Why is the Cre loxP system used in conditional knockout mice?
The Cre/lox system is one of the most powerful and versatile tools developed for mouse genetics. It gives mouse researchers sophisticated control over the location and timing of gene expression. Cre/lox is usually used to make knockout alleles, but it can also be used to activate gene expression.
What is loxP sequence?
LoxP (locus of X(cross)-over in P1) sites are 34-base-pair long recognition sequences consisting of two 13-bp long palindromic repeats separated by an 8-bp long asymmetric core spacer sequence.
How does Cre loxP work?
General principle of Cre-loxP system Concerning the mechanism of Cre-loxP system, a single Cre recombinase recognizes two directly repeated loxP site, then the Cre excises the loxP flanked (floxed) DNA, thus creating two types of DNA with circular, excised and inactivated gene Y (Figure 1A).
What is a loxP mouse?
A Cre mouse contains a Cre recombinase transgene under the direction of a tissue-specific promoter; a loxP mouse contains two loxP sites that flank a genomic segment of interest, the “floxed” locus. Typically, Cre and loxP mice are produced by using transgenic technology (Nagy 2000).
How big is a LoxP site?
34 base pairs
The loxP site is 34 base pairs (bp) in length and consists of two 13 bp inverted repeats separated by an 8 bp spacer region.
What is the Cre recombinase result when the LoxP sites have the same orientation on one DNA strand?
If lox sites are in the same orientation flanking a sequence, recombination will result in the excision of the sequence, deleting it from its original locus.
How much effect is there on the surrounding genes by the loxP sequence which is left after recombination has taken place?
4. How much effect is there on the surrounding genes by the loxP sequence which is left after recombination has taken place? Explanation: As the recombination has taken place, the loxP sequence remains there. The remaining loxP sequence has no effect on the surrounding genes.
What does it mean to get floxed?
Many of them describe a devastating and progressive condition, encompassing symptoms ranging from psychiatric and sensory disturbances to problems with muscles, tendons and nerves that continue after people have stopped taking the drugs. They call it being ‘floxed’.
What is a floxed mouse?
The Cre-lox system allows for the deletion of a gene in mouse ES cells and its inversion or complete inactivation. This process is called “floxing” since it combines flanking the gene with the help of its adjacent two loxP sites and the Cre-lox recombination system.
What is an FRT site?
FRT sites are used often (at least in Drosophila) for inducing deletions or “flipping out” of markers in transgenic constructs. When there are two FRTs sequences in tandem, after inducing flippase the DNA sequence that is between these two sites will be deleted.
What is a conditional knockout mouse?
A conditional Knockout mouse defines an animal model in which a gene of interest can be: Inactivated in specific cell types in a certain tissue; other cell types and tissues exhibit an unmodified, functional gene expression.
How are knockin mice made?
To produce knockout mice, researchers use one of two methods to insert artificial DNA into the chromosomes contained in the nuclei of ES cells. Both methods are carried out in vitro, that is in cultured cells grown in laboratory conditions.
How do you recover from Floxed?
Neuropathy can be treated with medications, technology and supplements. medications used to treat and manage associated neuropathy include gabapentin, lidocaine, imipramine, carbamazepine, and nutritional support. Supplements such as carnosine and alpha lipoic acid plus red light therapy and hyperbaric can help.
What are conditional knockout mice?
Conditional knockout mice (also known as Flox mice) are mice whose target genes contain paired loxp sites. After mating them with Cre mice, target genes can be knocked out in specific tissues or cells. If Flox mice are mated with Cre mice widely expressing Cre or expressing Cre only in germ cells, systemic KO mice can be obtained.
Is conditional gene knockout by Cre/loxP reversible?
Conditional gene knockout (cKO) mediated by the Cre/LoxP system is indispensable for exploring gene functions in mice. However, a major limitation of this method is that gene KO is not reversible.
How do you reverse CKO in mice with multiple alleles?
More importantly, in mice bearing both alleles and also expressing the Cre and FLP recombinases, sequential function of the two enzymes should lead to deletion of the target gene, followed by restoration of its expression, thus achieving reversible cKO.
How can we prevent knock-out errors in animal experiments?
The inadvertent generation and interpretation of data of nonspecific or global knock-out animals can be detrimental to an experiment. However, these errors can be tracked and avoided with appropriate genotyping and careful monitoring.