Is Cre recombinase reversible?
All recombination events mediated by FLP or Cre are reversible. Because recombination can only occur between target sites of the same sequence in FLEx vectors, the DNA sequence that should be inverted is flanked by two pairs of target sites that differ in their sequence.
How does the CRE loxP system work?
How does it work? As alluded to above, the Cre recombinase catalyzes the site specific recombination event between two loxP sites, which can be located either on the same or on separate pieces of DNA. Both 13bp repeat sequences on a single loxP site are recognized and bound by a Cre protein, forming a dimer.
How does Cre loxP system work?
Is Cre recombinase a restriction enzyme?
LoxP-directed cloning: use of Cre recombinase as a universal restriction enzyme. Biotechniques.
How does FLP recombinase work?
The Flp protein, much like Cre, is a tyrosine family site-specific recombinase. This family of recombinases performs its function via a type IB topoisomerase mechanism causing the recombination of two separate strands of DNA. Recombination is carried out by a repeated two-step process.
What is LoxP site?
LoxP sites are directional 34 bp sequences made up of two 13 bp recognition sites separated by an 8 bp spacer region. The sequences don’t occur naturally in any known genomes other than the P1 bacteriophage, and are long enough that they are unlikely to occur by chance.
How is Cre activity controlled by tamoxifen metabolite?
Cre/ER recombinases remain unexpressed until activated by the TAM metabolite, which provides external and time-specific control of Cre activity. This system is used to address the function of specific genes 3, 9, 10, 11, including those relating to immune cells.
What are the parameters of Cre loxP recombination in vivo?
Three parameters mainly determine the specific and effective Cre-loxP recombination in vivo: (1) the dose and duration of TAM that determine the activity of the potent metabolites 4-OH-TAM and END, (2) the level of Cre ERT2 expression and (3) the chromatin structure and epigenetic status of the floxed alleles.
What is the pharmacokinetic profile of tamoxifen in mice?
In mice, the pharmacokinetic profile of TAM appears significantly different, mainly due to the lower expression and activity levels of P450 isoenzymes 10, 11. Cell type-specific and temporally controlled recombination of floxed target genes is achieved by the Cre ERT2 /loxP system.
How is the inducible Cre / loxP transgenic system used?
The inducible Cre/loxP estrogen receptor (ER) transgenic system is used to study both ubiquitous and tissue-specific gene function through the expression of Cre recombinase fused to the G525R mutant form of the mouse estrogen receptor 1 (Esr1) to create a Cre/ER fusion protein 1, 2.