The Mouse as a Model Organism Flashcards
What is the most commonly used model organism?
Mice are the most widely used model organism in science, far outstripping any other species.
They are not only used as a whole organism, but immortalised mouse cell lines are also used for research.
Why are mice useful model organisms?
Mice are useful models due to their closer relationship with humans than most others. Most human genes have mouse orthologues, despite mice possessing only 40 chromosomes as opposed to our 46. Our genomes are also organised in a similar manner, with genes clustered into functional groups.
What is the advantage of mouse-human genetic similarity for use as a model organism?
The genetic similarity means that many human genetic diseases also have mouse counterparts. Some mutations, such as one that leads to leptin deficiency, are remarkably well conserved between the species, and others can be recreated through targeted mutation.
What are the two types of mutation study?
The forward genetic approach or the reverse.
A forward genetic approach is based on observation of a phenotype being tracked to a gene allele or mutation that causes it, where the reverse approach (or gene-driven approach) involves creating random mutations and observing the phenotype.
What are the two most commonly used inbred strains?
129, an agouti line and C57BL/6 which has black skin.
What methods can be used to induce random mutagenesis for screens? Why is this useful for screening for mutants?
It is often introduced by DNA damaging chemical agents such as ENU or EMS, or using ionising radiation – particularly UV. Gene trapping can also be used.
Random mutagenesis such as this has the advantage of being unbiased and also high throughput – many mutations can be created quickly. However it is difficult to identify the mutation that causes the phenotype produced.
What is gene trapping?
P̶u̶t̶t̶i̶n̶g̶ ̶t̶i̶n̶y̶ ̶m̶o̶u̶s̶e̶t̶r̶a̶p̶s̶ ̶i̶n̶ ̶t̶h̶e̶ ̶n̶u̶c̶l̶e̶u̶s̶ The use of transposons to disrupt genes in a random way for high-throughput mutagenic studies.
What is gene trapping, and how does it work?
A high-throughput random mutagenesis method used to knockout genes.
Gene trap vectors contain a gene trapping cassette composed of a splice site, reporter gene and polyA termination sequence. This inserts itself into the gene within an intron through homologous recombination, producing a truncated non-functional protein.
What are the advantages and disadvantages of gene trapping compared to other random mutagenesis methods?
Unlike mutagen use, this also reports upon its location within the genome through its provision of a gene-trap sequence tag allowing for rapid identification of the gene that has been knocked out to produce the phenotype.
However, the actual procedure is laborious and does not always produce a measurable phenotypic result depending on the intron targeted.
How are gene trap vectors used?
Gene trap vectors are inserted into the genome of a mouse by microinjection into the fertilised eggs taken from superovulating females.
These usually integrate into the genome during the first few cell divisions of pre-implantation development.
A number of different clones (founders) that have been produced like this must be analysed and positives bred together to produce phenotypically accurate offspring.
How does one produce a transgenic mouse?
- Clone and verify the targeting vector
- Insert into ES cells and select for recombinants, checking individual clones for desirable recombination
- Inject blastocyst with ES cells
- Implant blastocysts into pseudopregnant females
- Chimera breeding and germline transmission
- Breeding and analysis
- Phenotypic analysis
How does homologous recombination work as a gene editing technique?
This is characterised by flanking regions of homology that allow the vector to insert itself into that sequence, replacing what did exist.
Thus by using two homology arms with a different mid-section to the original region the gene or intron/other sequence can be edited.
How can homologous recombination be used to insert sequences?
The homology arms are reversed leading to the vector looping around and into the site. You’ll just have to picture it I’m way too tired to explain it better than that.
What enzyme does the Cre-loxP system depend upon?
This uses the Cre site-specific Recombinase enzyme that was extracted from the P1 phage.
What does Cre site-specific Recombinase do?
This recognises pairs of a 34bp sequence called loxP. When a pair of them are both bound by Cre the sequence in between them is looped and cleaved out, leaving a single loxP sequence left.
After excision they are referred to as post-Cre recombinant loci.
