. See www.wormbase.org
2.) Zebra Fish
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As a vertebrate, the zebrafish, Danio rerio, is more closely related to humans than are
yeast, worms or flies.
Many features of zebrafish development have been characterized, including early
embryonic patterning, early development of the nervous system, and aspects of cell fate
and lineage determination. The embryos are transparent and accessible throughout
development. In live embryos, the same specific cell or even cellular processes can, in
many cases, be identified from individual to individual, affording a high level of
precision in characterizing the effect of developmental or genetic perturbation.
Techniques for ablation and transplantation of individual cells have been used to explore
questions about induction and cell fate, and continue to be refined.
There are also a growing number of molecular markers to facilitate developmental
studies.
Because of their relatively short reproductive cycle, the large number of progeny that can
be produced, and the relatively small space needed to maintain large numbers of
offspring, the zebrafish is an efficient vertebrate model system for genetic analysis.
It is possible to generate haploid progeny, which are viable to the point where many
recessive embryonic phenotypes can be identified, and also homozygous diploid progeny
that carry only maternal (or paternal) genetic information.
A genetic map of approximately two - three centimorgan resolution is available, and
mutations can be readily placed on the map. Positional cloning of genes identified by
mutation has recently been accomplished.
There are several promising methods for transformation and insertional mutagenesis
which are now being developed.
One disadvantage of the zebra fish is that its genome is large as a result of whole genome
duplication
3.) Mouse
 The mouse has been used a model organism for genetics research since the early 1900s.
A major advantage of the mouse is, quite simply, that it is a mammal like ourselves and
can be used to model a wide range of human genetic disorders.
 Mice are hardy and easy to breed and maintain under laboratory conditions and have a
relatively short generation time for a vertebrate(2-3 months).
 Compared with other mammals they also have large litter sizes (6-12) and the males have
high re-mating frequency. Valuable strains of mice can be stored as frozen embryos.
 A major disadvantage for developmental genetics is that mice develop in utero and this
combined with the difficulty in breeding and maintaining very large numbers of mice
make it impractical to perform the sort of F2 screens for developmental mutants that have
been done in the zebrafish.
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4.) Drosophila
Within a few years of the rediscovery of Mendel's rules in 1900, Drosophila melanogaster (the
so-called fruit fly) became a favorite "model" organism for genetics research.
Some of the reasons for its popularity:
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The flies are small and easily reared in the laboratory.
They have a short life cycle The figure shows the various stages of the life cycle (not all
drawn to the same scale). A new generation of adult flies can be produced every two
weeks.
They are fecund; a female may lay hundreds of fertilized eggs during her brief life span.
The resulting large populations make statistical analysis easy and reliable.
The giant ("polytene") chromosomes in the salivary (and other) glands of the mature
larvae.
o
o
These chromosomes show far more structural detail than do normal
chromosomes, and
they are present during interphase when chromosomes are normally invisible.
More recently, Drosophila has proven in other ways to have been a good choice:
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Its embryo grows outside the body and can easily be studied at every stage of
development.
The blastoderm stage of the embryo is a syncytium (thousands of nuclei unconfined by
cells) so that, for example, macromolecules like DNA injected into the embryo have easy
access to all the nuclei.
The genome is relatively small for an animal (less than a tenth that of humans and mice).
Mutations can targeted to specific genes.
5. The cat as an animal model for human hereditary disease:
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over 200 heritable genetic defects identified, many of which are homologous to human
inborn errors.
Specific metabolic defects have been identified underlying many of these feline diseases
and derived cat strains homozygous for many of these mutations are maintained in
veterinary clinical centers for development of model therapy.
whereas genes associated with some feline disorders have been characterized, and even
corrective gene therapy strategies have been examined for some disorders, the genes
associated with the majority of feline disorders have yet to be identified.
Multiple animal models from diverse evolutionary backgrounds may be required to fully
characterize many molecular pathologies. As an example, lack of dystrophin in muscular
dystrophic human, mouse, cat and dog elicit very different clinical phenotypes
Murine models may prove inadequate for analysis of some quantitative characters, as
generations of inbreeding may have eliminated multigenic diversity at crucial modifying
loci
32
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Spontaneously generated models will continue to have value in animal models,
particularly for pathologies that are rare in humans and hence poor candidates for
traditional mapping strategies
The cat is frequently used as an animal in laboratory experimentation ranging from
gastroenterology to ophthalmology. As an example, in the field of vision research, a vast
amount of knowledge has been gained through elaborate investigations including
physiological and morphological aspects of the cat retina contributing to the value of the
cat as a model for retinitis pigmentosa.

Domestic cats are subject to epidemics of two viruses, FeLV and FIV, that cause
immunodeficiencies and neoplasias, providing a powerful animal model for leukemia and
AIDS. These viruses and other feline pathogens provide a good opportunity to investigate
the interaction of host immune response and fatal infectious disease through studies of
the major histocompatibility complex, T-cell receptor loci, immunoglobulin genes and
other loci that participate in immune response.

a high degree of linkage conservation between the cat and human genomes (3 to 4 times
more conserved than mouse vs. human), permitting a reconstruction of primitive
mammalian genome organization.
Increased understanding of genomic organization of the cat will contribute to our
understanding of mammalian genome evolution and whether there is adaptive rationale to
genomic organization as has been observed in the MHC, HOX and globin complexes.
high degree of conserved synteny between human and cat leads to powerful comparative
inference in gene mapping exercises.
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6.) Fugu Rubripes (Pufferfish)

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Fugu rubripes is one of at least 100 species of pufferfish. It has about the same number of
genes and regulatory regions as the human, but these elements are embedded in only 365
million bases as compared with the 3 billion that make up human DNA. With far less
noncoding DNA to sort through, finding Fugu genes and their controlling sequences
should be a much easier task.
Although separated by evolution nearly half a billion years ago, both genomes retain the
record of distant common ancestors.
The Fugu genome project was initiated in 1989 by Sydney Brenner and his colleagues
Greg Elgar, Sam Aparicio and Byrappa Venkatesh. In 1993, this team showed that the
genome of the Fugu is only 390 Mb which is about eight times smaller than the 3000 Mb
human genome, yet it contains a similar repertoire of genes to humans (Brenner et al.,
1993). The compact nature of the genome is largely due to scarcity of dispersed repetitive
sequences (<10%). Fugu was proposed as a model vertebrate genome to understand the
more complex human genome and other vertebrate genomes.
Studies have shown that the intergenic regions and introns in the Fugu are highly
compressed and uncluttered with repetitive sequences; the average gene density is about
one gene per 10 kb and that the gene order over short range is conserved between the
Fugu and humans.
33
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Apart from gene discovery, Fugu is also useful for identifying and characterizing gene
regulatory elements. Since the intergenic and intronic regions are compact in the Fugu, it
is easy to scan the non-coding sequences for identifying conserved putative regulatory
elements.
The Fugu and human lineages diverged about 450 million years ago and a comparison
between these two distant genomes provides interesting insight into the evolutionary
changes that have shaped the two vertebrates. Several changes in the genomic
organization of the two genomes involving segmental duplications, chromosomal
rearrangements and 'loss' and 'gain' of introns have been identified.
How closely related are mice and humans? How many genes are the same?
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Mice and humans (indeed, most or all mammals including dogs, cats, rabbits, monkeys,
and apes) have roughly the same number of nucleotides in their genomes -- about 3
billion base pairs. This comparable DNA content implies that all mammals contain more
or less the same number of genes, and indeed our work and the work of many others have
provided evidence to confirm that notion.
only a few cases in which no mouse counterpart can be found for a particular human
gene, and for the most part we see essentially a one-to-one correspondence between
genes in the two species. The exceptions generally appear to be of a particular type -genes that arise when an existing sequence is duplicated.
Gene duplication occurs frequently in complex genomes; sometimes the duplicated
copies degenerate to the point where they no longer are capable of encoding a protein.
However, many duplicated genes remain active and over time may change enough to
perform a new function.
Since gene duplication is an ongoing process, mice may have active duplicates that
humans do not possess, and vice versa. These appear to make up a small percentage of
the total genes. The number of human genes without a clear mouse counterpart, and vice
versa, won't be significantly larger than 1% of the total. Nevertheless, these novel genes
may play an important role in determining species-specific traits and functions.
The most significant differences between mice and humans are not in the number of
genes each carries but in the structure of genes and the activities of their protein products.
Gene for gene, we are very similar to mice. What really matters is that subtle changes
accumulated in each of the approximately 30,000 to 35,000 genes add together to make
quite different organisms.
Genes and proteins interact in complex ways that multiply the functions of each. In
addition, a gene can produce more than one protein product through alternative splicing
or post-translational modification; these events do not always occur in an identical way in
the two species. A gene can produce more or less protein in different cells at various
times in response to developmental or environmental cues, and many proteins can
express disparate functions in various biological contexts. Thus, subtle distinctions are
multiplied by the more than 30,000 to 35,000 estimated genes.
Similarities between mouse and human genes range from about 70% to 90%, with an
average of 85% similarity but a lot of variation from gene to gene (e.g., some mouse and
human gene products are almost identical, while others are nearly unrecognizable as
close relatives).
34
What are the comparative genome sizes of humans and other organisms being studied?
organism
estimated size
estimated number of
genes
average gene density
Human
3000 million
bases
~30,000
1 gene per 100,000
bases
M. Musculus (mouse)
3000 million
bases
30,000
1 gene per 100,000
bases
Drosophila (fruit fly)
135.6 million
bases
13, 061
1 gene per 13,781
bases
Arabidopsis (plant)
100 million bases
25,000
1 gene per 4000 bases
C. elegans
(roundworm)
97 million bases
19, 099
1 gene per 5079 bases
S. cerevisiae (yeast)
12.1 million bases 6034
1 gene per 2005 bases
E. coli (bacteria)
4.67 million bases 3237
1 gene per 1443 bases
H. influenzae (bacteria) 1.8 million bases
1740
1 gene per 1034 bases
Genome size does not correlate with evolutionary status, nor is the number of genes
proportionate with genome size.
Describe what is meant by a “candidate gene approach”
To infer the underlying genetic basis for phenotypic variation, 2 basic approaches are used:
1. Linkage: The genotypes measured are used only as markers of a chromosomal segment.
You systematically scan the entire genome of various affected members of families using
regularly spaced, polymorphic DNA markers.
2. The Candidate Gene Approach: here loci studied are based on prior knowledge of
potential involvement in the phenotype. Thus, this tests the effects of genetic variants of a
potentially contributing gene in an association study and tries to answer the question: is
one allele of a candidate gene more frequently seen in people with the disorder than in
people without?
Strategies for the Candidate Gene Approach
1. Select Candidate Gene:
When gene families perform the same function, or enzymes in the same metabolic pathway,
animal models, comparative genomics, genome scans.
2. Choose a DNA polymorphism:
Identify existing genetic variants.
Try to determine which of the variants are functionally important.
The variants need to occur at a sufficient frequency.
Sometimes, SNPs are used. They are informative because of Linkage Disequilibrium.
35
3. Testing the Candidate Gene
Test randomly selected cases and controls in an association study.
Advantages of the Candidate Gene Approach:

Easy to get large sample sizes, because it doesn’t require large families, use case-control
or small families

Decreased effect of disease heterogeneity

Don’t need to make assumptions about the mode of inheritance.

More effective in the detection of genes underlying common and more complex diseases.
Disadvantages:

May result in spurious associations if cases and controls aren’t appropriately matched.

Researchers must already have an understanding of the mechanisms underlying the
disease
RESOURCE:
An interesting paper that summarizes things quite nicely and it isn’t just related to candidate
genes. It essentially talks about linkage and association, but maybe it’ll help.
http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6T4T-46YV8SJ-5B&_cdi=4983&_orig=search&_coverDate=10%2F31%2F2002&_qd=1&_sk=999389998&view
=c&wchp=dGLbVtzzSkzV&_acct=C000050484&_version=1&_userid=1022551&md5=7d3dad5cc91c04bf16a2156
36ca31640&ie=f.pdf
Remember….
As Dr. Simpson said in class, the Fierce gene can now be used as a candidate gene to look at
aggressive behaviour in humans!
Discuss why genetic research into areas like behaviour or intelligence often evokes public
concern
Public’s concerns
 Tendency of media and public to oversimplify things. Eg. Headlines proclaim smart
gene found, designer babies, GATTACA on the way!
 Behaviour and intelligence are very subjective concepts and very difficult to quantify.
Intelligence is a classic example. Is intelligence the ability to solve a certain type of
problem? The ability to make one's way successfully in the world? The ability to score
well on an IQ test?
 Very difficult to separate from environmental effects.
 There is a public concern that genetic causes of behaviours will be used to excuse antisocial or criminal behaviours.
 Threatens people’s concept of free will.
36
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If we find a "gay gene" or “dumb gene” will it mean greater or lesser tolerance? Will it
lead to proposals that those affected by the "disorder" should undergo treatment to be
"cured" and that measures should be taken to prevent the birth of other individuals so
afflicted?
Raises real possibility of eugenics, an extremely distasteful concept to most.
Concerns of discrimination against or stigmas attached to those carrying certain
genotypes.
Genetic research into these areas evokes public concern not so much because of what the
research will find, but more, the use of that information.
1. The use of genetic testing for SELECTION purposes is of great concern.
I)
Prenatal testing: the fear here is that in the future, certain brhavioural traits
such as intelligence, sexual orientation and personality traits may be used as
the basis for embryo selection in IVF programs.
II)
Education: Streaming children in school based on intelligence and aptitude.
III)
Employment: screening of employees and jobseekers to exclude those that
the employer may consider ‘undesirable’.
IV)
Insurance: Use of genetic info to estimate risk (eg. Predisposition to suicidal
tendency may be significant from the eyes of the life insurance companies)
2. The use of genetic testing and LEGAL IMPLICATIONS
I)
Criminal responsibility: Claim of diminished legal responsibility for one’s
actions based on their genetic makeup.
II)
Sentencing: The use of genetic information in determining degree of
blame/punishment.
III)
Criminal disposition: Labeling some one as a criminal before they
have committed the crime just because they have a certain genotype.
Assigned Papers:
Cases, O., Seif, I., Grimsby, J., Gaspar, P., Chen, K., Pournin, S., Müller, U., Aguet, M.,
Babinet, C., Shih, J. C. & De Maeyer, E. (1995) Aggressive behavior and altered
amounts of brain serotonin and norepinephrine in mice lacking MAOA. Science 268,
1763-1766.
What did they do?
 Created a transgenic knock-out mouse model for MAOA gene and tested for aggressive
behaviour.
 MAOA and B are mitochondrially located enzymes that inactivate neuroactive amines
such as serotonin, dopamine, and norepherin.
How did they make their transgenic mice?
37
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
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Injected an IFN-B minigene into one-cell embryos of mice. Resulted in many transgenic
lines, one of which displayed X-linked recessive abnormal behaviour of mouse pups
(called Tg8).
Southern blot hybridization and PCR identified a single minigene copy.
This transgene was found to be transmitted by X-linked inheritance.
RT-PCR identified IFN-B transcripts in the spleen and testis but not the brain.
Brunner, H. G., Nelen, M., Breakefield, X. O., Ropers, H. H. & van Oost, B. A. (1993)
Abnormal behavior associated with a point mutation in the structural gene for monoamine
oxidase A. Science 262, 578-580.
Background:
 Studies of aggressive behaviour have implicated altered metabolism of serotonin,
dopamine, noradrenaline.
 Hypothesize that genetic defects in metabolism of these neurotransmitters could affect
aggressive behaviour.
What did they do?
 Investigating a large kindred with males affected by borderline mental retardation and
aggressive tendencies.
 X-linked disorder that has been assigned to p11-p12 region of X. near MAOA and
MAOB.
 Hypothesis: affected males have selective MAOA deficiency.
 Established skin fibroblast cultures and assayed for MAOA activity (how?).
 Found greatly reduced MAO activity in affected males.
 Used cDNA synthesis, PCR and sequencing to look for mutations in the MAOA gene in
these individuals.
 Found three synonymous SNPs and one non-conservative non-synonymous SNP (C936T
Glutamine -> stop).
 Conducted linkage analysis between clinical phenotype and the mutation.
What did they find?
 Complete and selective deficiency of MAOA in affected males. No effect on carrier
females.
 Describe possible link between this genotype and criminal/anti-social behaviour (next
paper continues with this hypothesis).
 Suggest 2 possible mechanisms:
o Reduced MAOA activity results in decreased 5-hydroxyindole-3-acetic acid (5HIAA) which has previously been linked to impulsive aggression.
o Suppresses REM sleep.
Conclusions and comments:
 study suggests a relation between MAOA deficiency and aggressive behaviour in males.
38

Questions raised:
o How frequent is MAOA deficiency in the population?
o How complete does the deficiency have to be for an effect on behaviour to be
observed?
o How can animal models be used to investigate the neurochemical alterations
induced by MAOA deficiency?
Caspi, A., McClay, J., Moffitt, T. E., Mill, J., Martin, J., Craig, I. W., Taylor, A. & Poulton,
R. (2002) Role of genotype in the cycle of violence in maltreated children. Science 297, 851854.
Background:
 Children who are abused are more likely to display anti-social or criminal tendencies.
 This association is very significant but is not a rule and many maltreated children go on
to lead non-criminal lives.
 The authors propose that there may be genetic factors that make some children more
susceptible to maltreatment than others.
What did they do?
 Looked for an association between functional polymorphisms on the monoamine oxidase
A (MAOA) gene and the development of antisocial behaviour among maltreated
children.
 MAOA located on X chromosome and is therefore an X-linked gene.
 MAOA encodes an enzyme that metabolizes neurotransmitters (eg. Dopamine).
Deficiencies in MAOA have already been linked with aggression.
 Maltreatment is also known to have lasting neurochemical effects in human children but
the involvement of MAOA is not yet known.
 In rats, it has been shown inhibition of MAOA (by phenelzine) prevents them from
habituating to stress.
 Hypothesis: MAOA genotype can moderate the influence of childhood maltreatment on
neural systems implicated in anti-social behaviour.
 Tested whether anti-social behaviour can be predicted by interaction between a gene
(MAOA) and environment (maltreatment). G x E interaction
 Looked at a well-characterized VNTR polymorphism at the promoter of MAOA, known
to affect expression.
 Used a 1037 individual birth cohort of 26 year olds broken into three groups: not
maltreated, probably maltreated, severely maltreated.
How did they assess antisocial behaviour?
 Used four outcome measures: 1) Adolescent conduct; 2) convictions for violent crimes;
3) personality disposition towards; 4) symptoms of antisocial personality disorder
 Using moderated regression analysis, scored individuals on a composite antisocial index,
comprising the four factors.
What did they find?
39
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Main effect of MAOA expression on antisocial behaviour not significant, whereas main
effect of maltreatment was significant.
However, the G x E interaction was significant. The effect of maltreatment on antisocial
tendencies was significantly stronger among males with lower MAOA activity.
Maltreated males with high MAOA levels were not more prone to adolescent conduct
disorder, adult violent conviction, disposition towards violence, or antisocial personalities
than non-maltreated males whereas maltreated males with low MAOA levels were much
more likely to engage in these activities.
What do they conclude?
 A functional polymorphism of MAOA gene moderates the impact of early childhood
maltreatment on the development of antisocial behaviour in males.
 Demonstrates the importance of considering both genetics and environment when
searching for cause of complex phenotypes.
 May help identify children at most risk of developing antisocial tendencies after
maltreatment.
 Investigation of MAOA role in neurotransmitter system may help us to understand
mechanisms for why stressful experiences are converted into antisocial behaviour in
some but not others.
 MAOA gene is a strong risk predictor: Individuals in the cohort with low MAOA and
maltreatment combination accounted 12% of group but 44% of criminal convictions.
85% of individuals with the genotype who were severely maltreated developed some
form of antisocial behaviour.
Ikemoto, K., Kitahama, K., Seif, I., Maeda, T., DeMaeyer, E. & Valatx, J. L. (1997)
Monoamine oxidase B (MAOB)-containing structures in MAOA-deficient transgenic mice.
Brain Res 771, 121-132.
What is the difference between MAOA and MAOB?
 subtypes encoded by two different genes, closely linked on X chromosome.
 70% similarity in amino acid sequence
 Different but overlapping substrate specificity
 Both metabolize DA and tyramine. MAOA is better for 5-HT.
 MAOB found in serotonergic and histaminergic and astrocytes. MAOA found in
noradrenergic neurons.
 Difficult to distinguish MAOB structures from MAOA ones.
What did they do?
 Utilize Tg8 mouse strain described in previous paper to describe MAOB-containing
structures.
 Essentially stained brain samples of control mice and Tg8 mice with an inhibitor of
MAOA or MAOB and visualized under magnification to observe which parts of brain
specifically express on or the other.
40
What did they find?
 Found only MAOB-containing structures in Tg8 line, confirming the absence of MAOA
activity in this mutant.
 Identified specific MAOA and MAOB structures in mouse brain.
 Identify numerous MAOB neurons and MAOB structures.
 Found differences from other species MAOB structures.
Young KA, Berry ML, Mahaffey CL, Saionz JR, Hawes NL, Chang B, Zheng QY, Smith
RS, Bronson RT, Nelson RJ, Simpson EM. 2002. Fierce: a new mouse deletion of Nr2e1;
violent behaviour and ocular abnormalities are background-dependent. Behav Brain Res.
132(2):145-58
What did they do?
 Report a new mutation named ‘fierce’ (frc) that includes a spontaneous deletion of
Nr2e1. Mutations in Nr2e1 are implicated in developmental defects of the anterior brain,
aggressive behaviour, and severe impairment of retinal and optic nerve development.
 Studied their mutation on three genetic backgrounds: C57BL/6J, 129P3/JEms and
B6129F1.
 Comprehensively examine behaviour, brain, and eye phenotypes and identify some
background-dependent and -independent elements.
 Crossed heterozygotes for the deletion mutation. Thus, 25% of offspring will be
homozygous mutants. No heterozygous phenotype was observed and thus, the mutation
is recessive and heterozygous littermates can be used as controls.
 Monitored growth, brain morphology, retinal histology, retinal morphometry, performed
electroretinograms, and behavioural tests, sensorimotor tests, aggression tests, mating
tests, hormonal tests (testosterone, corticosterone).
What did they find?
 fierce mutation characterized by violent behaviour, stunted growth, hypoplasia of
forebrain, and lack of maternal instincts.
 Fierce mice show significantly reduced growth. C57BL/6J mice significantly more
affected than B6129F1 mice.
 Fierce mice have abnormal brain development.
 Fierce mice have impaired ocular development.
 No effect on auditory function was detected.
 Fierce mice lack maternal behaviour, are ‘hard to handle’, and violently aggressive.
Mothers abandon their nests, they attack their littermates and attack mating partners.
 Fierce mice show deficits in standardized sensorimotor tests.
 Fierce mice show abnormal mating behaviour. Males are more prone to attack than
mount.
 Corticosterone and testosterone are not abnormal in fierce mice.
41
What do they conclude?
 Sufficient backcrossing is required to make sure the correct genotype is associated with
the phenotype.
 Identification of traits consistent across all backgrounds is suggestive of fundamental
genes.
 Through genetic purification background specific effects can be detected, indicating the
possible presence of modifier genes. For example, overt hydrocephalus was seen only in
frc mice on the C57BL/6J background.
Abrahams BS, Mak GM, Berry ML, Palmquist DL, Saionz JR, Tay A, Tan YH, Brenner S,
Simpson EM, Venkatesh B. 2002. Novel vertebrate genes and putative regulatory elements
identified at kidney disease and NR2E1/fierce loci.
Genomics. 80(1):45-53.
What did they do?
 Compared genomic sequences for H. sapiens and F. rubripes to learn more about NR2E1.
What did they find?
 Gene order and orientation is conserved closely between Human and Zebrafish but highly
(10x) compressed in the latter. Compression is achieved by shorter introns, highly
reduced intergenic regions, and a general reduction in repetitive elements.
 Also found reduced lengths (not numbers) of LINEs and SINEs in mice compared to
humans.
 Found numerous conserved elements suggestive of regulatory elements.
 NR2E1 protein highly conserved between species.
 NR2E1 expression localized to brain and eye tissue. Expression conserved between
species, lending further evidence to presence of conserved regulatory elements between
F. rubripes, H. sapiens, and M. Musculus.
 Used TESS (http://www.cbil.upenn.edu/tess/index.html) to examine conserved elements
close to the 5’ transcriptional start site for DNA motifs known to bind vertebrate proteins.
 Found several motifs, one of which (CE-10A) was also found to be present at multiple
loci proximal to other genes implicated in brain development. Suggest that these may be
regulators of brain transcripts.
 Identified a gene, LACE1, a novel vertebrate gene upregulated during lactation.
Confirms existing prediction by ensemble.
 Identification of estrogen receptor binding sites proximal to LACE1 implicates possible
hormonal control.
 Also identified possible alternative transcript (missing exon 3) with alternate functional
role (possibly involved in embryonic development.
What do they conclude?
 Demonstrate the value of interspecies comparisons for discovering new genes and
conserved regions with functional significance.
42
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

Limiting study to conserved elements dramatically reduces sequence that needs to be
examined and is more cost-effective.
By examining the locus in three species instead of only two, the risk of false positives is
greatly reduced.
Conservation within non-coding regions is unrelated to conservation within coding
regions and varies from case to case. In only some cases, highly conserved proteins are
accompanied by highly conserved non-coding regions (eg. NR2E1). This can either
reflect evolutionary age or preservation of regulatory elements.
Useful links:
http://www.cellmigration.org/res_resourcekomousetext.htm
Definitions:
X-linked inheritance - Males have only one allele of (almost) every gene on the X chromosome, so a
recessive mutation in one of those genes may cause disease. Inheritance of the disease is said to be Xlinked. Examples include haemophilia and X-linked colour blindness.
Genetic heterogeneity - A similar phenotype being caused by different mutations. Most commonly
used for a similar phenotype being caused by mutations in different genes. Allelic heterogeneity refers to
different mutations in the same gene.
Phenocopy - A trait which appears to be identical to a genetic trait, but which is caused by nongenetic factors.
Etiology - A branch of knowledge dealing with causes. More specifically, the study of the causes of
abnormal conditions or disease.
Multifactorial - A trait that is determined by the interaction between a number of genes and
environmental factors.
Quantitative Trait Loci (QTL) – Genetic loci containing allelic variants responsible for some
phenotype that can be quantitatively measured.
43