FRT in genetics stands for Flippase Recognition Target. It's a short DNA sequence that serves as a target site for the enzyme Flippase (Flp), enabling site-specific recombination, similar to the Cre-lox system.
Understanding FRT and Flp Recombination
Here's a breakdown of FRT and its role in genetic manipulation:
-
Flippase (Flp): This is a recombinase enzyme derived from the 2μ plasmid of the yeast Saccharomyces cerevisiae. Flp recognizes and binds to FRT sites.
-
FRT (Flippase Recognition Target): This is a short (typically ~34 base pair) DNA sequence specifically recognized by the Flp recombinase. Different FRT variants with varying recombination efficiencies have been developed (e.g., FRT, FRT3, FRT5, FRT11).
-
Recombination Process: When Flp encounters two FRT sites within a DNA molecule, it catalyzes a recombination event between them. The outcome depends on the location and orientation of the FRT sites.
Outcomes of Flp-FRT Recombination
The following scenarios are possible, based on the position and orientation of the FRT sites:
-
Excision: If two FRT sites flank a DNA sequence on the same DNA molecule and are in the same orientation, Flp will excise the intervening sequence, leaving a single FRT site behind. This is useful for removing selectable markers or conditional genes.
-
Inversion: If two FRT sites are on the same DNA molecule but in opposite orientations, Flp will invert the DNA sequence between them.
-
Chromosomal Translocation/Integration (Less Common): If FRT sites are located on different DNA molecules, Flp can catalyze a reciprocal exchange between them. This is less common due to the need for the molecules to be in close proximity.
Applications of the Flp-FRT System
The Flp-FRT system is a powerful tool for:
-
Conditional Gene Expression: By flanking a gene with FRT sites, researchers can control its expression. Crossing an organism containing the FRT-flanked gene ("floxed" gene) with an organism expressing Flp recombinase results in excision of the gene in cells where Flp is active. This allows for spatial and temporal control of gene expression.
-
Removal of Selectable Markers: During genetic engineering, selectable marker genes (e.g., antibiotic resistance genes) are often used to identify cells that have successfully incorporated the desired DNA. After selection, these markers are often no longer needed and can be removed using the Flp-FRT system.
-
Chromosome Engineering: The Flp-FRT system can be used for more complex chromosome manipulations, such as creating deletions, duplications, or inversions.
-
Transgene Integration and Cassette Exchange: Flp-FRT can be used to insert transgenes into specific genomic locations or to exchange one DNA cassette for another.
Example
Imagine a gene you want to study, but only in the brain of a mouse. You could:
- "flox" the gene by flanking it with FRT sites.
- Create a mouse strain that expresses Flp recombinase only in the brain (using a brain-specific promoter driving Flp expression).
- Cross these two strains. In the offspring, the gene will only be deleted (or altered, depending on experimental design) in brain cells due to the Flp activity.
