FLP-FRT Recombination
Encyclopedia
In genetics
, FLP-FRT recombination is a site-directed recombination
technology used to manipulate an organism's DNA under controlled conditions in vivo. It is analogous to Cre-Lox recombination
. It involves the recombination of sequences between short Flippase Recognition Target (FRT) sites by the Flippase recombination enzyme (FLP or Flp) derived from the 2µm plasmid of the baker's yeast
Saccharomyces cerevisiae
.
The 34bp long FRT site sequence is :
5'-GAAGTTCCTATTCtctagaaaGTATAGGAACTTC-3'.
Flippase (flp) binds to the 13-bp 5'-GAAGTTCCTATTC-3' and to the reverse complement of 5'-GTATAGGAACTTC-3' (5'-GAAGTTCCTATAC-3').
The FRT site is cleaved just before 5'-tctagaaa-3', the 8bp asymmetric core region, on the top strand and behind this sequence on the bottom strand.
Several variant FRT sites exist. Recombination can occur between two identical FRT sites but generally not between non-identical FRT sites
Many available constructs include the sequence
5'-GAAGTTCCTATTCC-3'
immediately upstream the FRT site
(resulting in 5'-GAAGTTCCTATTCCGAAGTTCCTATTCtctagaaaGTATAGGAACTTC-3')
but this sequence is dispensable for recombination.
Because the recombination activity can be targeted to only one target organ, or a low level of recombination activity can be used to consistently alter the DNA of only a subset of cells, FLP-FRT can be used to construct genetic mosaics
in multicellular organisms. Using this technology, the loss or alteration of a gene can be studied in one target organ of interest, even if experimental animals could not survive the loss of the gene in other organs. The effect of altering a gene can also be studied over time, by using an inducible promoter to trigger the recombination activity late in development - this prevents the alteration from affecting overall development of an organ, and allows single cells lacking the gene to be compared to normal neighboring cells in the same environment.
Genetics
Genetics , a discipline of biology, is the science of genes, heredity, and variation in living organisms....
, FLP-FRT recombination is a site-directed recombination
Site-specific recombination
Site-specific recombination, also known as conservative site-specific recombination, is a type of genetic recombination in which DNA strand exchange takes place between segments possessing only a limited degree of sequence homology...
technology used to manipulate an organism's DNA under controlled conditions in vivo. It is analogous to Cre-Lox recombination
Cre-Lox recombination
Cre-Lox recombination is a special type of site-specific recombination developed by Dr. Brian Sauer initially for use in activating gene expression in mammalian cell lines and transgenic mice . Subsequently, the laboratory of Dr...
. It involves the recombination of sequences between short Flippase Recognition Target (FRT) sites by the Flippase recombination enzyme (FLP or Flp) derived from the 2µm plasmid of the baker's yeast
Baker's yeast
Baker's yeast is the common name for the strains of yeast commonly used as a leavening agent in baking bread and bakery products, where it converts the fermentable sugars present in the dough into carbon dioxide and ethanol...
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes...
.
The 34bp long FRT site sequence is :
5'-GAAGTTCCTATTCtctagaaaGTATAGGAACTTC-3'.
Flippase (flp) binds to the 13-bp 5'-GAAGTTCCTATTC-3' and to the reverse complement of 5'-GTATAGGAACTTC-3' (5'-GAAGTTCCTATAC-3').
The FRT site is cleaved just before 5'-tctagaaa-3', the 8bp asymmetric core region, on the top strand and behind this sequence on the bottom strand.
Several variant FRT sites exist. Recombination can occur between two identical FRT sites but generally not between non-identical FRT sites
Many available constructs include the sequence
5'-GAAGTTCCTATTCC-3'
immediately upstream the FRT site
(resulting in 5'-GAAGTTCCTATTCCGAAGTTCCTATTCtctagaaaGTATAGGAACTTC-3')
but this sequence is dispensable for recombination.
Because the recombination activity can be targeted to only one target organ, or a low level of recombination activity can be used to consistently alter the DNA of only a subset of cells, FLP-FRT can be used to construct genetic mosaics
Mosaic (genetics)
In genetic medicine, a mosaic or mosaicism denotes the presence of two populations of cells with different genotypes in one individual who has developed from a single fertilized egg...
in multicellular organisms. Using this technology, the loss or alteration of a gene can be studied in one target organ of interest, even if experimental animals could not survive the loss of the gene in other organs. The effect of altering a gene can also be studied over time, by using an inducible promoter to trigger the recombination activity late in development - this prevents the alteration from affecting overall development of an organ, and allows single cells lacking the gene to be compared to normal neighboring cells in the same environment.