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1

How can we avoid lethality during gene knockout in early embryogenesis?

Chimeric mice - aggregation of mutant and wild type embryo, or injection of mutant ES cells into wild type blastocyst.

 

(not sure how the first example would work as it implies that there is already a mutant mouse that is capable of living with the mutation?)

 

Cre-loxP technology to make a tissue specific knockout. Cre-loxP allows you to inactivate a specific gene, in a specific tissue at a specifi stage of development of your choosing. 

2

What is Cre?

A site-specific recombinase enzyme from the P1 phage, it recognises a 34 bp DNA sequence called loxP.

 

LoxP is conventionally denoted as a little triangle

 

Phage uses the LoxP site of a bacterium to integrate its own DNA into the bacterium genome.

 

If the two loxP sites are facing the same direction the recombinase enzyme snips out the DNA inbetween. The loop of free DNA is then degraded and lost. What is left in the DNA sequence is one loxP site.

3

What identical system to the cre-loxP exists?

FLP serves the same purpose as Cre as a recombinase enzyme. Instead of loxP however, FLP acts upon the sites called FRT. 

 

FLP is derived from the yeast S. cerevisiae

4

What are the principles of cre-recombinase in action. i.e how do they work?

If we want to knockout a gene in a specific tissue then we need a line/strain of mice that expresses cre recombinase from a tissue specific promoter

We then need a second strain that has loxP sites around the gene of interest. The gene is said to be 'floxed'

 

When these two mice breed, after a couple of generations then there should be offspring that have the gene knocked out in the cells that cre-recombinase is expressed.

5

How do we integrate loxP sites into the mice?

Conditional targetting by homologous recombination in ES cells - (electroporate targeting vector into ES cells, inject into blastocysts creating chimeras nd breed these to get heterozygous floxed mice.

There are additional FRT sites. This is becuase keeping the neomycin resistance gene in the targetting vector may affect the function of the gene. In order to remove the neomycin resistance gene it is possible to have the FRT sites surrounding the gene and use the FLP recombinase to remove it. 

These mice are then bread with mice that express the FLP recombinase from a constitutive promoter i.e expressed in all cells such as b-actin promoter - This is in contrast to the tissue specific promoter that we are using for cre recombinase. 

 

As we can see there is positive and negative selection by using the neomycin resistance gene as well as the thymidine kinase gene that makes the cell sensitive to gangciclovir.

6

How do we target cre-recombinase to the tissue of interest?

Drive Cre from the promoter of a gene that

Is only expressed in the tissue you want (quite rare) OR

Is expressed in areas that overlap with your tissue of interest

 

OR

 

Create a ‘Designer’ Promoter…

 

Make Transgenic Mouse carrying Cre on your Promoter,  and Check Expression

7