L16: Knock-out Gene Mutations Flashcards
Name common model organisms
- Mus musculus (house mouse)
- Xenopus sp (clawed frog)
- Danio rerio (zebrafish)
- Drosophila m (fruit fly)
- C. elegan (neamtode worm)
Why is the mus musculus a popular model organism?
- 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
What 3 licenses are required for animal experiments?
Personal for research, project for the study and establishment for site of study conducted
97% of animal research in the UK is done on what animals?
Mice, rats, fish and birds
What are the main approaches for mouse models
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
Explain the transgenic approach in mouse models
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
How is germline transmission of transgene tested following gene targeted transgenic approach?
- correctly targeted cells micro-injected in mouse blastocysts
- blastocysts transferred to psuedo preganant mouse
- chimeric offspring identified and mated
What does the transgenic approach require?
A simple vector, gene of interest, relevant promoter, 3’ protein tag for detection and poly A tail
Name advantages of the transgenic approach
- cheap and easy to make
- multiple founders are generated
Name cons of transgenic approach
- can’t control site of integration of genome
- wt gene product is still present so may interfere and influence phenotype
Explain the gene targeting approach for KO mice
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
What is gene trapping?
A high-throughput approach used to introduce insertation mutations across genome in mouse ESCs
What does insertion of a gene trap vector result in?
- disrupts gene function
- reports gene expression
- provides convenient tag for identification of insertions site
How is gene trap targeted inactivation through recombination acheived?
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
Name the main purposes of loxP and FRT sites
- loxP allows straightforward KOs to be generated, with cre recombinase allowing for targeted deletion
- they both allow conditional KOs to be generated
How do FRT and loxP sites generate conditional KOs
- 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
What is an advantage of gene targeting for KO mice?
Genetraps are available for virtually all genes
Name cons of gene targeting for KO mice
- can be lethal or have no phenotype
- may not accurately model a known human disease
Explain the use of knock in technology
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
Name pros of knock in technology
- CRISPR/cas9 has revolutionised this approacher & is cheaper
- genomically relevant mouse model of human disease
Name cons of knock in technology
- traditionally time consuming
- expensive
- remaining loxP site may interfere with mRNA stability
Name 3 examples of disease modelling using a variety of transgenics
- 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
Explain the generation of a targeted KI mouse to model human genetics (bone disease)
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
Explain the generation of transgenic ‘phenocopies’ to provide proof of concept for understanding disease mechanisms
e. g. to investigate if stress causes MED
- thyroglobulin expressed in cartillage cells and phenocopy found thus proving theory
