Knock-out mice and conditional mice

Question 1

What are knockout mice?

Answer 1

-The creation of a null (completely non-functioning) mutation for a specific gene

Question 2

What are knockout mice sometimes used to model?

Answer 2

-Sometimes used to model a human loss of function mutation/disease

Question 3

How are knockout mice usually made?

Answer 3

-Usually done by removing all coding exons, or essential exons near the 5’ end of the gene

Question 4

How are mouse models of cystic fibrosis created?

Answer 4

• 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

Question 5

What happens to the mouse mutants of cystic fibrosis and due to what?

Answer 5

• Both mutants die shortly after birth

- But from intestinal obstruction, not lung problems

Question 6

What is Cre?

Answer 6

-Cre is a site-specific recombinase

Question 7

What is loxP?

Answer 7

34bp recognition site of Cre

Question 8

What is the structure of loxP?

Answer 8

-2 inverted (palindromic) repeats and core GCATACT

Question 9

What does the effect of cre-loxP recombination depend on?

Answer 9

-Effect depends on the orientation of the loxP sites

Question 10

What happens if the 2 loxP sites are placed in the same orientation on the same piece of DNA?

Answer 10

• 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

Question 11

What does adding cre recombinase to 2 loxP sites present on 2 different sites of DNA in the cre-loxP system lead to?

Answer 11

-Adding Cre recombinase will lead to crossing over of sequences

Question 12

What does adding cre recombinase to 2 loxP sites present on the same piece of DNA in the cre-loxP system lead to?

Answer 12

-Adding Cre recombinase will cause recombination between the 2 loxP sites and removal of intervening sequence

Question 13

What is a conditional allele referred to as?

Answer 13

Conditional allele which is referred to as a floxed gene

Question 14

Do mammals have cre-recombinase?

Answer 14

-Mammals don’t have Cre-recombinase

Question 15

How are loxP sites inserted into DNA?

Answer 15

• 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

Question 16

What are the 3 ways that expression of Cre can be driven?

Answer 16

• 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

Question 17

How is Cre made conditional?

Answer 17

• 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

Question 18

What is the FLP-FRT system and how does it work?

Answer 18

The FLP-FRT system is similar to the Cre-lox system. FLP recognises FRT sequences that flank a genomic region of interest

Question 19

What is a possible workflow for creating a mouse model of human genetic disease?

Answer 19

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?