Lecture Homberg

Question 1

Transgenic rodent models

Question 2

What does knockout and transgenic mean?

Answer 2

Knockout means that a gene has been inactivated / not functional

Transgenic is the overarching type with conditional knockout while knockout is specific gene knockout

Question 3

Purchasing knockout mice

Answer 3

• collaborate with researcher who has made a knockout mouse
• Not always easy (specific rules and regulations)

so, then

• buy existing knockout mice at Jackson laboratory
• let company generate a knockout mouse for you (jackson laboratory, sage labs, etc.. ) –> more costly - 25000 to 30000 euros

Question 4

Import of animals and GGO

Answer 4

GGO = Genetisch Gemodificeerde Organismen

When using GGO animals (generated by the insertion of foreign DNA) you need a GGO permit

–> Health status of host and home animal facility are compared by veterinary person

–> Animals are transported (air, road)

–> Animals are placed in quarantaine and tested for micro-organisms: 6-8 weeks and several 1000euros

–> When no additional micro-organisms are detected, the animals are allowed

Whole procedure: several months&raquo_space; hard to be competitive in EU

Question 5

Breeding

Answer 5

Determine the best breeding scheme

Rule: breeding animals have to be crossed every generation with commercially purchased wild-type animals having the same genetic background to prevent inbreeding. Best to use heterozygous animals for this outbreeding.

Question 6

Getting experimental animals +/- X +/- (best option)

Answer 6

+/- X +/- =

25% +/+
50% +/-
25% -/-

Advantage: all mothers same genotype.

Disadvantage: overshoot +/- among offspring and genotyping is necessary

Calculation: 1/8 male -/-

Question 7

Getting experimental animals -/- X -/- or +/+ X +/+

Answer 7

100% -/-
100% +/+

Advantage: no overhoot.

Disadvantage: mothers having different genotypes (influence of maternal care)

Calculation: 1/2 male -/-

Question 8

what is -/-

Answer 8

homoygous knockout

Question 9

+/-

Answer 9

heterozygous knockout (one allele has the gene, and the other does not)

Question 10

+/+

Answer 10

wild-type

Question 11

Certain genes affect maternal care

Answer 11

You are not sure if you are looking at genotype or also maternal care effect

Question 12

What are ‘bad’ mothers and why?

Answer 12

when -/- sometimes mothers are bad, so then you take +/- X -/- and +/- X +/+

50% +/- and 50% -/- or +/+

Advantage: mothers same genotype.

Disatvantage: Overshoot +/- animals.

Calculation: 1/8 male -/-

Question 13

How does breeding work?

Answer 13

Put male and females together in a cage. Sometimes 2 female and 1 male.

usually 2 weeks later the female is pregnant.

Also depends on age (older than 6 month might be more difficult).

Males are fertile longer (over 1 year), but animals are not allowed to be in the animal facility when older than 1 year.

Question 14

Pregnancy in rats and mice

Answer 14

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

Question 15

planning breeding

Answer 15

you want to know when the mother got pregnant . Female only gets pregnant if she is estrous. There is an impedance apparatus which measures voltage (the voltage changes when the female is estrous). then you put female with male in cage over night. Validation is done if female has vaginal plug, either sticking in vagina or falling down. The it is likely that she is pregnant.

Question 16

How to detect female vs male in pups

Answer 16

Distance between the urine tube and sex organs (closer in females compared to males) - only when very young ca. 1 week of age

then later female develops nipples

Question 17

Genotyping

Answer 17

do yourself (PCR and visualization of the size of bands on a gel

send DNA samples to a company : LGC Genomics

Now exact location and sequence where your mutation cause also if you send it to company you need to design primers. otherwise you have to sequence the wild-type and mutated gene

Question 18

How are mouse models made originally

Answer 18

Homozigous recombination:

female rat is given hormones –> super-ovulating agouti female
bred with stud agouti male.

Female is pregnant and in blastocyst stage the embryo is isolated. Remove embryonic stem cells from gray-fur blastocyst and grown in tissue culture.

Question 19

Homologous recombination in EPSCs

Answer 19

1. Targeting vector design
2. Selection for recombination
3. Determination of homologous recombinants
4. injection into E3.5 host blastocyst
5. Transfer into pseudo-pregnant foste mother, birth of chimeras
6. Breed for germline transmission

Question 20

Backcrossings

Answer 20

20 backcrossings to get the mutation and the mouse model that you are interested in (up to 2 years)

animal consists of mixture of cells from different parents. usually for host where the embrionic stem cells that are mutated are introduced to

Make continuous breeding the mutation mouse with the strain you want

Question 21

Why back-crossings

Answer 21

Crossover in chromosomes. Recombination until it get more and more life the wild-type, but keeping the mutation.

Question 22

Backcrossing reexplained

Answer 22

mouse with mixed cells, you cross it with mouse line you are interested in.

You get offspring: you pick the one where the mutation is in in the germline (other is discarted), and then you cross it again with the mouse line you are interested in. You can genotype to make sure the animal contains the mutation, and then you repeat in many generations. you only conserve the mutation but the rest is of the strains you want

Question 23

Strain differences - mouse

Answer 23

Animal models are differently sensitive to an SSRI, so choosing the right model is essential. background mutation has an influence of how genotype

Question 24

Serotonin transporter knockout in different inbred mouse strains

Answer 24

Tested in two tests for anxiety

homozygous knockout vs wild-type

If you want to see difference and introduce anxiety, it is better to use mouse line that is not already anxious. Otherwise you do not see the effect of your genotype

Question 25

RAT genetics

Answer 25

NO ES cells available for rat, mouse is the exception.

In rat they are deviding too fast (so fast, that there is no time to introduce a mutation)

129S female line in mice (not deviding so fast)

Question 26

why are mice often used?

Answer 26

More to choose from

Question 27

How can rat knockout rats be made?

Answer 27

Zinc Finger technology: triplets of base pairs that can recognise a certain piece of the genome. They are connected to nucleasis brought to gene of interest and make a cut.
Zinc finger proteins are naturally capable of binding to short sequences of DNA (usually about 3 base pairs long). The cool part of this technology is that by modifying the structure of the zinc finger protein, we can make it bind to a very specific sequence of DNA.

Non-Homologous End-Joining (NHEJ) –> gene disruption in 70% of NHEJ events by out of frame deletions

Homologous Repair –> targeted insertion : SUCCESSFUL

You always have to check if you inserted the mutation

Question 28

disatvantage of zinc fingers

Answer 28

• Very expensive
• you cannot be sure mutation was inserted

Question 29

CRISPR Cas9

Answer 29

RNA guiding molecule that brings nucleasis in the location of genome where you want to make a cut and then same repair mechanism as in Zink fingers

• much cheaper and better compared to zink fingers

Question 30

CRISPR Cas9 and generating transgenics

Answer 30

put together in a tube:

Cas9 mRNA
guiding sgRNA
(with genome of interest where cut needs to be made)

you can add DNA ligase inhibitor

Scr7-mixture zygote injection

Different typer of repair take place (HDR or NHEJ)

two cell stage and transfer (pseudo-pregnant)

Question 31

Lox P - Cre system

Answer 31

2 components:

gene of interesn inbetween your loxP sites and you have a promoter with a Cre enzime

If you combine the two: loxP sites come together, and everything in-between is cut away –> leading to non-functional gene, i.e. gene function is disrupted.

key of optogenetics, chemogenetics, etc…..

Other manipulations are possible

Question 32

RAT targeted ENU-mediated mutagenesis

Answer 32

used when other alternatives where not existing but knockout rats were needed

ENU is a chemical compound that introduces mutations in fast deviding cells (specifically in sperm of male).

Serotonine knockout (little mutation is sufficiant)

There is random mutations and some animals have multiple mutations

Also very costly because it was not very common to sequence genes.

critical factors: mutation frequency, mutation detection efficiency

–> generated knockout/rats (first knockout rat model in the world)

Question 33

SERT knockout rat models and how to validate them (demonstrate that the gene is absent or not functional)

Question 34

Premature stopcodon in serotonin transporter gene

Answer 34

TGC was made into TGA (subcodon) through chemical mutagenesis

Protein (serotonin transporter) mutation was so that only a small part could be made and the body therefore removed it.

Question 35

How to validate that the serotonin transporter was not there

Answer 35

–> Measuring mRNA levels of serotonin transporter (theoretically can be there, but the levels was almost none) –> all mRNA that is not useful is removed by the body

–> demonstrate on protein level:

first antibodies (but quality of antibodies was questioned)

autoradiography –> use of ligand to bind to target and radioactive compound:

wild-type +/+ (a lot of dark (serotonin transporter)

+/- less than wild type

-/- no dark (no serotonin transporter)

mycrodialysis: measure extracellular neurotransmitters in the brain.

Serotonine levels were higher in the knockout

Serotonine transporter (reuptake of serotonine from synaptic cleft to nerve terminal) if transporter is missing there is more accumulation of serotonin in the synaptic cleft

SSRI was added –> immediate rise of serotonine level in wild-type (no reuptake anymore)

Does not work for knockout because there is nothing to bind to (target - transporter - is gone)

Behavioral validation: measuring anxiety (time spent on open arms of + maze) –> knockout rats were more anxious

Very robust phenotype

Question 36

TPH2 knockout rats

Answer 36

TPH2: enzime to synthetize serotonine

Done with Zinc Finger technology. Targeted particular sequence, piece was cut out so then the gene was not functional

Authors validated model at protein level (Immunostaining on Raphe nuclei) for TPH (enzime that is knocked out), DCC (decarboxylase) - also in serotonin enzime

Intracellular serotonin levels –> TPH2 knockouts the serotonin levels were suuuper low (opposite to serotonine knockouts where serotonine level is super high in synaptic cleft)

This model shows aggressive behaviour (fighting, mounting, chasing) –> very obvious phenotype : not so easy to handle for the researcher

Question 37

Drd1 mutant rats

Answer 37

Just one amino acid change due to ENU mutogenesis.

In second transmembrane domain a hydrophobic isoleucine was turned into hydrophilic serine.

Usually D1 receptors are closed and only opens with G-protein coupling. Due to mutation, the receptor is always open/active. So there is constant activity.

autoradiography D1 –> in homozygous you still see staining, so it is not a total knockout

on cellular level : cloned the D1 receptor and put in cell system. Tagged with other protein that could be recognised by antibodies.

fixed condition: animals were dead (cell membrane is permeable (antibodies can get in))

–> antibody binding to cloned D1 receptor

living cells(where they cannot get in

–> no binding to D1 receptor

conclusion: since receptor is always active, it is internalized (not on membrane). Receptor is not removed, but it is not functional (i.e. functional knockout)

animals were severely impaired in learning

Question 38

DAT knockout rats

Answer 38

validation at protein and DNA level (antibodies against dopamine transporters)

Very clear phenotypes (knockout has much lower body weight –> potential reason could be that the rats are super active) –> model for ADHD

Dopamine transporter is doing the same as in serotonin transporter. mycrodialisis shows very high dopamine levels

Show a lot of hiperactivity, but also a lot of anhedonia

Question 39

Summary

Answer 39

• Homologous recombination technology does not work for rats. However, several alternative technologies have been developed by which it is as easy to make knckout rats as mice. You have to know these different technologies.
• Besides technologies to make transgenic mice and rats there are also technologies to manipulate gene expression in a specific tissue or during a specific period in life (CriSPR-CAS9, LoxP-Cre)
• The effect of gene knockout on behaviour is dependent on the background strain used
• Knockout rats are well suited for extensive behavioural phenotyping
• comparison of different genetic models provides deeper understanding of how a molecule shapes behaviour
• Knockout rats bring the opportunity to understand how gene variance contributes to behaviour, but not to understand gene function (because of compensatory changes)