Mouse Models of Human Disorders

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Mouse Models of Human Disorders

• Prepared by
• Rohan Palmer
• Institute of Behavioral Genetics
• University of Colorado at Boulder
• Rohan.Palmer_at_Colorado.edu

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Goals

• Understanding Genetic Influences on a Trait
• Accounting for Variation in an Observed Trait
• Why have animal models?
• Response to Selection Study
• Types of Mouse Models of Human Disorders
• Transgenic Models
• Mutagenic Models

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Lecture Goal Exam Question

• You are reading a Times article with a friend and
  you come across the
• following statement
• The study hypothesized that some of these
  susceptibility factors may be
• allelic variants of genes that govern embryonic
  serotonin neuron
• development and that these alleles may contribute
  to behavioral disorders
• by adversely increasing or decreasing serotonin
  system activity.
• The article is about finding ways to create mouse
  models for human DCGs.
• Your classmate has not yet taken Dr. Careys
  Behavioral Genetics course but
• fortunately you have. Describe to him/her what
  an allelic variant is and at
• how it might affect a behavioral disorder.
  Lastly, your friend looks at you as if
• you were crazy and says, How does a mouse study,
  help US? Based on what
• you have taken away from this course, convince
  your friend about the
• relevance and validity of mouse models. (20
  Points)

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Heritability

• The extent to which variation on a trait in a
• given population at a given point in time is
• attributable to genetic variation between
• individuals.
• VP VG VE
• h2 VG/VP

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Comparison Terminologies

• Synteny The co-localization of genes on
• chromosomes of related species.
• Homolog The situation where nucleic
• acid or protein sequences are similar
• because they have a common
• evolutionary origin. Often used loosely
• to indicate that sequences are very
• similar.
• Ortholog gene sequences are similar
• between species.
• Paralog gene sequences are similar
• within a species.

Adapted from the Department of Energy
genomics.energy.gov
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Why Create Animal Models?

• Pros
• Test many hypothesizes
• No human genetics ethical dilemma
• Shortens length of study
• (Avg. gestational period is 19 days)
• (Females become fertile in 3 weeks)
• (Approximately 40 days for turnover giving
  approximately 365/40 generations annually)

• Cons
• Creating a representative phenotype is
  difficult
• Ethical dilemma (Animal Rights Institutional
  Animal Care and Use Committee (IACUC)
• Can be very expensive.
• Difficult to replicate.

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Mouse Genome Informatics
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Determining Genetic Influences on a Trait

• Response to 30 generations
• of selection for Open-Field
• Activity in Lab Mice (DeFries et al. 1978)

• Strain A collective of animals from the same
  species that are genetically identical.
• Response to selection DeFries et al. 1978
• It is possible to observe variation within and
  between strains of mice.
• Variation on a measured trait within a stain is
  indicative of environmental influences on that
  trait.
• Variation on a measured trait between strains is
  indicative of genetic influences on that trait.
• For eg. The A/J inbred strain is susceptible to
  arthritis while the C57BL/6J strain is resistant.
  (Source Adapted from Bioinformatics and
  Functional Genomics, p. 685)
• C57BL/6J mice consume far greater amounts of
  alcohol than DBA/2J mice.

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Why does Emotionality in mice matter?

• Humans
• Includes anxiety and neuroticism
• Think about what happens to you when you are
  anxious?

• Rodents
• Exploration, activity, anxiety, fear and
  emotionality/reactivity are measured in an open
  field apparatus (OFA)

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Why does Emotionality in mice matter?

• Humans
• Includes anxiety and neuroticism
• Think about what happens to you when you are
  anxious?
• (Sweaty, elevated heart rate, preoccupied
  thoughts, urge to defecate, involuntary movement,
  Immobile)

• Rodents
• Low activity high defecation scores define
  emotionality measures are genetically correlated
• Animals with high defecation scores are inactive

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Breeding Selection Experiment
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Differences within and between strains
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Differences btw Selected Lines
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Peculiar Association?
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What Can We Do With Mouse Models
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Database of Mouse Models
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Disease Genes and Polymorphisms

• The Basics
• Typically transgenic mice are used to show how
  the over-expression of a gene product affects
  physiology, behavior, etc.
• Costs 3000 per mouse
• Relatively straightforward procedure.
• Transgenic mouse can be made in 3-6 months.

• Targeted Transgenesis in Mice
• - Methodology
• - Example
• - Issues
• Targeted Mutagenesis in Mice
• - Types of mutagenic mice
• - Methodology - Example
• - Issues
• Definitions
• Transgenesis The transfer of human genes into
  animals or cultured cells, esp. to produce
  molecules with therapeutic value.
• Mutagenesis The occurrence or induction of
  mutation

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Disease Genes and Polymorphisms

• Targeted Transgenesis in Mice
• - Methodology
• - Example
• - Issues
• Targeted Mutagenesis in Mice
• - Types of mutagenic mice
• - Methodology - Example
• - Issues

Adapted from Hedari Laboratory, Wayne State Univ.
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Disease Genes and Polymorphisms

• Alzheimers Transgenic Mice

The APP transgenic mouse has been the most
popularly used model with neuritic plaques
conspicuously displayed by the age of 12-13
months of age

• Targeted Transgenesis in Mice
• - Methodology
• - Example
• - Issues
• Targeted Mutagenesis in Mice
• - Types of mutagenic mice
• - Methodology - Example
• - Issues

The development of transgenic mice that are
doubly transgenic for APP and presenilin-1 (PS-1)
yielded mice that developed plaques at a much
earlier age than the singly transgenic APP mouse.
Pictures of Alzheimer's Pathology
Stain Campbell-Switzer Silver Method
Excert from
Neuroscience Associates Labs http//www.neuroscien
ceassociates.com/Stains/alzheimers.htm
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Disease Genes and Polymorphisms

• Issues
• Integration of transgene is random.
• It may disrupt the function of another gene
• May integrate into a part of the genome where
  gene expression is suppressed.
• May integrate into a part of the genome under the
  control of a locus control region.
• Number of copies cannot be controlled.

• Targeted Transgenesis in Mice
• - Methodology
• - Example
• - Issues
• Targeted Mutagenesis in Mice
• - Types of mutagenic mice
• - Methodology - Example
• - Issues
• Definitions
• Locus Control Region (LCR) region of the
  chromosome that is responsible for the expression
  of linked genes to physiologically appropriate
  levels in specific tissues at developmentally
  appropriate time points.

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Disease Genes and Polymorphisms

• Issues
• Localization of expression cannot be controlled
  completely.
• Over time, the transgene is frequently shut
  off.
• These issues can be overcome by generating
  several transgenic lines using the same construct
  and comparing the data across lines.

• Targeted Transgenesis in Mice
• - Methodology
• - Example
• - Issues
• Targeted Mutagenesis in Mice
• - Types of mutagenic mice
• - Methodology - Example
• - Issues

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Disease Genes and Polymorphisms

• The Basics
• Targeted mutant mice Alteration of gene of
  interest through homologous recombination
• The gene of interest can be eliminated (knock-out
  or null mutant) or altered (knock-in).
• Loss of gene function or alteration of function
  is typically evaluated in KO and KI mice

• Targeted Transgenesis in Mice
• - Methodology
• - Example
• - Issues
• Targeted Mutagenesis in Mice
• - Types of mutagenic mice
• - Methodology - Example
• - Issues
• Definitions
• Mutagenesis The occurrence or induction of
  mutation

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Disease Genes and Polymorphisms

• The gene of interest can be conditionally
  deleted or altered.
• Very labor intensive and technically challenging
  methods are involved.
• Typical time to generate a null-mutant mouse is
  1-3 years.

• Targeted Transgenesis in Mice
• - Methodology
• - Example
• - Issues
• Targeted Mutagenesis in Mice
• - Types of mutagenic mice
• - Methodology - Example
• - Issues

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Disease Genes and Polymorphisms

• Knock-out Remove gene or exon of gene that
  renders the gene product non-functional.
• Knock-in Replace the natural gene or exon with
  an altered gene or exon that alters the function
  of the gene product.
• Congenic Introgression of a locus of interest
  from another strain selecting for a given marker
  in the donor strain.

• Targeted Transgenesis in Mice
• - Methodology
• - Example
• - Issues
• Targeted Mutagenesis in Mice
• - Types of mutagenic mice
• - Knock-out/ Knock-in/ Conditional
• Knock-out/ Inducible expression /
• Congenic
• - Methodology - Example
• - Issues
• Definition
• Introgression Infiltration of the genes of one
  species into the gene pool of another through
  repeated backcrossing of an interspecific hybrid
  with one of its parents.

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Disease Genes and Polymorphisms

• Conditional KO Gene is modified so that it is
  deleted only under specific conditions.
• Inducible/Tissue specific expression Gene
  expression is regulated by a drug supplement
  (typically a chemical not endogenous to the
  mouse).

• Targeted Transgenesis in Mice
• - Methodology
• - Example
• - Issues
• Targeted Mutagenesis in Mice
• - Types of mutagenic mice
• - Knock-out/ Knock-in/ Conditional
• Knock-out/ Inducible expression
• - Methodology - Example
• - Issues

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Disease Genes and Polymorphisms

• Targeted Transgenesis in Mice
• - Methodology
• - Example
• - Issues
• Targeted Mutagenesis in Mice
• - Types of mutagenic mice
• - Knock-out/ Knock-in/ Conditional
• Knock-out/ Inducible expression
• - Methodology - Example
• - Issues
• Definitiion
• Chimera - an animal that has two or more
  different populations of genetically distinct
  cells that originated in different zygotes

Adapted from the Fred Hutchinson Cancer Research
Center
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Disease Genes and Polymorphisms

• Gene targeting in the mouse

• Targeted Transgenesis in Mice
• - Methodology
• - Example
• - Issues
• Targeted Mutagenesis in Mice
• - Types of mutagenic mice
• - Knock-out/ Knock-in/ Conditional
• Knock-out/ Inducible expression
• - Methodology - Example
• - Issues

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Disease Genes and Polymorphisms

• Problems with KOs and KIs
• Targeting vector components (such as the marker
  used) may affect the expression of genes close to
  the mutated gene.
• Potential developmental effects, such as
• Altered development
• Compensatory mechanisms
• Knock-out is lethal

• Targeted Transgenesis in Mice
• - Methodology
• - Example
• - Issues
• Targeted Mutagenesis in Mice
• - Types of mutagenic mice
• - Knock-out/ Knock-in/ Conditional
• Knock-out/ Inducible expression
• - Methodology - Example
• - Issues

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Disease Genes and Polymorphisms

• Problems with KOs and KIs
• Genetic background No behavioral trait is
  determined by a single gene.
• It is expensive and labor intensive.
• Mice take a long time to create.

• Targeted Transgenesis in Mice
• - Methodology
• - Example
• - Issues
• Targeted Mutagenesis in Mice
• - Types of mutagenic mice
• - Knock-out/ Knock-in/ Conditional
• Knock-out/ Inducible expression
• - Methodology - Example
• - Issues

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Disease Genes and Polymorphisms

• Conditional targeted mutation Use of Cre
  recombinase to delete genes in a temporal or
  spatial
• manner.
• Cre recombinase is an enzyme that when in contact
  with LOXP sites flanking a region of DNA, it can
  alter the DNA between them.

• Targeted Transgenesis in Mice
• - Methodology
• - Example
• - Issues
• Targeted Mutagenesis in Mice
• - Types of mutagenic mice
• - Knock-out/ Knock-in/ Conditional
• Knock-out/ Inducible expression
• - Methodology - Example
• - Issues

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Disease Genes and Polymorphisms

• Inducible Transgenic Mice
• The inserted gene is activated at a desired
  developmental stage.
• An novel transcription factor designed to bind
  to the gene and cause expression in the presence
  of an activator compound is used.

• Targeted Transgenesis in Mice
• - Methodology
• - Example
• - Issues
• Targeted Mutagenesis in Mice
• - Types of mutagenic mice
• - Knock-out/ Knock-in/ Conditional
• Knock-out/ Inducible expression
• - Methodology - Example
• - Issues

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Disease Genes and Polymorphisms

• Problems of conditional KOs and inducible
  transgenics
• Limited availability of tissue-specific mice to
  knock-out or induce expression in a tissue
  specific fashion.
• Conditional knockout may not delete the gene in
  all of the desired cells.
• Gene induction/repression may not be efficiently
  regulated.

• Targeted Transgenesis in Mice
• - Methodology
• - Example
• - Issues
• Targeted Mutagenesis in Mice
• - Types of mutagenic mice
• - Knock-out/ Knock-in/ Conditional
• Knock-out/ Inducible expression
• - Methodology - Example
• - Issues

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Disease Genes and Polymorphisms

• NEIL 1 KO mouse model of
• Metabolic Syndrome (Vartanian et al, 2006)

• Targeted Transgenesis in Mice
• - Methodology
• - Example
• - Issues
• Targeted Mutagenesis in Mice
• - Types of mutagenic mice
• - Knock-out/ Knock-in/ Conditional
• Knock-out/ Inducible expression
• - Methodology - Example
• - Issues
• Definitions
• Dyslipidemia abnormal concentrations for
  lipoproteins in the blood.

Deletion of the DNA glycosylase NEIL1 gene in
mice results in severe obesity, fatty liver,
dyslipidemia, and insulin resistance symptoms
that are consistent with metabolic syndrome in
humans (a collective title for obesity, insulin
resistance, dyslipidemia, and hypertension in
humans)
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• Voluntary Ethanol Consumption in Chrna6 and
  Chrnb3 Knockout Mice (Vidable,. L, et al. 2008
  Ehringer Lab)

Human studies suggest that a6 ß3 nAch receptors
are associated with alcohol. Alcohol modulates
binding.
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• Voluntary Ethanol Consumption in Chrna6 and
  Chrnb3 Knockout Mice(Ehringer Lab, Summer 08)

Chrna6 Knockouts Results
Chrnb3 Knockouts Results
Blue Male Green - Female
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Disease Genes and Polymorphisms

• Examples of transgenic mice
• Inducible Breast Cancer
• Inducible Memory
• Molecular defects of Alz.
• No Immune System

• Targeted Transgenesis in Mice
• - Methodology
• - Example
• - Issues
• Targeted Mutagenesis in Mice
• - Types of mutagenic mice
• - Knock-out/ Knock-in/ Conditional
• Knock-out/ Inducible expression
• - Methodology - Example
• - Issues

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