View source for Floxing

{{Short description|Genetic engineering technique}}
{{About|the genetics term|the nickname for severe reactions to quinolone antibiotics|Quinolone antibiotics}}
[[File:Floxing Flow Chart.svg|thumb|This figure depicts how Floxing is used in scientific research for spatial and temporal control of gene expression.]]
In genetic engineering, '''floxing''' refers to the [[insertional mutagenesis|insertion]] of a [[DNA]] sequence (which is then said to be '''floxed''') between two ''[[Cre-Lox recombination|LoxP]]'' sequences, creating an artificial [[gene cassette]] which can then be conditionally [[deletion (genetics)|deleted]] (knocked out), [[chromosomal translocation|translocated]], or [[chromosomal inversion|inverted]] in a process called [[Cre-Lox recombination]].<ref>
{{cite journal | vauthors = Nagy A | s2cid = 2916710 | title = Cre recombinase: the universal reagent for genome tailoring | journal = Genesis | volume = 26 | issue = 2 | pages = 99–109 | date = February 2000 | pmid = 10686599 | doi = 10.1002/(SICI)1526-968X(200002)26:2<99::AID-GENE1>3.0.CO;2-B | doi-access = free }}
</ref> [[Recombinant DNA|Recombination]] between LoxP sites is catalysed by [[Cre recombinase]]. The term "floxing" is a portmanteau constructed from the phrase "flanking/flanked by LoxP".
The floxing method is essential in the development of scientific model systems as it allows researchers to have spatial and temporal alteration of gene expression.<ref name="HayashiMcMahon20022">{{cite journal | vauthors = Hayashi S, McMahon AP | title = Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse | journal = Developmental Biology | volume = 244 | issue = 2 | pages = 305–18 | date = April 2002 | pmid = 11944939 | doi = 10.1006/dbio.2002.0597 | doi-access = free }}</ref> The Cre-Lox system is widely used to manipulate gene expression in model organisms such as mice in order to study human diseases and drug development.<ref name=":4">{{Cite book|title=Mouse genetics : methods and protocols|others=Singh, Shree Ram,, Coppola, Vincenzo|date=26 July 2014 |isbn=9781493912155|location=New York, NY|oclc=885338722}}</ref> For example, using the Cre-Lox system, researchers are able to study [[oncogene]]s and [[tumor suppressor]] genes and their role in the development and progression of [[cancer]] in mouse models.<ref name=":3">{{Cite book|title=Genetically Engineered Mice for Cancer Research |doi=10.1007/978-0-387-69805-2|year=2012|isbn=978-0-387-69803-8|last1=Green|first1=Jeffrey E.|last2=Ried|first2=Thomas |s2cid=40599715 | name-list-style = vanc }}</ref>
== Uses in research ==
Floxing a gene allows it to be deleted (knocked out),<ref>{{cite book | vauthors = Friedel RH, Wurst W, Wefers B, Kühn R | chapter = Generating Conditional Knockout Mice | title = Transgenic Mouse Methods and Protocols | series = Methods in Molecular Biology | volume = 693 | pages = 205–31 | date = 2011 | pmid = 21080282 | doi = 10.1007/978-1-60761-974-1_12 | isbn = 978-1-60761-973-4 }}</ref><ref>{{Citation|last1=Sakamoto|first1=Kazuhito|title=Generation of Conditional Knockout Mice|date=2014|work=Mouse Genetics|volume=1194|pages=21–35|editor-last=Singh|editor-first=Shree Ram|publisher=Springer New York|doi=10.1007/978-1-4939-1215-5_2|pmid=25064096|isbn=9781493912148|last2=Gurumurthy|first2=Channabasavaiah B.|last3=Wagner|first3=Kay-Uwe|series=Methods in Molecular Biology |editor2-last=Coppola|editor2-first=Vincenzo | name-list-style = vanc }}</ref> translocated or inserted<ref name=":1">{{cite journal | vauthors = Imuta Y, Kiyonari H, Jang CW, Behringer RR, Sasaki H | title = Generation of knock-in mice that express nuclear enhanced green fluorescent protein and tamoxifen-inducible Cre recombinase in the notochord from Foxa2 and T loci | journal = Genesis | volume = 51 | issue = 3 | pages = 210–8 | date = March 2013 | pmid = 23359409 | pmc = 3632256 | doi = 10.1002/dvg.22376 }}</ref> (through various mechanisms in Cre-Lox recombination).
The floxing of genes is essential in the development of scientific model systems as it allows spatial and temporal alteration of gene expression. In layman's terms, the gene can be knocked-out (inactivated) in a specific tissue ''[[in vivo]]'', at a specific time chosen by the scientist. The scientist can then evaluate the effects of the knocked-out gene and identify the gene's normal function.<ref>{{cite journal | vauthors = Hall B, Limaye A, Kulkarni AB | title = Overview: generation of gene knockout mice | journal = Current Protocols in Cell Biology | volume = Chapter 19 | pages = Unit 19.12 19.12.1–17 | date = September 2009 | pmid = 19731224 | pmc = 2782548 | doi = 10.1002/0471143030.cb1912s44 }}</ref> This is different from having the gene absent starting from conception, whereby inactivation or loss of genes that are essential for the development of the organism may interfere with the normal function of cells and prevent the production of viable offspring.<ref>{{Cite journal|last1=Rodrigues|first1=João V.|last2=Shakhnovich|first2=Eugene I.| name-list-style = vanc |date=2019-08-01|title=Adaptation to mutational inactivation of an essential E. coli gene converges to an accessible suboptimal fitness peak |journal=bioRxiv|language=en|pages=552240|doi=10.1101/552240|doi-access=free|arxiv=1902.06630}}</ref>
=== Mechanism of deletion ===
[[File:CreLoxP_experiment.png|thumb|297x297px|A model experiment in genetics using the Cre-lox system: the premature stop sequence present in floxed mice is removed only from cells that express Cre recombinase when the mice are bred together.]]
Deletion events are useful for performing [[Genome editing|gene editing]] experiments through precisely removing segments of or even whole genes. Deletion requires floxing of the segment of interest with loxP sites which face the same direction. The Cre recombinase will detect the unidirectional loxP sites and excise the floxed segment of DNA.<ref name=":0">{{cite journal | vauthors = Schwenk F, Baron U, Rajewsky K | title = A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells | journal = Nucleic Acids Research | volume = 23 | issue = 24 | pages = 5080–1 | date = December 1995 | pmid = 8559668 | pmc = 307516 | doi = 10.1093/nar/23.24.5080 }}</ref> The successfully edited clones can be selected using a selection marker which can be removed using the same Cre-LoxP system.<ref name=":0" /> The same mechanism can be used to create [[Conditional gene knockout|conditional alleles]] by introducing an [[FLP-FRT recombination|FRT/Flp]] site which accomplishes the same mechanism but with a different enzyme.
=== Mechanism of inversion ===
Inversion events are useful for inactivating a gene or DNA sequence without actually removing it, and thereby maintaining a consistent amount of genetic material. The inverted genes are not often associated with abnormal [[phenotype]]s, meaning the inverted genes are generally viable.<ref>{{Cite journal|last1=Griffiths|first1=Anthony JF|last2=Miller|first2=Jeffrey H.|last3=Suzuki|first3=David T.|last4=Lewontin|first4=Richard C.|last5=Gelbart|first5=William M.| name-list-style = vanc |date=2000|title=Inversions|url=https://www.ncbi.nlm.nih.gov/books/NBK22042/|journal=An Introduction to Genetic Analysis. 7th Edition }}</ref> Cre-LoxP recombination that results in inversion requires loxP sites flanking the gene of interest, with the loxP sites oriented towards each other as [[inverted repeats]]. By undergoing Cre recombination, the region flanked by the loxP sites will become inverted, i.e. re-inserted in the same position but in reverse orientation;<ref name="A rapid in vitro method to flip bac">{{cite journal | vauthors = Xu J, Zhu Y | title = A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid | journal = BMC Biotechnology | volume = 18 | issue = 1 | pages = 52 | date = August 2018 | pmid = 30170595 | pmc = 6119287 | doi = 10.1186/s12896-018-0462-x | doi-access = free }}</ref> this process is not permanent and can be reversed.<ref>{{cite journal | vauthors = Oberdoerffer P, Otipoby KL, Maruyama M, Rajewsky K | title = Unidirectional Cre-mediated genetic inversion in mice using the mutant loxP pair lox66/lox71 | journal = Nucleic Acids Research | volume = 31 | issue = 22 | pages = 140e–140 | date = November 2003 | pmid = 14602933 | pmc = 275577 | doi = 10.1093/nar/gng140 }}</ref>  
=== Mechanism of translocation ===
Translocation events occur when the loxP sites flank genes on two different DNA molecules in a unidirectional orientation. Cre recombinase is then used to generate a translocation between the two DNA molecules, exchanging the genetic material from one DNA molecule to the other, forming a simultaneous translocation of both floxed genes.<ref name="A rapid in vitro method to flip bac"/><ref>{{Cite book |last1=Griffiths|first1=Anthony JF|last2=Miller|first2=Jeffrey H.|last3=Suzuki|first3=David T.|last4=Lewontin|first4=Richard C.|last5=Gelbart|first5=William M. | name-list-style = vanc |date=2000|chapter =Translocations|url=https://www.ncbi.nlm.nih.gov/books/NBK21947/|title =An Introduction to Genetic Analysis | edition = 7th  }}</ref>
== Common applications in research ==
[[Cardiomyocytes]] (heart muscle tissue) have been shown to express a type of Cre recombinase that is highly specific to cardiomyocytes and can be used by researchers to perform highly efficient recombinations. This is achieved by using a type of Cre whose expression is driven by the [[Myosin heavy chain|<math>\alpha</math>-myosin heavy chain promoter (<math>\alpha</math>-MyHC)]]. These recombinations are capable of disrupting genes in a manner that is specific to heart tissue ''in vivo'' and allows for the creation of conditional knockouts of the heart, mostly for use as controls.<ref>{{cite journal | vauthors = Pugach EK, Richmond PA, Azofeifa JG, Dowell RD, Leinwand LA | title = Prolonged Cre expression driven by the α-myosin heavy chain promoter can be cardiotoxic | journal = Journal of Molecular and Cellular Cardiology | volume = 86 | pages = 54–61 | date = September 2015 | pmid = 26141530 | pmc = 4558343 | doi = 10.1016/j.yjmcc.2015.06.019 }}</ref> For example, using the [[Cre recombinase]] with the [[Myosin heavy chain|<math>\alpha</math>]]-MyHC promoter causes the floxed gene to be inactivated in the heart alone. Further, these knockouts can be made inducible. In several mouse studies, [[tamoxifen]] is used to induce the expression of [[Cre recombinase]].<ref name="HayashiMcMahon20022"/> In this case, [[Cre recombinase]] is fused to a portion of the mouse [[estrogen receptor]] (ER) which contains a mutation within its [[Ligand binding domain|ligand binding domain (LBD)]]. The mutation renders the receptor inactive, which leads to incorrect localization through its interactions with chaperone proteins such as heat shock protein 70 and 90 ([[Hsp70]] and [[Hsp90]]). Tamoxifen binds to Cre-ER and disrupts its interactions with the chaperones, which allows the Cre-ER fusion protein to enter the nucleus and perform recombination on the floxed gene.<ref>{{cite journal | vauthors = Danielian PS, Muccino D, Rowitch DH, Michael SK, McMahon AP | title = Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase | journal = Current Biology | volume = 8 | issue = 24 | pages = 1323–6 | date = December 1998 | pmid = 9843687 | doi = 10.1016/s0960-9822(07)00562-3 | doi-access = free | bibcode = 1998CBio....8.1323D }}</ref><ref>{{Cite book|title=Transgenesis techniques : principles and protocols|date=2002|publisher=Humana Press|others=Clarke, Alan R.|isbn=9781592591787|edition=2nd|location=Totowa, NJ|oclc=50175106}}</ref> Additionally, [[Cre recombinase]] can be induced by heat when under the control of specific heat shock elements (HSEs).<ref>{{Cite book|title=Cancer and zebrafish : mechanisms, techniques, and models|others=Langenau, David M.|date=10 May 2016 |isbn=9783319306544|location=Switzerland|oclc=949668674}}</ref><ref>{{cite journal | vauthors = Kobayashi K, Kamei Y, Kinoshita M, Czerny T, Tanaka M | title = A heat-inducible CRE/LOXP gene induction system in medaka | journal = Genesis | volume = 51 | issue = 1 | pages = 59–67 | date = January 2013 | pmid = 23019184 | doi = 10.1002/dvg.22348 | s2cid = 25211137 }}</ref>
== References ==
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[[Category:DNA]]
[[Category:Genetics techniques]]