FLP-FRT recombination

Last updated
Site-specific recombinase Flp
Identifiers
Organism Saccharomyces cerevisiae
SymbolFLP1
UniProt P03870
Search for
Structures Swiss-model
Domains InterPro
Simplified Flp-FRT Recombinase Mechanism.png

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 flippase (Flp) derived from the 2 μ plasmid of baker's yeast Saccharomyces cerevisiae .

Contents

The 34bp minimal FRT site sequence has the sequence

5'GAAGTTCCTATTCtctagaaaGAATAGGAACTTC3'

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 identicalFRTs but generally not among non-identical ("heterospecific") FRTs. [2] [3]

Biological function

In yeast, this enzyme corrects decreases in 2 μ plasmid copy number caused by rare missegregation events. It does so by causing recombination between the two inverted repetitions on the 2 μ plasmid during DNA replication. This changes the direction of one replication fork, causing multiple rounds of copying in a single initiation. [4]

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 one 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 than 100-fold. [5] 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. [5]

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

5'GaAtagGaacttc3' [5]

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. [6]

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.

Biochemical structure of Flp and mechanism of action

Biochemically relevant structure and active site

The Flp protein, much like Cre, is a tyrosine family site-specific recombinase. [7] This family of recombinases performs its function via a type IB topoisomerase mechanism causing the recombination of two separate strands of DNA. [7] Recombination is carried out by a repeated two-step process. The initial step causes the creation of a Holliday junction intermediate. The second step promotes the resulting recombination of the two complementary strands. As their family name suggests, a highly conserved tyrosine nucleophile cleaves the DNA strands. [7] The nucleophilic properties of the tyrosine attack and bind to the 3'-phosphate at the point of DNA cleavage. [7] The resulting 5'-hydroxyl group of the cleaved DNA acts as the nucleophile and attacks the 3'-phosphate on the complementarily cleaved DNA strand, resulting in successful recombination. [7] Outside of the tyrosine residue, there is a conserved catalytic pentad. This pentad is made up of a lysine (Lysβ), two arginines (Arg I and II), a histidine (His-II), and a histidine/tryptophan (His/Trp-III) that comprises a mandatory and highly conserved constellation of residues for the active site of Flp and Cre (along with other IB topiosomerases). [7] These other residues are crucial to the correct orientation of Flp binding and positioning on the DNA strands.

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. [8] After recombination was confirmed by western-blotting and the mutated FLP genes were sequenced, this eighth generation FLP protein (FLPe) was transfected into mammalian cell culture, and recombination in mammalian cells was confirmed. [8] This variant of FLP only has 4 amino acid substitutions: P2S, L33S, Y108N, and S294P.

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. [9] This has been seen from fruit flies to mice and is indiscriminate against specific chromosomes (somatic and sex) or cell types (somatic and germline). [9] [10]

Determination of cell lineages

Before the publication of Dymecki et al. (1998), Cre recombinase had been used for cell-fate mapping of neuronal progenitors in mice using the En2 promoter. [11] Thus, the authors of Dymecki et al. (1998) theorized that FLP recombinase could be utilized in a similar fashion with a similar efficacy as Cre recombinase in mice. The authors created a two transgenic mouse lines: a neuronal Wnt1::Flp fusion line and a line that possessed the FRT cassette flanking the 18th exon of tm1Cwr. [9] The authors chose this exon for excision because if it is excised, its results in a null phenotype. The authors mated the two lines and allowed the progeny to reach adulthood before the progeny were sacrificed. RNA extraction was performed on neuronal, muscular, enteric, and tail tissue. Reverse-transcription PCR and northern blotting confirmed the excision of the 18th exon of tm1Cwr abundantly in the brain tissue and moderately in muscular tissue (due to Scwhann cells within the muscle). As expected, excision was not seen in other tissues. The authors saw equal, if not better, efficiency of the FLP recombinase in cell-fate determination than Cre recombinase.

In Drosophila melanogaster (Fruit Fly)

To date, Flp recombinase has been utilized many times in D. melanogaster. A comparison of Flp recombinase to Cre recombinase in D. melanogaster was published by Frickenhaus et al. (2015) [12] . The authors of Frickenhaus et al. (2015) had a two-fold objective: characterize and compare the efficacy of Flp recombinase "knock-out" to Cre recombinase "knock-out" and RNAi knockdown and reveal the function of cabeza (caz), the fly ortholog to FUS, in the neurons and muscle tissue of D. melanogaster. FUS has been strongly implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia in humans [12] . The authors used an elav-Gal4/UAS-Flp or Cre system to express the recombinase specifically in neurons and a Mef2-Gal4/UAS-Flp or Cre system to express it specifically in muscles. [12] The authors conclude that the Flp recombinase "knock-out" tool is more effective than both RNAi and Cre recombinase for the purpose of knocking out specific genes in specific tissues or cell lines due to the lack of the leaky expression seen in both the Cre protein and the RNAi transcript. Also, the authors witnessed a toxicity to the Cre protein that is not seen with the Flp protein. [12]

In Danio rerio (Zebrafish)

The efficacy of the FLPe recombinase system was evaluated in zebrafish by Wong et al. (2009). [13] Embryos, that were hemizygous for a FRT-flanked enhanced green fluorescent protein (EGFP) downstream of a muscle-specific promoter, were injected with the FLPe protein. Without FLPe, these embryoes should express EGFP in all muscle tissue, and if crossed with a wild type strand, 50% of the resulting progeny should also express EGFP in muscle tissue. Embryoes, injected with FLPe, had significantly reduced expression of EGFP in muscle tissue, and mosaicism was seen also. [13] When these embryoes reached adulthood, they were mated with a wild type strain, and the resulting clutches had significantly less progeny that expressed the EGFP in the muscle tissue (0-4%). [13] These results show, that not only is FLPe highly effective in somatic cells, it is also highly effective in the germline of zebrafish.

In plants

Creation of "phytosensors" or "sentinels" in Arabidopsis thaliana and Tobacco

Phytosensors are genetically modified plants that can report the presence of biotic or abiotic contaminants. [14] The production of these engineered plants have great agricultural and laboratory promise. However, creating an appropriate reporter vector has shown to be problematic. cis-regulatory elements play a major role in the transcriptional activation of genes in plants, and many are not well understood. Many phytosensors either under express their reporter genes or report false-positives due to synthetic promoters. [14] The authors of Rao et al. (2010) utilized the FLP recombinase tool for the production of a highly efficient phytosensors. The authors used a heat shock promoter to induce the production of FLP while a FRT-flanked vector separated the CaMV 35S promoter from the beta-glucuronidase gene (GUS). When the plants were exposed to heat-shock, FLP-induction led to the excision of the FRT-flanked vector, effectively moving the GUS gene directly downstream of the CaMV 35S promoter. The activation of GUS led to the leaves of the plants to change from green to blue; thus, the phytosensor effectively reported stress to the model system! [14]

With Cre-recombinase

Production of the gate-way-ready inducible MiRNA (GRIM) expression system

RNA interference (RNAi) has caused a paradigm shift in the expression of genes and potential gene knockouts in Eukaryotes. Before the production of the GRIM expression system, the creation of RNAi vectors was expensive and time consuming. The vectors were produced by the traditional copy-and-paste molecular cloning method. [15] Garwick-Coppens et al. (2011) developed a much more efficient method for the production of RNAi vectors in which expression of the RNAi can be knocked-in using Cre-recombinase and knocked-out using Flp-recombinase. The novel GRIM Expression System allows for the much faster generation of expression vectors containing artificial RNAi constructs. [15] The authors went on to show that their expression system works quite effectively in human embryonic kidneys (HEK) cells, a common human immortalized cell line in molecular research.

See also

Related Research Articles

<span class="mw-page-title-main">Library (biology)</span> Collection of genetic material fragments

In molecular biology, a library is a collection of genetic material fragments that are stored and propagated in a population of microbes through the process of molecular cloning. There are different types of DNA libraries, including cDNA libraries, genomic libraries and randomized mutant libraries. DNA library technology is a mainstay of current molecular biology, genetic engineering, and protein engineering, and the applications of these libraries depend on the source of the original DNA fragments. There are differences in the cloning vectors and techniques used in library preparation, but in general each DNA fragment is uniquely inserted into a cloning vector and the pool of recombinant DNA molecules is then transferred into a population of bacteria or yeast such that each organism contains on average one construct. As the population of organisms is grown in culture, the DNA molecules contained within them are copied and propagated.

Cre-Lox recombination is a site-specific recombinase technology, used to carry out deletions, insertions, translocations and inversions at specific sites in the DNA of cells. It allows the DNA modification to be targeted to a specific cell type or be triggered by a specific external stimulus. It is implemented both in eukaryotic and prokaryotic systems. The Cre-lox recombination system has been particularly useful to help neuroscientists to study the brain in which complex cell types and neural circuits come together to generate cognition and behaviors. NIH Blueprint for Neuroscience Research has created several hundreds of Cre driver mouse lines which are currently used by the worldwide neuroscience community.

Site-specific recombinase technologies are genome engineering tools that depend on recombinase enzymes to replace targeted sections of DNA.

<span class="mw-page-title-main">Cre recombinase</span> Genetic recombination enzyme

Cre recombinase is a tyrosine recombinase enzyme derived from the P1 bacteriophage. The enzyme uses a topoisomerase I-like mechanism to carry out site specific recombination events. The enzyme is a member of the integrase family of site specific recombinase and it is known to catalyse the site specific recombination event between two DNA recognition sites. This 34 base pair (bp) loxP recognition site consists of two 13 bp palindromic sequences which flank an 8bp spacer region. The products of Cre-mediated recombination at loxP sites are dependent upon the location and relative orientation of the loxP sites. Two separate DNA species both containing loxP sites can undergo fusion as the result of Cre mediated recombination. DNA sequences found between two loxP sites are said to be "floxed". In this case the products of Cre mediated recombination depends upon the orientation of the loxP sites. DNA found between two loxP sites oriented in the same direction will be excised as a circular loop of DNA whilst intervening DNA between two loxP sites that are opposingly orientated will be inverted. The enzyme requires no additional cofactors or accessory proteins for its function.

In biology, a gene cassette is a type of mobile genetic element that contains a gene and a recombination site. Each cassette usually contains a single gene and tends to be very small; on the order of 500–1,000 base pairs. They may exist incorporated into an integron or freely as circular DNA. Gene cassettes can move around within an organism's genome or be transferred to another organism in the environment via horizontal gene transfer. These cassettes often carry antibiotic resistance genes. An example would be the kanMX cassette which confers kanamycin resistance upon bacteria.

Recombineering is a genetic and molecular biology technique based on homologous recombination systems, as opposed to the older/more common method of using restriction enzymes and ligases to combine DNA sequences in a specified order. Recombineering is widely used for bacterial genetics, in the generation of target vectors for making a conditional mouse knockout, and for modifying DNA of any source often contained on a bacterial artificial chromosome (BAC), among other applications.

Recombinases are genetic recombination enzymes.

P1 is a temperate bacteriophage that infects Escherichia coli and some other bacteria. When undergoing a lysogenic cycle the phage genome exists as a plasmid in the bacterium unlike other phages that integrate into the host DNA. P1 has an icosahedral head containing the DNA attached to a contractile tail with six tail fibers. The P1 phage has gained research interest because it can be used to transfer DNA from one bacterial cell to another in a process known as transduction. As it replicates during its lytic cycle it captures fragments of the host chromosome. If the resulting viral particles are used to infect a different host the captured DNA fragments can be integrated into the new host's genome. This method of in vivo genetic engineering was widely used for many years and is still used today, though to a lesser extent. P1 can also be used to create the P1-derived artificial chromosome cloning vector which can carry relatively large fragments of DNA. P1 encodes a site-specific recombinase, Cre, that is widely used to carry out cell-specific or time-specific DNA recombination by flanking the target DNA with loxP sites.

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 at least a certain degree of sequence homology. Enzymes known as site-specific recombinases (SSRs) perform rearrangements of DNA segments by recognizing and binding to short, specific DNA sequences (sites), at which they cleave the DNA backbone, exchange the two DNA helices involved, and rejoin the DNA strands. In some cases the presence of a recombinase enzyme and the recombination sites is sufficient for the reaction to proceed; in other systems a number of accessory proteins and/or accessory sites are required. Many different genome modification strategies, among these recombinase-mediated cassette exchange (RMCE), an advanced approach for the targeted introduction of transcription units into predetermined genomic loci, rely on SSRs.

The Tn3 transposon is a 4957 base pair mobile genetic element, found in prokaryotes. It encodes three proteins:

<span class="mw-page-title-main">Transplastomic plant</span>

A transplastomic plant is a genetically modified plant in which genes are inactivated, modified or new foreign genes are inserted into the DNA of plastids like the chloroplast instead of nuclear DNA.

RMCE is a procedure in reverse genetics allowing the systematic, repeated modification of higher eukaryotic genomes by targeted integration, based on the features of site-specific recombination processes (SSRs). For RMCE, this is achieved by the clean exchange of a preexisting gene cassette for an analogous cassette carrying the "gene of interest" (GOI).

DNA adenine methyltransferase identification, often abbreviated DamID, is a molecular biology protocol used to map the binding sites of DNA- and chromatin-binding proteins in eukaryotes. DamID identifies binding sites by expressing the proposed DNA-binding protein as a fusion protein with DNA methyltransferase. Binding of the protein of interest to DNA localizes the methyltransferase in the region of the binding site. Adenine methylation does not occur naturally in eukaryotes and therefore adenine methylation in any region can be concluded to have been caused by the fusion protein, implying the region is located near a binding site. DamID is an alternate method to ChIP-on-chip or ChIP-seq.

The Gateway cloning method, invented and commercialized by Invitrogen since the late 1990s, is the cloning method of the integration and excision recombination reactions that take place when bacteriophage lambda infects bacteria. This technology provides a fast and highly efficient way to transport DNA sequences into multi-vector systems for functional analysis and protein expression using Gateway att sites, and two proprietary enzyme mixes called BP Clonase and LR Clonase. In vivo, these recombination reactions are facilitated by the recombination of attachment sites from the lambda/phage chromosome (attP) and the bacteria (attB). As a result of recombination between the attP and attB sites, the phage integrates into the bacterial genome flanked by two new recombination sites. The removal of the phage from the bacterial chromosome and the regeneration of attP and attB sites can both result from the attL and attR sites recombining under specific circumstances.

<span class="mw-page-title-main">Floxing</span> Sandwiching of a DNA sequence between two lox P sites

In genetics, floxing refers to the sandwiching of a DNA sequence between two lox P sites. The terms are constructed upon the phrase "flanking/flanked by LoxP". Recombination between LoxP sites is catalysed by Cre recombinase. Floxing a gene allows it to be deleted, translocated or inverted in a process called Cre-Lox recombination. The floxing of genes is essential in the development of scientific model systems as it allows researchers to have spatial and temporal alteration of gene expression. Moreover, animals such as mice can be used as models to study human disease. Therefore, Cre-lox system can be used in mice to manipulate gene expression in order to study human diseases and drug development. For example, using the Cre-lox system, researchers can study oncogenes and tumor suppressor genes and their role in development and progression of cancer in mice models.

<span class="mw-page-title-main">Genetic engineering techniques</span> Methods used to change the DNA of organisms

Genetic engineering techniques allow the modification of animal and plant genomes. Techniques have been devised to insert, delete, and modify DNA at multiple levels, ranging from a specific base pair in a specific gene to entire genes. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.

<span class="mw-page-title-main">Reverse genetics</span> Method in molecular genetics

Reverse genetics is a method in molecular genetics that is used to help understand the function(s) of a gene by analysing the phenotypic effects caused by genetically engineering specific nucleic acid sequences within the gene. The process proceeds in the opposite direction to forward genetic screens of classical genetics. While forward genetics seeks to find the genetic basis of a phenotype or trait, reverse genetics seeks to find what phenotypes are controlled by particular genetic sequences.

No-SCAR genome editing is an editing method that is able to manipulate the Escherichia coli genome. The system relies on recombineering whereby DNA sequences are combined and manipulated through homologous recombination. No-SCAR is able to manipulate the E. coli genome without the use of the chromosomal markers detailed in previous recombineering methods. Instead, the λ-Red recombination system facilitates donor DNA integration while Cas9 cleaves double-stranded DNA to counter-select against wild-type cells. Although λ-Red and Cas9 genome editing are widely used technologies, the no-SCAR method is novel in combining the two functions; this technique is able to establish point mutations, gene deletions, and short sequence insertions in several genomic loci with increased efficiency and time sensitivity.

Susan M. Dymecki is an American geneticist and neuroscientist and director of the Biological and Biomedical Sciences PhD Program at Harvard University. Dymecki is also a professor in the Department of Genetics and the principal investigator of the Dymecki Lab at Harvard. Her lab characterizes the development and function of unique populations of serotonergic neurons in the mouse brain. To enable this functional dissection, Dymecki has pioneered several transgenic tools for probing neural circuit development and function. Dymecki also competed internationally as an ice dancer, placing 7th in the 1980 U.S. Figure Skating Championships.

<span class="mw-page-title-main">GESTALT</span> Method for lineage tracing using CRISPR-Cas9-edited barcodes

Genome editing of synthetic target arrays for lineage tracing (GESTALT) is a method used to determine the developmental lineages of cells in multicellular systems. GESTALT involves introducing a small DNA barcode that contains regularly spaced CRISPR/Cas9 target sites into the genomes of progenitor cells. Alongside the barcode, Cas9 and sgRNA are introduced into the cells. Mutations in the barcode accumulate during the course of cell divisions and the unique combination of mutations in a cell's barcode can be determined by DNA or RNA sequencing to link it to a developmental lineage.

References

  1. Zhu XD, Sadowski PD (September 1995). "Cleavage-dependent ligation by the FLP recombinase. Characterization of a mutant FLP protein with an alteration in a catalytic amino acid". The Journal of Biological Chemistry. 270 (39): 23044–54. doi: 10.1074/jbc.270.39.23044 . PMID   7559444.
  2. Schlake T, Bode J (November 1994). "Use of mutated FLP recognition target (FRT) sites for the exchange of expression cassettes at defined chromosomal loci". Biochemistry. 33 (43): 12746–51. doi:10.1021/bi00209a003. PMID   7947678.
  3. Turan S, Kuehle J, Schambach A, Baum C, Bode J (September 2010). "Multiplexing RMCE: versatile extensions of the Flp-recombinase-mediated cassette-exchange technology". Journal of Molecular Biology. 402 (1): 52–69. doi:10.1016/j.jmb.2010.07.015. PMID   20650281.
  4. Reynolds AE, Murray AW, Szostak JW (October 1987). "Roles of the 2 microns gene products in stable maintenance of the 2 microns plasmid of Saccharomyces cerevisiae". Molecular and Cellular Biology. 7 (10): 3566–73. doi: 10.1128/mcb.7.10.3566 . PMC   368010 . PMID   3316982.
  5. 1 2 3 Senecoff JF, Rossmeissl PJ, Cox MM (May 1988). "DNA recognition by the FLP recombinase of the yeast 2 mu plasmid. A mutational analysis of the FLP binding site". Journal of Molecular Biology. 201 (2): 405–21. doi:10.1016/0022-2836(88)90147-7. PMID   3047402.
  6. Turan S, Bode J (December 2011). "Site-specific recombinases: from tag-and-target- to tag-and-exchange-based genomic modifications". FASEB Journal. 25 (12): 4088–107. doi: 10.1096/fj.11-186940 . PMID   21891781. S2CID   7075677.
  7. 1 2 3 4 5 6 Ma CH, Kwiatek A, Bolusani S, Voziyanov Y, Jayaram M (April 2007). "Unveiling hidden catalytic contributions of the conserved His/Trp-III in tyrosine recombinases: assembly of a novel active site in Flp recombinase harboring alanine at this position". Journal of Molecular Biology. 368 (1): 183–96. doi:10.1016/j.jmb.2007.02.022. PMC   2002523 . PMID   17367810.
  8. 1 2 Buchholz F, Angrand PO, Stewart AF (July 1998). "Improved properties of FLP recombinase evolved by cycling mutagenesis". Nature Biotechnology. 16 (7): 657–62. doi:10.1038/nbt0798-657. PMID   9661200. S2CID   21298037.
  9. 1 2 3 Dymecki SM, Tomasiewicz H (September 1998). "Using Flp-recombinase to characterize expansion of Wnt1-expressing neural progenitors in the mouse". Developmental Biology. 201 (1): 57–65. doi: 10.1006/dbio.1998.8971 . PMID   9733573.
  10. Golic MM, Rong YS, Petersen RB, Lindquist SL, Golic KG (September 1997). "FLP-mediated DNA mobilization to specific target sites in Drosophila chromosomes". Nucleic Acids Research. 25 (18): 3665–71. doi:10.1093/nar/25.18.3665. PMC   146935 . PMID   9278488.
  11. Zinyk DL, Mercer EH, Harris E, Anderson DJ, Joyner AL (May 1998). "Fate mapping of the mouse midbrain-hindbrain constriction using a site-specific recombination system" (PDF). Current Biology. 8 (11): 665–8. Bibcode:1998CBio....8..665Z. doi: 10.1016/S0960-9822(98)70255-6 . PMID   9635195.
  12. 1 2 3 4 Frickenhaus M, Wagner M, Mallik M, Catinozzi M, Storkebaum E (March 2015). "Highly efficient cell-type-specific gene inactivation reveals a key function for the Drosophila FUS homolog cabeza in neurons". Scientific Reports. 5: 9107. doi:10.1038/srep09107. PMC   5390904 . PMID   25772687.
  13. 1 2 3 Wong AC, Draper BW, Van Eenennaam AL (April 2011). "FLPe functions in zebrafish embryos". Transgenic Research. 20 (2): 409–15. doi:10.1007/s11248-010-9410-9. PMC   3051101 . PMID   20552273.
  14. 1 2 3 Rao MR, Moon HS, Schenk TM, Becker D, Mazarei M, Stewart CN (2010-09-13). "FLP/FRT recombination from yeast: application of a two gene cassette scheme as an inducible system in plants". Sensors. 10 (9): 8526–35. Bibcode:2010Senso..10.8526R. doi: 10.3390/s100908526 . PMC   3231192 . PMID   22163670.
  15. 1 2 Garwick-Coppens SE, Herman A, Harper SQ (November 2011). "Construction of permanently inducible miRNA-based expression vectors using site-specific recombinases". BMC Biotechnology. 11: 107. doi: 10.1186/1472-6750-11-107 . PMC   3252340 . PMID   22087765.