MN: Animal Models for psychological and neurological development
Lecture 1
Rats and mice are not the same:
Rats are used for behavioral pharmacology/toxicology, they are more expensive, higher cognitive
functions, bigger and thus more tissue, social and easy to handle, outbred (not the same genetic
background).
Mice have genetic tools, they are cheaper and less housing space, they have lower cognitive
functions, they are smaller and thus less tissue, they are less social, and they are inbred (same
genetic background, it is probably less translatable to humans because humans are heterogenic).
The brain of animals is organized in the same way, but it is not the exact same. You cannot say that a
mouse has the same disease, but you can look at e.g. memory. For these basic functions, rodent
models can be used.
Face validity: similarity in symptoms between humans and animals (there could however be other
reasons to get the symptoms)
Predictive validity: similarity in drug effects (drug is working in both, however some mice don’t
respond to certain drugs, which is the same for patients)
Construct validity: similarity in neural substrates (there are also challenges here, in humans you look
for instance in fMRI and in mice on the cellular level, can you draw conclusions then?)
It is very hard to start animal experiments, it takes a lot of weeks and steps to go through.
Lecture 2
Things to take into account before starting the animal experiment
are allowed to enter the animal facility.
Whole procedure: several months > hard to be competitive in EU.
You also have to think about the genotypes of the animals. You have the WT animal (+/+), +/- animals
and the -/- animals (full knockouts). You can breed the heterozygous animals where you will get a
mixture of genotypes in the offspring (25% +/+, 50% +/-, 25% -/-).
Advantages: all mothers have the same genotype (which is only +/-).
Disadvantage: you have an overshoot of +/- among offspring and genotyping is necessary.
You can also do this the other way around (so -/- with -/- or +/+ with +/+)
Advantage: no overshoot
Disadvantage: mothers having different genotypes (+/+ or -/-) (influence of maternal care)
,You can also do +/- times -/- or +/- times +/+ which will give 50% +/- and either 50% -/- or +/+
Advantage: mothers have the same genotype (+/-, the male will be -/- or +/+)
Disadvantage: overshoot +/- animals
Animal pregnancy
You have to plan the breeding: sometimes you want to know when the female is getting pregnant.
Usually, after 2 weeks the female rat is pregnant. When a female animal is older than 6 months and
has never delivered a litter before, the chance that she gets pregnant decreases. Males remain fertile
for a long time, over the age of 1 year, animals are not allowed to be in the animal facility when
being older than 1 year.
The pregnancy is 19-20 days for mice, 21-22 days for rats. At 3-4 weeks after birth the pups are
weaned, and can be earclipped for genotyping.
An impedance apparatus will check for the estrous phase of the female which will say whether the
female is pregnant when the score is higher than 5.
The difference between male and female pups is as follows: the distance between the genitals and
the uterus is bigger in males than in females.
Generating genetic models
You can use PCR and visualization of the size
of bands on a gel. You can also send your
DNA samples to a company.
How genetic models are generated: the first
method is homologous recombination
method only in mice. Here you firstly ensure
that the vector design will cross with the
genome. In this way you get ES cells with the
recombination (so you have KO and WT
ESCs). Then both cells are injected into E3.5
host blastocyst which is then brought into
the pseudo-pregnant mother. The offspring
can then have the mutation (25%), be WT
(25%) or be a chimera (50%). The last step is
to breed for germline transmission. This can
be seen in the figure.
Problems: multiple insertions in the DNA: too
much protein, insertion into a life-necessary gene:
lethality, insertion into a gene leading to gene-
silencing: no protein, insertion in a different area
can lead to differential gene regulation,
background genotype can influence the effect of
the mutation.
Backcrossing
You have the chimeric mouse but it is only
interesting when the germ cells have this
mutation. When you cross the chimeric mouse
model and cross it with a black WT mouse, you get
offspring and you select the transgenic mice and
, cross them again with the black mice. For every generation, the chance of getting the transgenic
mouse will increase. The WT mice are discarded after the breeding.
In rats the ES cells already start to divide before you insert the vector with the mutation. This
technology thus doesn’t work for rats, only for mice.
Generating genetic models with other techniques
Zinc Finger: they use endonucleases that can cut the DNA at a very specific location in the genome.
These nucleases are coupled to the Zinc Fingers (triplets of base pairs which are specific to a
particular location in the genome). You can then have two types of repairs. The first is homologous
repair where you show the template and based on the template, the DNA is repaired with a piece of
the template (targeted insertion). The other one is non homologous end-joining (NHEJ) where the
DNA tries to fix itself but in this way can delete certain genes by accident (knockout).
CRIPSR Cas9: the difference is that this system uses an RNA guiding molecule. The Cas9 (nuclease)
will cut the DNA at the place where the RNA guiding molecule can bind to. So you put in Cas9 mRNA,
sgRNA and DNA ligase inhibitor. Then you inject this mixture in the zygote where you will either see
HDR or NHEJ. Then you let the mouse grow in the mother and you get the mutated version.
Tet on/off: it consists of a trans activator component with a promoter and TetR. This trans activator
can travel to another part of the genome and can then bind to another promoter and either turn on
or off transcription of the gene of interest. When you have Tet-off and when doxycycline is present,
the trans activator cannot bind to the promoter. Tet-on and doxycycline is present: there will be
activation.
Lox P – Cre: you have a LoxP and a Cre mouse that you cross. The offspring will have both genes. Cre
will cut out the area within two LoxP sites and will remove it from the DNA. In this way you can cut
out certain genes from the DNA.
Rat targeted ENU-mediated mutagenesis : you give this compound to male and there will be
mutations in the sperm. When you mate the males and females with each other, the offspring would
get mutated. The problem was that you didn’t know where the mutation was. This could be solved
by genotyping every offspring rat (but one animal could have more mutations than the other one)
and you can eventually see which gene was mutated in the knockout rats.
Lecture 3
Experiment with SERT knockout rats: Northern blot, autoradiography
These knockout rats have high serotonin levels in their brain (the transporter normally removes
serotonin from the synaptic cleft). TPH2 is the enzyme that is synthesizing serotonin. So you can
compare SERT knockout with TPH2 knockout rats.
SERT knockout rats are made by
changing TGC to TGA (stop codon)
where the proton translation will
stop. This small piece of protein is
thus not being made in the
serotonin transporter. A northern
blot analysis was used in order to
show mRNA levels. From this
northern blot you see that the
serotonin mRNA levels are very
low. Furthermore, they did
autoradiography of the rat brain
