Transgenic Mice Flashcards
Why use mice as transgenic models
We share 95% of genes
Short life cycle so rapid breeding
What 3 licences are needed for animal research
Personal
Project
Establishment
What does transgenic/ko/ki need to deliver dna
A vector construct (with selection genes, promoters and regulators)
For non specific trans genesis what is vector injected into first with the insert
Mouse oocytes/eggs
What happens after oocytes are injected in standard trans genesis
They are delivered into a pseudo pregnant mouse and then all offsprings are screened for the insert
What is issue with random standard trans genesis
Need to screen all offspring which takes time
Random integration means you can’t control the gene expression levels
It could interfere with endogenous genes affecting phenotype
What is targeted gene trans genesis vector injected into first
Embryonic stem cells
How are embryonic stem cells screened for target insert/vector
Antibiotic resistance gene in the vector allows selection of embryonic sc
What are correct stem cells injected into after selection and what happens after
Blastocysts (mix of normal cells and embryonic sc)
Injected into pregnant mouse
Offspring chimeras are mated to test germ line transmission of the gene/vector
What is added to vectors to detect gene in offspring
A tag to detect it
Which antibiotic resistance gene is usually used for selection of e sc
Neomycin
What techniques are used to screen embryonic sc
PCR or southern blot
How is vector inserted into embryonic stem cells causing a possible knock out
Homologous recombination and then this integrates the neomycin gene disrupting normal mouse genome
Which vectors are used to knock out all possible genes of mice
Gene traps
What are the 2 sites on gene traps which cause homologous recombination (to inactivate gene)
Frt and loxp
Which enzyme causes homologous recombination between 2 loxP sites on gene traps
Cre recombinase
Which enzyme causes hr of 2 frt sites
Flippase
What happens if you cross a mouse with a gene trap vector and a mouse with cre recombinase
The gene will be knocked out via loxp homologous recombination
What happens if you cross a mouse who’s gene was knocked out via cre recombinase lox p with a mouse with flippase
It reverts it back to a functioning gene (reverts deletion)
How can the cre lox system be used for conditional knock outs
If you cross mouse with the gene trap and flocked allele with lox p sites with mouse with cre recombinase under a tissue specific promoter eg heart
The cre recombinase only knocks gene out in heart
What is good about cre lox system
Can be conditional which stops lethal effects
How can you knock in genes eg putting a mutated gene in
Crispr hdr
Which cells are in cartilage growth plate which allow for proliferation and hypertrophy bone growth
Chondrocytes
How was a mouse used to model MED skeletal disease
Med mutation induced into mouse via homologous recombination HDR
Found mouse shorter
What did they find after knocking in med mutation into mice
Induced apoptosis of chondrocytes, reduced proliferation, er build up of misfolded proteins
What is building of misfolded proteins in er called
Er stress (cause of med and dwarfism)
What is the signalling cascade in er which is in response to misfolded proteins
Upr
Unfolded protein response
Which mutant gene was expressed in mice under cartilage specific promoters to show er stress in chondrocytes
Mutant thyroglobulin, found it accumulated and caused er stress in chondrocytes caused dwarfism/med
Was upr knockout in mice found to help dwarfism or make them even shorter
Even shorter without misfolded signalling
Upr was favoured for chondrocytes
Which drugs were given to mice which reverted dwarfism
Drugs that broke down misfolded proteins causing er stress
