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Overview
Models in laboratory research
The unique position of the mouse as a model
Mouse genetics 101
Genetic engineering for hypothesis testing
Genetic variation
Why Use Models?
Allows for controlled experiments
Environmental variables can be controlled
Dosage or exposures can be controlled
Experiments can be replicated
Phenotypes
Genes
Environment
Chapel Hill
Stochastic
Processes
Effects of Genes Can Be Complex
Gene
Pleiotropy
Phenotype
Heterogeneity
Phenotypes
Genes
Unique Position of the Mouse
Histology
Gene Content
gataccagattagt
agttagagttgaga
gtccgctagatcgc
Strain: A/J
ID: XXXX
Tissue: col
Genetics
Genetic
Tools
Molecular
Profile
Biologic
Tools
Proteome
Content
Mutagenesis
Physiology
DETECTION
CHARACTERIZATION
VALIDATION
Engineering
Tools
Transgenics
Knockouts
Knockins
Why Mice As an Experimental Organism?
Hardy
Requires little space
Short life cycle
Easily bred
High fecundity
Mammalian species
Large amount of phenotypic variation
Easy to genetically engineer
Evolutionary Relationships
Humans
Mice
Xenopus
D. melanogaster
C. elegans
1000
900
800
700
600
500
400
300
200
100
0 myr bp
The Mus Species Group
domesticus
musculus
castaneus
bactrianus
The
M. musculus
group
macedonicus (Greece>Iran>Israel)
spicilegus (Austria.Ukraine>Bulgaria)
spretus (Spain/N. Africa)
caroli (Indonesia)
cookii (India/S.E. Asia)
cervicolor (Nepal/S.E. Asia)
booduga (India/Burma/Pakistan)
dunni (India/Sumatra)
6.0
5.0
4.0
3.0
2.0
1.0
0 myr bp
Genetic Stocks
Outbred
• segregating many alleles
• ex, Swiss mice, Wistar rats
Hybrid
• isogenic until bred
• ex, B6D2, B6SJL
Inbred
• genetically identical (isogenic)
• ex, C57BL/6J
Mutant/Engineered
• specific defects
• may or may not be ‘genetically clean’
Exercise A
You have developed a compound that you think will help
to prevent rejection of transplanted hearts, and you
want to test this experimentally.
The experiment will involve heart grafts between a
donor and recipient mouse (whose own heart is not
removed) with a control group and one treated with the
compound.
The following mouse strains are available: outbred ICR
and CD1 and inbred C57BL/6J, A/J, and FVB/NJ.
Which strains will you use as donor and recipient?
Why?
Exercise B
You also need to test the potential toxicity of the
compound, and will want to do a long-term study with
control and treated mice. You know it is not acutely
toxic.
Being a toxicologist, you reason that in this case since
you wish to model humans who are genetically
heterogeneous, you decide to use outbred genetically
heterogeneous ICR mice, the strategy used by virtually
all toxicologists.
Do you decide to go with your initial intuition? Why?
Identify the ‘Experimental Unit’
The unit of randomization
The experimental units must be capable
of being assigned to different
treatments
Could be:
a cage of animals
a single animal
an animal for a period of time
a well in a tissue culture dish
The Problem With Genetic Heterogeneity
Treated
Control
beagle
mouse
horse
gerbil
lion
cat
rabbit
chicken
crow
frog
hamster
beaver
dog
toad
A Better Design
Treated
Control
beagle
mouse
horse
gerbil
lion
cat
rabbit
beagle
mouse
horse
gerbil
lion
cat
rabbit
A Better Design
Treated
Control
C57BL/6J
A/J
FVB/NJ
DBA/2J
SWR/J
SJL/J
BALB/cJ
C57BL/6J
A/J
FVB/NJ
DBA/2J
SWR/J
SJL/J
BALB/cJ
Variable Results With Heart Transplants
‘We transplanted hearts of young … ICR
into … recipient CD1. An outbred strain
was selected since such animals are
usually heartier and easier to handle …
We are puzzled by our results … palpable
heart beats were evident in the saline
group long after acute rejections … were
expected … Results in the experimental
groups varied considerably …’
Exercise C: Power Calculations for Sample Size
Barbiturate sleeping time
Strain
BALB/c (inbred)
ICR (outbred)
Mean
Std. Dev.
40
40
4
15
What sample size would be needed to
detect a 10% change in mean, with a 90%
power and 5% significance level using a
2-sample t-test?
Data from Jay (1955) Proc Soc exp Biol Med 90:378
Power Calculations for Sample Size
BALB/c (inbred)
ICR (outbred)
23
297
Types of Genetic Crosses
Cross
Backcross
Matings
Uses
A/a X A/A
linkage analysis;
production of congenic
strains
A/a X a/a
A/A X A/A
a/a X a/a
Maintenance of an
inbred strain
Intercross
A/a X A/a
Linkage analysis
Outcross
A/A X a/a
Initial step in strain
production and linkage
analysis; production of F1
hybrids
Incross
a1/a2 X a3/a4
Making an Inbred Strain
% Homozygosity
P
0
50
100
F1
Independent
Assortment
Inbred
F2
F20
98.7%
Inbred Strain
20 or more generation of brother x sister mating
Isogenic
Homozyogus
Phenotypically uniform
Long-term stability
Unique strain characteristics
International distribution
Easily identifiable
‘immortal clone of genetically identical individuals’
EgfrWa5/+
C57BL/6J
129S1/SvImJ
BALB/cJ
‘The introduction of inbred strains into
biology is probably comparable in
importance with that of the analytical
balance into chemistry’
Gruneberg (1952)
Outbred Stocks
Widely used
Characteristics not widely understood
Advatages
• cheap
• easily available (no alternative for some species)
• breed well
• outbred like humans (?????)
Disadvantages
• unknown genetics (heterozygosity)
• subject to genetic change (inbreeding, drift, selection)
• lack of reliable background information
• genotype not internationally distributed
• not histocompatible
• not easily identifiable
Outbred Mice
CF1
ICR
CD1
MF1
Swiss-Webster
B6D2
Engineered Models
Allows controlled experimental testing of
• specific genes
• specific environmental conditions
or exposures
Ideally suited to test specific hypothesis
generated from human population studies
or other laboratory findings
Engineered Models
Transgenics
• usually used to over-express genes
• can be global or tissue-specific
• can be temporally regulated
Knockouts/knockins
• usually used in inactivate genes
• can be global or tissue-specific
• can be temporally regulated
• can introduce genes into a foreign locus
• can make amino acid modifications
Terminology
Transgenic
(carries foreign DNA;
may or may not be mosaic;
two parents)
Mosaic
(may or may not carry
foreign DNA;
two parents)
Chimeric
(may or may not
carry foreign DNA;
more than two parents)
Mosaic vs Chimeric Progeny
Mosaic
Chimeric
Pre-implantation Mouse Development
Fertilization
E0.5
Activation of embryonic genome
E1.5
Compaction
E2.5
Blastocoelic fluid
accumulation
E3.5
TE and endoderm
differentiation
Transgenic Production
Flush fertilized
oviduct E0.5
pro-nuclear stage
DNA
pro-nuclear
fusion
recover
test for expression
and phenotype
test for germline
transmission
implant into
pseudopregnant females
founder
Gene Targeting in ES Cells
electroporate
selection
analyze
colonies
test for
germline
transmission
cross
heterozygotes
Analyze offspring
for phenotype
P 1 2
test offspring
for chimerism
implant
make
chimeras
Humanized Mice
Gene knock-ins have been generated to introduce a
variety of human genes that have relevance to
toxicology: CYP, MHC, etc.
Humanized Mice
Another approach to humanizing mice is tissue
replacement via inter-species chimeras. This has been
achieved for liver by using immunodeficient mice on a
liver toxic transgenic background (urokinase-type
plasminogen activator) by injecting human liver cells IV.
Up to 90% of the mouse hepatocytes can be replaced
by human hepatocytes.
Exercise D
You obtain a mouse line from your collaborator that
carries a knockout in PPAR-gamma. The collaborator
made the mice by gene targeting in 129 strain derived
ES cells. He made chimeras with the cells by
blastocyst injection into C57BL/6J embryos. After
birth, he bred the chimeras to C57BL/6J mice and
then intercrossed heterozygous carriers to make the
PPAR-gamma knockout homozygous line.
You need wild-type controls for your experiment. What
mice to you use for this? Why?
Making a Congenic Strain
Donor Recipient
P
0
% Homozygosity
50
100
F1
Independent
Assortment
Residual
Heterozygosity
N2
N3
N10
Congenic
99.8%
Congenic
1
2
3
Co-isogenic
4
5
1
2
3
4
5
Genetic Variation
Significant extant genetic (and thus phenotypic)
variation exists across mouse strains
Can be used to identify a more ‘accurate’ model
of specific human exposures or responses
Phenotypic variation across inbred mouse strains
is as great or greater than humans for virtually
any trait
Azoxymethane Induction of Mouse Colorectal Tumors
4 X 1 weekly IP injections (10mg/kg)
Tumor
22 week latency period
Azoxymethane
CH3
N
N
CH3
N
CH2OH
O
Methylazoxymethanol
CH3 N
O
Methyldiazonium
(alkylating agent)
Carbonium
(methylating agent)
+
CH3
H3C
N
+
N
+ N2
Normal
....one
mouseisstrain
is not
Just as
one person
not representative
representative
of population....
the human species
of the human
AKR/J
SWR/J
A/J
Colorectal Cancer Susceptibility
100
75
Swiss strains
Castle’s strains
Strains from Asia
Other strains
C57-related strains
Wild-derived strains
50
25
0
Strain
mouse populations > human populations
Total mouse SNPs = ~40M
musculus, domesticus, castaneous
mouse populations > human populations
Total mouse SNPs = ~40M
musculus, domesticus, castaneous
Total human SNPs = <20M
mouse populations > human populations
Total mouse SNPs = ~65M
musculus, domesticus, castaneous
+ spretus
Total human SNPs = <20M
Why would human and mouse
biology be similar?
Evolutionary conservation!!
- genome (gene content,
arrangement and sequence)
- structure (gross and molecular
anatomy)
- function (physiology and
molecular circuits)
Why would human and mouse
biology be similar?
Evolutionary conservation!!
- Phenotypic differences are
due to a finite set of key genes
- “Key” means nodes in
regulatory networks