DB3002 Model Organisms The Mouse

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DB3002Model Organisms - The Mouse

• J Martin Collinson
• School of Medical Sciences
• University of Aberdeen
• m.collinson_at_abdn.ac.uk Tel F55750

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Mice used in the lab
The standard lab mouse has been derived from
wild strains of the common field or house mouse
Mus musculus. Other species, such as Mus
castaneus and Mus spretus, have also been taken
into the lab. They can hybridise with M.
musculus and are used primarily for genetic
mapping.
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Why is the mouse used as an experimental animal
in developmental biology?

• It is a mammal, like humans
• Other lab mammals include rat, macaque, opossum,
  dog, cat, sheep. Some of these are better for
  physiological, neuroscience, or medical
  experiments.
• But mice are small (cheaper to house and feed),
  breed quickly, fully sequenced, and facilitate
  powerful genetic analyses.
• Embryology resembles human. Can genetically
  mutate them to create models of human disease

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Sequence homology to humans
Sequence homology not as good as monkeys, but
much better than chick, fish, frogs
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Mouse genetics

• Developed out of mouse fancy
• Breeding unusual-looking mice for fun was popular
  pastime for Victorian gentlemen selecting for
  spontaneous mutations. The fancy continues today.

Brown
Splotch and Steel
Some fancy mice have been characterised as
mutations in important developmental genes.
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Inbred Strains

• We are all individuals all genetically
  different hair and skin colour, body shape,
  physiology.
• For controlled experiments in developmental
  biology, we normally want all the mice to be
  genetically identical.
• So a number of inbred mouse strains have been
  created such that each individual of the same
  strain should be genetically the same.
• Brother-sister matings (mice dont mind)

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Inbreeding

• At every locus (gene) a wild population of
  animals will be polymorphic, i.e. lots of
  different alleles for any particular gene.
  Contributes to genetic variation in population.
• Inbreeding reduces genetic variation by
  increasing homozygousity at each locus (gene).
• After about 10 generations of brother-sister
  matings from consequetive litters, every mouse in
  the colony will be effectively genetically
  identical.
• Doing this lots of times, starting with different
  wild founder mice has allowed the creation of
  dozens of different inbred mouse strains.
• You might want to think about how inbreeding
  reduces genetic variation in the population and
  why it is bad in wild populations.

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Some inbred lines
Balb/c the common white mouse CBA/Ca
agouti 129/Sv used for ES cells C57Bl/6
black Differences in gene alleles, single
nucleotide polymorphisms (SNPs), and
microsatellite markers between different inbred
strains have been very well mapped we know how
they differ genetically. They breed OK and are
reasonably healthy, but not as healthy as outbred
mice. So outbred, genetically variable strains
are also maintained. e.g. CD1, CF1, Swiss
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Breeding system of mice and early development is
largely like human
Sperm meets egg (oocyte)
Fertilised egg zygote 1-cell embryo
Morula
Blastocyst
Outer layer of blastocyst, the trophectoderm,
contributes to the placenta, the inner cell mass
makes the embryo and some extra-embryonic tissues
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Dont try and remember all this Important to
see that the mouse embryo (the apple green bits)
develops in a cone shape to begin with, and
undergoes gastrulation in a complicated 3-D
system. Not just complicated, but hidden in
uterine walls difficult to study The 3D
arrangement is not like frogs, chickens, fish,
and is even different from humans, but the
tissues and genes involved are the same.
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Towards the end of gastrulation, the embryo
starts to turn, from lying on its stomach, to
form the familiar curled up embryo
shape. Although 3D arrangements at
gastrulation are very different for different
vertebrates, they converge on a common Bauplan
arrangement even mammals have gill sltis at this
stage and then subsequently diverge away again
to form their various body shapes.
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The periods shown here, from head-fold stage at
E8.5, to birth after E18.5, are most easy to
access (dissection relatively straightforward),
and are the periods when most organogenesis is
occurring. Hence most mouse developmental
studies look at these stages.
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Timed matings
Mating happens at night and embryogenesis is
internal how do you know when the embryos are
at the right stage to study? When mice mate,
male leaves a seminal plug in female can check
for it every morning. 12 noon on the day plug is
found is taken as half a day of gestation
(E0.5). E3.5 blastocyst stage E5.5
gastrulation E8.5 E9.5 embryo turning,
closure of neural tube, heart starts to
pump. E12.5 limbs developing, CNS starts to
make connections and become functional E19 E20
born. Motor functions and CNS not yet fully
formed. Much of development is postnatal.
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Accessing the mouse embryo

• In vitro embryo culture
• now relatively straightforward between E8.5 and
  E11.5.
• Can treat embryos pharmacologically or
  electroporate DNA in
• In utero surgery
• Possible to open up mum, and operate on embryos,
  then sew mum up.

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X-inactivation
Males XY. Females XX therefore to maintain gene
dosage, early in embryogenesis every cell in the
female heritably shuts down one of its X
chromosomes (either the maternal or paternal one)
(can see it condensed as Barr body). Every
time a cell undergoes mitosis, the same X
chromosome (maternal or paternal) is condensed in
the daughter cells.
Cornea of female mouse heterozygous for an
X-linked LacZ expressing transgene in of female
mouse. Some cells silence the LacZ transgene
early in development. Some silence the LacZ-.
After X-Gal staining (turns LacZ cell blue) see
clones of blue and white cells that reveal tracks
of cell migration across cornea
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Imprinting

• We inherit 2 copies of each gene, one paternal
  and maternal.
• For most genes, both the paternal and maternal
  alleles can be expressed.
• But for a subset of genes, only the maternal or
  paternal allele is expressed, the other one is
  permanently silenced (e.g. by methylation).
• E.g.
• insulin-like growth factor 2 (IGF2) only the
  paternal copy of the gene is ever expressed
• but for its receptor, IGF2r only the
  maternal copy is ever expressed Dads allele is
  shut down.
• http//users.rcn.com/jkimball.ma.ultranet/BiologyP
  ages/I/Imprinting.html

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Imprinting

• Imprinting means that if experimentally you try
  to create an embryo by fertilising an oocyte
  with the nucleus of another oocyte (rather than a
  sperm), it will always die, because it lacks any
  expression of imprinted genes that require to be
  on the paternal chromosome.
• And vice versa.
• Failure to inherit paternal or maternal alleles
  may cause diseases and cancer.
• Mice are at the forefront of study of mechanisms
  (and reasons for) imprinting.

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The mouse as a modern genetic tool

• Transgenesis and gene knockout.
• Gene knockout by homologous recombination in ES
  cells. Research in mice pioneered the discovery
  and use of ES cells.

Embryonic Stem (ES) Cells are derived from the
inner cell mass (ICM) of the blastocyst, i.e. the
bit that forms the embryo.
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Culture, disaggregate, maintain (feeder layer or
LIF)
ES cells in culture
Blastocyst
Embryonic Stem (ES) Cells are derived from the
inner cell mass (ICM) of the blastocyst. They
can be maintained, with care, for a long
time. Leukemia Inhibitory Factor (LIF) prevents
their differentiation They are pluripotent can
be made to differentiate into many cell types in
culture, and will contribute to all parts of the
embryo if reintroduced into a blastocyst.
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It is possible to remove (knockout) genes from ES
cells and then use these knockout ES cells to
make knockout mice. Uses a process of crossing
over - homologous recombination - to replace a
functional gene with a non-functional piece of
DNA - the targeting vector. Homologous
recombination usually occurs during meiosis
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Suppose you want to replace this red bit of DNA
(in the cell) with a yellow bit of DNA that you
have made.
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Knocking out genes by homologous recombination in
ES cells - need to make a targeting vector
Endogenous gene
ATG
Targeting vector (a piece of DNA)
neoR
regions of homologous DNA sequence
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Bathe ES cells in media containing the targeting
vector Electroporate pass electric shock
through them.
Over the next couple of days, most ES cells will
expel or degrade any DNA they took up A small
proportion will integrate targeting vector into
their genome. A small proportion of those
cells that integrate targeting vector will do so
by homologous recombination. Select
electroporated cells using neomycin and
reintroduce into a blastocyst.
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Integration of target vector by homologous
recombination followed by positive/negative
selection
neoR
Functional gene has been removed from genome, and
as free-floating DNA will be chewed up by enzymes
in nucleus and lost. neoR stably replaces it.
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Use of ES cells to produce genetically modified
mice
selection
transgene electroporation
Analyse survivors and grow up clones of
successfully transfected cells
Implant into uterus of host female
Inject into ICM of host blastocyst (normally
Black)
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The recipient female is mated with a vasectomised
male. Over the next 2 days, her uterine wall
swells and vascularises, ready for implantation
of blastocysts
Uterine transfer of chimeric blastocysts
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The female gives birth to a litter of variably
chimeric mice
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Mate chimeric mice with wild-type
Some of the progeny will carry the required
genetic modification
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Summary

• The mouse is used in developmental biology
  partly because it is a small, cheap, mammal,
  which can be a good model for human genetic
  disease.
• It has been developed as a very sophisticated
  genetic tool that lends itself to forward and
  reverse genetic analysis.
• Disadvantages are practical and logistic it is
  still not as cheap or small as some other models,
  and there are greater ethical issues than other
  models.
• Also embryogenesis is internal, not easily
  experimentally accessible.
• Most work on signalling during early
  embryogenesis up to around gastrulation has been
  easier in frogs, fish, and chicks (but they are
  not as genetically accessible).