Model organisms: mice
• vertebrates!
• mice are ~ 3 inches long, can keep
many mice in a room.
• generation time is ~ 3 months, so
genetics can be done
• history - scientists have worked with
mice for 100 years
• genetic tools - can introduce extra
genes or remove a specific gene, then
study the effect on development
• Disadvantages: development inside
the mother, hard to see. Expensive!
Large Genome = 3 Gb
The mouse provides a superb model for
human development and disease because
we share virtually ALL of our genes
and use them in similar ways
Kit gene
Figure
1.22
Genetic analysis: creating transgenic mice
Problem:
Find a cell line that can grow in tissue
culture but also retains the potential to
become part of a real embryo.
Solution:
Embryonic stem cells
blastocyst
inner cell mass
Embryonic stem cells:
blastocyst-stage cells
(from inner cell mass)
that have been coaxed
into growing in culture
Blastocyst stage cells can be easily incorporated
into a different blastocyst stage embryo,
allowing production of chimeric mice
mom and
dad have
white fur
mom and
dad have
black fur
mouse with 4 parents!!
Fig. 8.26
A mouse with
3 of its parents
(6 total!)
Fig. 8.26
Adding a gene: Producing Transgenic Mice
Production of Transgenic Mice
Embryonic stem cells (ES
cells) are then incorporated
into blastocysts, with the
hope that they “go germline”.
If so, a line is created
Production of Transgenic Mice
Production of Transgenic Mice
Recipe to "knockout" a gene:
A normal cell has two copies of a gene (ie. BMP7)
RNA
Gene X
RNA
Gene X
1. Insert gene for resistance to the drug neomycin into the
middle of gene X, destroying its function. (Gene X is
contained in a DNA plasmid.)
2. Introduce gene X KO plasmid into ES cells and use
homologous recombination to replace one of the wildtype
copies of gene X with mutant gene.
Mario Cappechi
Neo resistance gene
No RNA
Gene X
RNA
Oliver Smithies
Gene X
Technique for Gene Targeting
#2
#1
#3
Now you have heterozygous ES cells--how do
you make a homozygous mutant mouse?
#4
#5
Now you have a chimeric mouse…
#6
#7
Sometimes the effects are dramatic!
Wild-type
BMP7 knockout
Morphological Analysis of Bmp7
Knockout Mice
Sometimes the effects are not dramatic
--no phenotype!
Mouse models of human disease
allow us to design and test new treatments
CFTR and cystic fibrosis
Oliver Smithies
remember
me?
Wildtype
Ultrabithorax mutant
The Homeotic genes in Drosophila
ANT-C
BX-C
Fig 6.35
Ed Lewis had predicted that the
homeotic genes would shape the body plans
of all animals
In vertebrates
the Hox genes
have been
duplicated,
creating
four clusters
Figure 8.30
Different Hox genes are expressed
at different places along the
anterior-posterior body axis
Knocking out
Hoxc8
Figure 8.30
Partial transformation of the first lumbar
vertebra into a thoracic vertebra by knockout of
the Hoxc8 gene
Genetic analysis
of Hox genes
is more
complicated
in mice
Knocking out
Hoxa10,
Hoxc10 &
Hoxd10
paralog group
Figure 8.30
The duplication of the Hox clusters means
that in the mouse, Hox genes work together
to give each body region its own identity
wildtype
Hoxa10 Hoxc10 Hoxd10
triple mutant
Figure 8.32
Lumbar vertebrae transformed to thoracic vertebrae
Remember the
segment-polarity
genes wingless and
engrailed?
Wg En
Retroviruses can also cause cancer by
inserting next to and thus
activating the expression of proto-oncogenes
retroviral insertion
sites in different tumors
Transcribe to mRNA
5 kilobases
exons
wnt-1 gene
Wnt-1, The mouse homolog of wingless,
is normally expressed at the
midbrain-hindbrain junction
Expresses Wnt-1
Expresses En-1
Wildtype brain
Structures lost in Wnt-1 mutant
Expresses Wnt-1
Expresses En-1
Wildtype brain
Brain of Wnt-1 mutant
Pax6
Rules of Evidence
What type of experiment is this?
Pax6 regulates eye development in
flies, squid, mice, and us
Normal eye
iris
no iris
Aniridia eye
small eye mutant mouse
When eyeless (Pax6 homolog) is expressed at
the ends of fly legs, extra eyes form there!
When squid Pax6 homolog is expressed at
the ends of fly legs, also see extra eyes!
ectopic eye (fly Pax6)
ectopic eye (squid Pax6)
The Pax-3 gene is altered in a
classic mouse mutation
Wild-type
Splotch mutant
Mutations in Pax3 lead to Waardenburg
Syndrome I.
•
•
•
•
dominant mutation
eyes can be different colors
white patch of hair (forelock)
deafness
Why Models Matter
The Example of Mutation of the Kit gene in humans and mice
“Piebaldism”
• Affected individuals are anemic, sterile, deaf, and lack pigment in certain skin cells
• Kit encodes a receptor tyrosine kinase and is required for cell proliferation in neural
crest, blood, and germ cells
Figure
1.22
White spotting
and Steel:
Connecting classic
mouse mutations to
stem cells and cancer
An example of stem cells:
the blood cell lineage
Cells lacking signal behave differently than cells lacking receptor
Mosaics can
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help us
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understand
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gene and thus
protein function
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Thanks, I
needed that!
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mutant
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mutant
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mutant
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If mutant cells lack signal,
they can be rescued by wildtype
neighbors which make signal.
What? I can't
hear you!
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mutant
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mutant
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mutant
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If mutant cells lack receptor,
they cannot be rescued by wildtype
neighbors which make signal.
White-spotting and Steel:
Which is signal and which is receptor??
Experiment #1
Put blood cells from Steel
homozygous mutant
embryos into a wild-type
host.
Experiment #2
Put blood cells from
White-spotting
homozygous mutant
embryos into a wildtype host.
The mutant
blood cells
migrated to the
bone marrow.
These mutant
blood cells did
not migrate to
the bone
marrow.
Steel is the Signal- Mutant cells can still receive information
White-spotting is the receptor- Mutant cells cannot receive
information
Steel encodes a diffusible ligand and White-spotting
(Kit) encodes its transmembrane receptor
tyrosine kinase
domain activated
when Steel binds,
phosphorylating
target proteins
PAX3 activates Mitf
melanin genes
Fig. 3.22
Mutations in MITF lead to Waardenburg
Syndrome II.