Knock-out mice and conditional mice Flashcards
What are knockout mice?
-The creation of a null (completely non-functioning) mutation for a specific gene
What are knockout mice sometimes used to model?
-Sometimes used to model a human loss of function mutation/disease
How are knockout mice usually made?
-Usually done by removing all coding exons, or essential exons near the 5’ end of the gene
How are mouse models of cystic fibrosis created?
- Cystic fibrosis caused by an inactivating mutation of the CTFR by insertion of exogenous gene sequences
- CTFR delta F508 mutation introduced by homologous recombination
- CTFR null mice have a more severe phenotype than deltaF508 mutations
What happens to the mouse mutants of cystic fibrosis and due to what?
- Both mutants die shortly after birth
- But from intestinal obstruction, not lung problems
What is Cre?
-Cre is a site-specific recombinase
What is loxP?
34bp recognition site of Cre
What is the structure of loxP?
-2 inverted (palindromic) repeats and core GCATACT
What does the effect of cre-loxP recombination depend on?
-Effect depends on the orientation of the loxP sites
What happens if the 2 loxP sites are placed in the same orientation on the same piece of DNA?
- Most commonly, the loxP sites are placed in the same orientation leading to a recombination of the 2 sties
- The recombination will lead to the removal of the intervening sequence between the loxP sites
What does adding cre recombinase to 2 loxP sites present on 2 different sites of DNA in the cre-loxP system lead to?
-Adding Cre recombinase will lead to crossing over of sequences
What does adding cre recombinase to 2 loxP sites present on the same piece of DNA in the cre-loxP system lead to?
-Adding Cre recombinase will cause recombination between the 2 loxP sites and removal of intervening sequence
What is a conditional allele referred to as?
Conditional allele which is referred to as a floxed gene
Do mammals have cre-recombinase?
-Mammals don’t have Cre-recombinase
How are loxP sites inserted into DNA?
- To insert loxP sites, the region of genomic DNA is cloned into bacteria and modified, then used to generate targeted embryonic stem cells by homologous recombination
- Targeted ES cells are used to generate mouse line with a floxed allele
- These mice are normal
- Then those floxed mice are crossed with a Cre mouse and that causes recombination between the 2 loxP sites in the same orientation and that’s now the deleted gene
What are the 3 ways that expression of Cre can be driven?
- It can be knocked in to an existing gene, so controlled by that gene’s promoter
- It can be driven by a promoter of its own-this could be expressed in all tissues or some and expressed all the time or at specific times
- It can be turned on/off by specific drugs that are given to mice
How is Cre made conditional?
- A fusion protein of Cre-ERT is used (expression driven in all tissues or in specific cell types)
- ERT is a modified version of the ligand binding domain of the oestrogen receptor with high affinity for tamoxifen
- ER is normally held in the cytoplasm by Hsp90
- Addition of tamoxifen causes Hsp90 dissociation and the Cre-ERT protein translocates to the nucleus where Cre mediates loxP recombination
What is the FLP-FRT system and how does it work?
The FLP-FRT system is similar to the Cre-lox system. FLP recognises FRT sequences that flank a genomic region of interest
What is a possible workflow for creating a mouse model of human genetic disease?
A possible workflow for creating a mouse model of human genetic disease:
- Disease associated gene identified in humans by genetic techniques
- Question what does the gene do and where its expressed
- Is there a mouse knockout already? Check IMPC
- Are there any known publications or phenotypes in the mouse
- Obtain or create specific mutants, usually starting with germline mutation
- Is a conditional mutation needed?
