L16: Knock-out Gene Mutations

Question 1

Name common model organisms

Answer 1

• Mus musculus (house mouse)
• Xenopus sp (clawed frog)
• Danio rerio (zebrafish)
• Drosophila m (fruit fly)
• C. elegan (neamtode worm)

Question 2

Why is the mus musculus a popular model organism?

Answer 2

• We share ~95% of our genes with mice
• They have short life cycle so useful for rapid breeding of strains
• However they’re relatively expensive to keep

Question 3

What 3 licenses are required for animal experiments?

Answer 3

Personal for research, project for the study and establishment for site of study conducted

Question 4

97% of animal research in the UK is done on what animals?

Answer 4

Mice, rats, fish and birds

Question 5

What are the main approaches for mouse models

Answer 5

Transgenic, knockout or knock in
- all approaches required a vector construct but level of complexity differs between each technique and whether it is targeted to a specifc part of mouse genome or not

Question 6

Explain the transgenic approach in mouse models

Answer 6

Can either be standard or gene targeted

• Standard: DNA (gene of interest) microinjected into pro-nucleus of fertilised mouse oocyte and transferred to psuedo pregnant recipient mouse. Offspring then screened for expression of transgene by DNA analysis
• Gene Targeted: isogenic transgene with drug selection gene introduced into ESCs. Drug selection used and surviving cells are screened for correct integration of transgene

Question 7

How is germline transmission of transgene tested following gene targeted transgenic approach?

Answer 7

• correctly targeted cells micro-injected in mouse blastocysts
• blastocysts transferred to psuedo preganant mouse
• chimeric offspring identified and mated

Question 8

What does the transgenic approach require?

Answer 8

A simple vector, gene of interest, relevant promoter, 3’ protein tag for detection and poly A tail

Question 9

Name advantages of the transgenic approach

Answer 9

• cheap and easy to make

- multiple founders are generated

Question 10

Name cons of transgenic approach

Answer 10

• can’t control site of integration of genome

- wt gene product is still present so may interfere and influence phenotype

Question 11

Explain the gene targeting approach for KO mice

Answer 11

Precise but requires complex vector and relies on homologous recombination between vector and host genome

• target vector is electroplated into ESCs establishing homologous recmbination
• then inserted into the correct place usually via PCR analysis
• cells which have transgene correctly inserted are then selected to be injected into pseudo pregnant mouse
• chimaeras bred

Question 12

What is gene trapping?

Answer 12

A high-throughput approach used to introduce insertation mutations across genome in mouse ESCs

Question 13

What does insertion of a gene trap vector result in?

Answer 13

• disrupts gene function
• reports gene expression
• provides convenient tag for identification of insertions site

Question 14

How is gene trap targeted inactivation through recombination acheived?

Answer 14

Using FRT and loxp, these sites mediate site- specific recombination through DNA recognition sites

• cre recombinase catalyses site specific recombination through 2 loxp sites
• flippase catalyses site specific recombination between FRT sites

Question 15

Name the main purposes of loxP and FRT sites

Answer 15

• loxP allows straightforward KOs to be generated, with cre recombinase allowing for targeted deletion
• they both allow conditional KOs to be generated

Question 16

How do FRT and loxP sites generate conditional KOs

Answer 16

• fix recombinased used for site-specific recombination between 2 FRT sites, forming a conditinal allele/ floxed allele
• this mouse is then crossed w a mouse expressing cre recombinase, causing the deletion of that allele
• results in fully deleted allele

Question 17

What is an advantage of gene targeting for KO mice?

Answer 17

Genetraps are available for virtually all genes

Question 18

Name cons of gene targeting for KO mice

Answer 18

• can be lethal or have no phenotype

- may not accurately model a known human disease

Question 19

Explain the use of knock in technology

Answer 19

Mostly used to introduce human mutation into mouse genome

• relies on homologous recombination or section of vector
• DNA that is introduced into mouse genome contains specific mutation

Question 20

Name pros of knock in technology

Answer 20

• CRISPR/cas9 has revolutionised this approacher & is cheaper
• genomically relevant mouse model of human disease

Question 21

Name cons of knock in technology

Answer 21

• traditionally time consuming
• expensive
• remaining loxP site may interfere with mRNA stability

Question 22

Name 3 examples of disease modelling using a variety of transgenics

Answer 22

• generation of a targeted KI mouse to model bone disease
• generation of transgenic phenocopies to provide proof of concept for understanding disease mechanisms
• use of mouse KOs to probe disease mechanisms

Question 23

Explain the generation of a targeted KI mouse to model human genetics (bone disease)

Answer 23

All long bone growth occurs in cartillage growth plates and driven by cell proliferation, hypertrophy and apoptosis
- KI mouse generated with matrilin-3 so it will displlay multiple epiphyseal dysplasia (MED)
- Found brown lumps: mutant protein was being misfolded and retained in ER
ER stress induces signalling casacade (unfolded protein response) that attempts to alleviate stress. Failure to do so can cause cell death
- reduced cell proliferation
- increased & spatially dysregulated apoptosis

Question 24

Explain the generation of transgenic ‘phenocopies’ to provide proof of concept for understanding disease mechanisms

Answer 24

e. g. to investigate if stress causes MED

- thyroglobulin expressed in cartillage cells and phenocopy found thus proving theory

Question 25

Explain the use of mouse KOs to probe disease mechanisms

Answer 25

e. g. is the ER stress response to protein misfolding more pathogenic than the mutant protein itself?
- generate model mouse that has Xbp1 branch of UPR ‘knocked out’ and bred with matn3 MED mouse
- results showed disease becomes more severe