FLP-FRT recombination

In genetics, Flp-FRT recombination is a site-directed recombination technology, increasingly used to manipulate an organism's DNA under controlled conditions in vivo. It is analogous to Cre-lox recombination but involves the recombination of sequences between short flippase recognition target (FRT) sites by the recombinase(Flp)derived from the 2 µm plasmid of baker's yeast Saccharomyces cerevisiae.

The 34bp minimal FRT site sequence has the sequence

5'GAAGTTCCTATTCtctagaaaGtATAGGAACTTC3'

for which flippase (Flp) binds to both 13-bp 5'-GAAGTTCCTATTC-3' arms flanking the 8 bp spacer, i.e. the site-specific recombination (region of crossover) in reverse orientation. FRT-mediated cleavage occurs just ahead from the asymmetric 8bp core region (5'tctagaaa3') on the top strand and behind this sequence on the bottom strand.[1] Several variant FRT sites exist, but recombination can usually occur only between two identical FRTs but generally not among non-identical ("heterospecific") FRTs.[2][3]

Mutations of the FRT Site Sequence

Senecoff et al. (1987) investigated how nucleotide substitutions within the FRT affected the efficacy of the FLP-mediated recombination. The authors induced base substitutions in either one or both of the FRT sites and tested the concentration of FLP required to observe site-specific recombinations. Every base substitution was performed on each of the thirteen nucleotides within the FRT site (example G to A, T, and C). First, the authors showed that most mutations within the FRT sequence cause minimal effects if present within only of the two sites. If mutations occurred within both sites, the efficiency of FLP is dramatically reduced. Second, the authors provided data for which nucleotides are most crucial for the binding of FLP and efficacy of the site-specific recombination. If the first nucleotide in both FRT sites is substituted to a cytosine (G to C), the third nucleotide is substituted for a thymine (A to T), or the seventh nucleotide is substituted for an adenosine (G to A), then the efficacy of the FLP-mediated site-specific recombination is reduced more 100-fold.[4] While a base substitution of any of the aforementioned nucleotides in only one of the FRT sites led to a ten-fold, ten-fold, and five-fold reduction of efficacy, respectively.[4]

Base Substitutions in the capitalized nucleotides led to the greatest reduction in FLP-mediated site-specific recombination (Wildtype x mutatnt and mutant x mutant):

5' GaAtagGaacttc 3'[4]

Many available constructs include an additional arm sequences (5'-GAAGTTCCTATTCC-3') one base pair away from the upstream element and in the same orientation:

5'GAAGTTCCTATTCcGAAGTTCCTATTCtctagaaaGtATAGGAACTTC3'

This segment is dispensable for excision but essential for integration, including Recombinase-mediated cassette exchange.[5]

Because the recombination activity can be targeted to a selected 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 a given target organ of interest, even in cases where experimental animals would not survive the loss of this gene in other organs("spatial control"). 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 ("temporal control") - this prevents the alteration

Application of FLP-Mediated Site-Specific Recombination

Initial Problems

Thermolability

Initial application of the FLP-FRT recombinase did not work in mammals. The FLP protein was thermolabile (denatured at elevated temperatures) and therefore was not useful in the mammalian model due to elevated body temperatures of these model systems. However, due to patents and restrictions on the use of Cre-Lox recombination, great interest was taken to produce a more thermostable FLP-FRT cassette. Some of the first results were produced by Buchholz et al. (1997) by utilizing cycling mutagenesis in Escherichia coli . In their research, the authors transfected E. coli cells with two plasmids: one coding for randomly mutated FLP proteins downstream of an arabinose promoter and another containing a lacZ gene promoter within a FRT cassette. The E. coli were grown on arabinose plates at 37 °C and 40 °C, and if recombination occurred, the lacZ expression would be attenuated, and the colonies would appear white. White colonies were selected from each generation and grown on a new arabinose plates at same previous temperatures for eight generations.[6] After recombination was confirmed by western-blotting and the mutated FLP genes were sequenced, the most effective FLP protein (FLPe) was transfected into mammalian cell culture, and recombination in mammalian cells was confirmed.[6]

Generation of Genetic Mosaics

Genetic mosaicism occurs within an organism when similar cell types express different phenotypes due to dissimilar genotypes at specific loci. Simply put, this occurs when one organism contains different genotypes, which is usually rare in nature. However, this can be easily (and problematically) produced using FLP-FRT recombination. If two different FRT sites are present within a cell, and FLP is present in appropriate concentrations, the FRT cassette will continue to be excised and inserted between the two FRT sites. This process will continue until the FLP proteins fall below the required concentrations resulting in cells within an organism possessing different genotypes.[7] This has been seen from fruit flies to mice and is indiscriminate against specific chromosomes (somatic and sex) or cell types (somatic and germline).[7][8]

See also

References

  1. ↑ Zhu XD, Sadowski PD (1995). "Cleavage-dependent Ligation by the FLP Recombinase". Journal of Biological Chemistry 270 (39): 23044–54. doi:10.1074/jbc.270.39.23044. PMID 7559444.
  2. ↑ Schlake T, Bode J (1994). "Use of mutated FLP recognition target (FRT) sites for the exchange of expression cassettes at defined chromosomal loci". Biochemistry 33 (43): 12746–12751. doi:10.1021/bi00209a003. PMID 7947678.
  3. ↑ Turan, S.; Kuehle, J.; Schambach, A.; Baum, C.; Bode, J. (2010). "Multiplexing RMCE: Versatile Extensions of the Flp-Recombinase-Mediated Cassette-Exchange Technology". J. Mol. Biol. 402 (1): 52–69. doi:10.1016/j.jmb.2010.07.015. PMID 20650281.
  4. 1 2 3 Senecoff, Julie F.; Rossmeissl, Peter J.; Cox, Michael M. (1988-05-20). "DNA recognition by the FLP recombinase of the yeast 2 μ plasmid: A mutational analysis of the FLP binding site". Journal of Molecular Biology 201 (2): 405–421. doi:10.1016/0022-2836(88)90147-7.
  5. ↑ Turan, S., Bode, J. (2011). "Site-specific recombinases: from tag-and-target- to tag-and-exchange-based genomic modifications". FASEB J. 25: 4088–4107. doi:10.1096/fj.11-186940. PMID 21891781.
  6. 1 2 Buchholz, Frank; Angrand, Pierre-Olivier; Stewart, A. Francis (1998-07-01). "Improved properties of FLP recombinase evolved by cycling mutagenesis". Nature Biotechnology 16 (7): 657–662. doi:10.1038/nbt0798-657.
  7. 1 2 Dymecki, Susan M.; Tomasiewicz, Henry (1998-09-01). "Using Flp-Recombinase to Characterize Expansion ofWnt1-Expressing Neural Progenitors in the Mouse". Developmental Biology 201 (1): 57–65. doi:10.1006/dbio.1998.8971.
  8. ↑ Golic, M M; Rong, Y S; Petersen, R B; Lindquist, S L; Golic, K G (1997-09-15). "FLP-mediated DNA mobilization to specific target sites in Drosophila chromosomes.". Nucleic Acids Research 25 (18): 3665–3671. ISSN 0305-1048. PMC 146935. PMID 9278488.


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