Download Animal Models of human DCG`s

Mouse Models of Human
Disorders
Prepared by
Rohan Palmer
Institute of Behavioral Genetics
University of Colorado at Boulder
[email protected]
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
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 DCG’s.
Your classmate has not yet taken Dr. Carey’s 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)
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
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
Why Create Animal Models?
Pros
1. Test many hypothesizes
2. No human genetics
ethical dilemma
3. Shortens length of study
1.
2.
(Avg. gestational period is 19 days)
(Females become fertile in 3 weeks)
(Approximately 40 days for turnover;
giving approximately 365/40
generations annually)
3.
4.
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.
Mouse Genome Informatics
Determining Genetic
Influences on a Trait
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.
Response to 30 generations
of selection for Open-Field
Activity in Lab Mice
(DeFries et al. 1978)
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)
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
Breeding & Selection Experiment
1st 10 litters
H1 line
L1 line
2nd 10 litters
H2 line
L2 line
Balb
The entire breeding
experiment
used a total of 14,184
mice
3rd 10 litters
C1 line
x
F1
B6
x
F2
F1
x
F30
F2
4th 10 litters
C2 line
Differences within and between strains
Differences btw Selected Lines
Peculiar Association?
What Can We Do With Mouse Models
Database of Mouse Models
Disease Genes and
Polymorphisms
•
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
•
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.
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.
Disease Genes and
Polymorphisms
Targeted Transgenesis in Mice
- Methodology
- Example
- Issues
Targeted Mutagenesis in Mice
- Types of mutagenic mice
- Methodology
- Example
- Issues
Alzheimer’s 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
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.neuroscienceassociates.com/Stains/alzheimers.htm
Disease Genes and
Polymorphisms
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.
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.
Disease Genes and
Polymorphisms
Targeted Transgenesis in Mice
- Methodology
- Example
- Issues
Targeted Mutagenesis in Mice
- Types of mutagenic mice
- Methodology
- Example
- Issues
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.
Disease Genes and
Polymorphisms
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
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
Disease Genes and
Polymorphisms
Targeted Transgenesis in Mice
- Methodology
- Example
- Issues
Targeted Mutagenesis in Mice
- Types of mutagenic mice
- Methodology
- Example
- Issues
• 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.
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 /
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.
• Knock-out: Remove gene or
exon of gene that renders
the gene product nonfunctional.
• 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.
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
• 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).
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
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
Gene targeting in the mouse
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
Problems with KO’s and KI’s
• 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
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
Problems with KO’s and KI’s
• Genetic background: No
behavioral trait is
determined by a single
gene.
• It is expensive and labor
intensive.
• Mice take a long time to
create.
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
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.
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
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.
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
Problems of conditional KO’s
and inducible transgenics
• Limited availability of tissuespecific 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.
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)
Voluntary Ethanol Consumption in Chrna6 and
Chrnb3 Knockout Mice
(Vidable,. L, et al. 2008; Ehringer Lab)
Human studies suggest that α6 & β3
nAch receptors are associated with
alcohol. Alcohol modulates binding.
Types & Forms of nAch Receptors
Two Bottle Paradigm: Alcohol vs. Water
Voluntary Ethanol Consumption in Chrna6 and
Chrnb3 Knockout Mice
(Ehringer Lab, Summer ‘08)
Chrnb3 Knockouts Results
Blue – Male; Green - Female
Chrna6 Knockouts Results
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
Examples of transgenic mice:
Inducible Breast Cancer
Inducible Memory
Molecular defects of Alz.
No Immune System
Alternate Resources