Recombinase cre lox and flp-frt

Introduction 
Cre-lox recombination
Cre-lox system- Cre recombinase , loxP site
FLP-FRT recombination
FLP-FRT system- FLP recombinase , FRT site
Mechanism of Cre-lox and FLP-FRT recombination
Binding
Synapsis , cleavage and strand exchange
Three type of arrangement
Inversion
Translocation/ Insersion
Deletion
Application of Cre-lox and FLP-FRT recombination
Disadvantage of FLP-FRT
Advantage and disadvantage of Cre-lox
Conclusion
References

1. FLP-FRT & Cre-lox Recombination
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By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2. FLP-FRT and Cre-lox Recombination
Synopsis
Introduction
Cre-lox recombination
Cre-lox system-Cre recombinase , loxPsite
FLP-FRT recombination
FLP-FRT system-FLP recombinase , FRT site
Mechanism of Cre-lox and FLP-FRT recombination
Binding
Synapsis, cleavage and strand exchange
Three type of arrangement
Inversion
Translocation/ Insersion
Deletion
Application of Cre-lox and FLP-FRT recombination
Disadvantage of FLP-FRT
Advantage and disadvantage of Cre-lox
Conclusion
References
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3. FRT –FLP and Cre lox recombination
INTRODUCTION
Cre-lox andFRT-FLP recombinationare site-specific recombination .
Widely used to carry out deletions, insertions, translocations and inversions in the
DNA of cells.
Site-specific recombination is the enzyme-mediated cleavage and ligation of two
defined deoxynucleotide sequences.
Site-specific recombination (SSR) involves specific sites for the catalysing action of
special enzymes called recombinases.
Cre, or cyclic recombinase, and flipase or FLP are such enzyme.
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4. FRT –FLP and Cre lox recombination
Cre-lox recombination
The system consists of a single enzyme, Cre recombinase that recombines a pair of
short target sequences called the Loxsequences.
The Cre enzyme and the original Loxsite called the LoxPsequence are derived from
a bacteriophageP1
The Cre-lox system
Discovered in P1 bacteriophageas part of this organism's normal viral live cycle by
Sauer and Henderson 1988.
The bacteriophageP1 uses Cre-lox recombination to circularize and facilitate
replication of its genomic DNA when reproducing.
It requires only two components:
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5. FRT –FLP and Cre lox recombination
a)Cre recombinase
The Cre protein (encoded by the locus originally named as "Causes recombination",
with "Cyclizationrecombinase" being found in some references) has 343 amino
acids.
The Cre protein consists of a linker and two domains: The larger carboxyl (C-
terminal) domain, and smaller amino (N-terminal) domain.
Amino terminal domain
-5 alpha helices
Carboxy terminal domain
-9 alpha helices and 3 beta strands.
-catalytic site consist of Arg, His , Trp, Tyr
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6. FRT –FLP and Cre lox recombination
b) Lox Psite
-Binding site for Cre
-34 bp
13bp 8bp 13bp
ATAACTTCGTATA -GCATACAT –TATACGAAGTTAT
Fig –lox site
FLP-FRT recombination
It is analogous to Cre-Lox recombination.
It involves the recombination of sequences between short Flippase
Recognition Target (FRT) sites by the Flippaserecombination enzyme
(FLP or Flp)
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7. FRT –FLP and Cre lox recombination
FLP-FRT system
The FLP recombinase and FRT site are derived from the 2µm
plasmid of the Saccharomycescerevisiae
FLP
Filpaseenzyme belong to tyrosine recombinase family.
Its catalytic site consist of Arg-His-Argand a Tyr that make a
nucleophilicattack.
FRT site
FRT site is 34bp long sequence.
8bp crossover region flanked by 13bp sequences.
5'-GAAGTTCCTATTCtctagaaaGTATAGGAACTTC-3'
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8. Mechanism of Cre –lox and FRT-FLP recombination
•Binding
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Fig: Binding of Cre subunit to lox site.

9. FRT –FLP and Cre lox recombination
Synapsis, Cleavage and Strand exchange
In order for recombination to occur, two DNA sites must associate to form a synaptic
complex, within which the cleavage and strand exchange reactions take place.
Cleavage-
Tyr perform nucleopilic attack on
phoshate Group of DNA resulting –
3’ phosphotyrosine linkage
and leaving 5’ –OH group
on one strand of double helices.,
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Fig: Active site of Cre; where Tyr perfom
nucleophilic attack.

10. FRT –FLP and Cre lox recombination
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Fig-mechanism of cre lox recombination

11. FRT –FLP and Cre lox recombination
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Three type of arrangement
•Inversion
If the loxPor FRT sites are oriented in opposite directions, recombinase
mediates the inversion of the floxedsegment.
•Translocation
If the loxP/ FRT sites are located on different chromosomes (trans
arrangement), recombinase mediates a chromosomal translocation.
•Deletion
•If the loxP/FRT sites are oriented in the same direction on a chromosome
segment (cisarrangement), recombinase mediates a deletion of the floxed
segment.

12. FRT –FLP and Cre lox recombination
Fig: recombination produces inversion, translocation, and deletion; depends on
orientation of lox site.
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13. FRT –FLP and Cre lox recombination
Application of Cre-lox and RLP-FRT recombination system
-Cre-loxPor Flp-FRT can be used to remove selection markers or modify
constructs.
-Generation of insertions or deletions and orientation of inserted fragment
depends on choice of homology regions
-DNA cloning in vitro
-clonalanalysis of gene function in Drosophila
-removal of FRT-flanked resistance cassettes or genomic segments in mice
-Cre system can be used for subcloningof fragments.
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14. FRT –FLP and Cre lox recombination
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Fig; Application of Cre-lox and Flp-FRT recombination

15. FRT –FLP and Cre lox recombination
Disadvantage of FLP-FRT
1) Flpis temperature-sensitive and less active above 30oC
2) Problems for in vivo experiments in vertebrates
3) Development of enhanced Flp(Flpe)
Advantage of Cre-lox
Simplicity ( no co-factor is required)
Fidelity –recombination is carried out such that there is no loss or gain of
nucleotide
Small 34 bpsite-which does not perturb the surrounding genes when positioned
in chromosomal DNA.
broad utility -acting on supercoiled, relaxed, or linear DNA substrates; functioning
over large megabasedistances; and functional in a wide range of cell types.
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16. FRT –FLP and Cre lox recombination
Disadvantage of Cre-lox
1) The disadvantages of Cre include recombination of DNA sequences
naturally occurring in yeast and mammalian genomes, via "cryptic" loxP-
like sites in vitro.
2)Crerecombinase can cause chromosomal
rearrangements/aberrations and increased number of sister chromatid
exchanges when expressed at very high levels
3) High level of Cre expression has also been shown to reduce cell
proliferation in mouse embryonic fibroblasts and is speculated to be
involved in causing cell-cycle arrest.
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17. FRT –FLP and Cre lox recombination
Conclusion
The Cre-lox and FLP-FRT system is used as a genetic tool to
control site specific recombination events in genomic DNA. This
system has allowed researchers to manipulate a variety of
genetically modified organisms to control gene expression, delete
undesired DNA sequences and modify chromosome architecture.
Advantage of this system is that no additional co-factor is
required.
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18. FRT –FLP and Cre lox recombination
References
Gene X by Benjamin and Lewine
Molecular bologyof the gene by Watson
http://jaxmice.jax.org/jaxnotes/archive/501c.html
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