Transgenic Mice

Question 1

What are the advantages of using transgenic mice?

Answer 1

• their DNA has 80% sequence identity to humans
• 90% similarity at the genome organization level
• only different is position of the centromere on chromosomes
• they have large ltters (~10 pups) so more likely to get geetic phenotype of interest
• plus a short gestation time –> less time consuming than other primates eg monkeys

Question 2

Give an example of how mouse models are used to help understand human cognition/disease?

Answer 2

• lots of mouse models of Alzheimers - can use conditional knock-outs/knock-ins to induce B-amyloid plaques to observe physical and behavioural effects
• addiction research - addicted rats have observable behaviours eg sensitized locomotion; rats also show CPP for morphine, amphetamine and cocaine; rats demonstrate craving by increased lever pressing in response to small dose of drug or drug cue (conditioned stimulus eg flashing light)
• eating disorders –> mice models with DA2Rs –> overexpression of these Rs in female mice was associated with over exercising inr esponse to calorific restriction (fasting) but not when there was no fasting –> demonstrating D2 receptors in the nucleus accumbens core neurons drives robust weight loss only in females during scheduled fasting –> anorexia nervosa potential therapeutic targets genetically target these dopaminergic pathways in the brain’s reward system!

Question 3

When might you use conditional KOs?

Answer 3

When the KO gene is essential for growth –> sometimes KO mice don’t survive so you can induce the KO at on a specific temporal scale, or only in specific tissues (eg only in heart tissues). You can check for this by labelling the KO gene with fluoro tags, and visualising where they are switched on. This is oftehn used for exploring the development of the cortex, for example to see which signalling molecules such as Arc or Synaptin are active at different times/locations.

Question 4

How would you ensure conditional KO is only switched on in specific heart tissue, and how would you identify this?

Answer 4

Place flox allele under specific heart tissue promotor region.
Incorporate GFP gene into the flox +/+ strain , so tissue with fluoresce under cre recombinase

Question 5

What controls do you need for conditonal KO mice models?

Answer 5

1. Cre recombinase, flox +/+ –> cre transgene control to see that the cre hasn’t integrated with random genes, cutting out the wrong gene (off-target effects) this would make a new phenotype that has nothing to do with the KO gene!
2. cre -/-, flox +/+ (homo flox control) –> to see if KO itself (w/o cre) causes any undesired changes in phenotype
3. Sometimes cre +/+ flox hetero -/+ –> sometimes to see if theres an intermediate phenotype

Question 6

How do you identify KO mice?

Answer 6

Diagnostic PCR with hetxhet offspring. 25% will be KO (genetic edit). You design primers to atatch to exons/region surrounding target gene. Then you ungergo PCR and you separate fragments via electrophoresis. Longer fragments won’t travel as far; shorter fragments will be ones with taregt gene removed.

Question 7

Draw the mating process for producing KO mice and conditional KO mice.

Answer 7

answers on lecture slides