Transgenic Mice

Question 1

Why use mice as transgenic models

Answer 1

We share 95% of genes

Short life cycle so rapid breeding

Question 2

What 3 licences are needed for animal research

Answer 2

Personal
Project
Establishment

Question 3

What does transgenic/ko/ki need to deliver dna

Answer 3

A vector construct (with selection genes, promoters and regulators)

Question 4

For non specific trans genesis what is vector injected into first with the insert

Answer 4

Mouse oocytes/eggs

Question 5

What happens after oocytes are injected in standard trans genesis

Answer 5

They are delivered into a pseudo pregnant mouse and then all offsprings are screened for the insert

Question 6

What is issue with random standard trans genesis

Answer 6

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

Question 7

What is targeted gene trans genesis vector injected into first

Answer 7

Embryonic stem cells

Question 8

How are embryonic stem cells screened for target insert/vector

Answer 8

Antibiotic resistance gene in the vector allows selection of embryonic sc

Question 9

What are correct stem cells injected into after selection and what happens after

Answer 9

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

Question 10

What is added to vectors to detect gene in offspring

Answer 10

A tag to detect it

Question 11

Which antibiotic resistance gene is usually used for selection of e sc

Answer 11

Neomycin

Question 12

What techniques are used to screen embryonic sc

Answer 12

PCR or southern blot

Question 13

How is vector inserted into embryonic stem cells causing a possible knock out

Answer 13

Homologous recombination and then this integrates the neomycin gene disrupting normal mouse genome

Question 14

Which vectors are used to knock out all possible genes of mice

Answer 14

Gene traps

Question 15

What are the 2 sites on gene traps which cause homologous recombination (to inactivate gene)

Answer 15

Frt and loxp

Question 16

Which enzyme causes homologous recombination between 2 loxP sites on gene traps

Answer 16

Cre recombinase

Question 17

Which enzyme causes hr of 2 frt sites

Answer 17

Flippase

Question 18

What happens if you cross a mouse with a gene trap vector and a mouse with cre recombinase

Answer 18

The gene will be knocked out via loxp homologous recombination

Question 19

What happens if you cross a mouse who’s gene was knocked out via cre recombinase lox p with a mouse with flippase

Answer 19

It reverts it back to a functioning gene (reverts deletion)

Question 20

How can the cre lox system be used for conditional knock outs

Answer 20

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

Question 21

What is good about cre lox system

Answer 21

Can be conditional which stops lethal effects

Question 22

How can you knock in genes eg putting a mutated gene in

Answer 22

Crispr hdr

Question 23

Which cells are in cartilage growth plate which allow for proliferation and hypertrophy bone growth

Answer 23

Chondrocytes

Question 24

How was a mouse used to model MED skeletal disease

Answer 24

Med mutation induced into mouse via homologous recombination HDR

Found mouse shorter

Question 25

What did they find after knocking in med mutation into mice

Answer 25

Induced apoptosis of chondrocytes, reduced proliferation, er build up of misfolded proteins

Question 26

What is building of misfolded proteins in er called

Answer 26

Er stress (cause of med and dwarfism)

Question 27

What is the signalling cascade in er which is in response to misfolded proteins

Answer 27

Upr

Unfolded protein response

Question 28

Which mutant gene was expressed in mice under cartilage specific promoters to show er stress in chondrocytes

Answer 28

Mutant thyroglobulin, found it accumulated and caused er stress in chondrocytes caused dwarfism/med

Question 29

Was upr knockout in mice found to help dwarfism or make them even shorter

Answer 29

Even shorter without misfolded signalling

Upr was favoured for chondrocytes

Question 30

Which drugs were given to mice which reverted dwarfism

Answer 30

Drugs that broke down misfolded proteins causing er stress