Floxing

In genetic engineering, floxing refers to the insertion of a DNA sequence (which is then said to be floxed) between two LoxP sequences, creating an artificial gene cassette which can then be conditionally deleted (knocked out), translocated, or inverted in a process called Cre-Lox recombination.[1] 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.[2] 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.[3] For example, using the Cre-Lox system, researchers are able to study oncogenes and tumor suppressor genes and their role in the development and progression of cancer in mouse models.[4]

## Contents

* 1 Uses in research
* 1.1 Mechanism of deletion
* 1.2 Mechanism of inversion
* 1.3 Mechanism of translocation
* 2 Common applications in research
* 3 References

## Uses in research

Floxing a gene allows it to be deleted (knocked out),[5][6] translocated or inserted[7] (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.[8] 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.[9]

### Mechanism of deletion

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 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.[10] The successfully edited clones can be selected using a selection marker which can be removed using the same Cre-LoxP system.[10] The same mechanism can be used to create conditional alleles by introducing an 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 phenotypes, meaning the inverted genes are generally viable.[11] 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;[12] this process is not permanent and can be reversed.[13]

### 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.[12][14]

## 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 α-myosin heavy chain promoter (α-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.[15] For example, using the Cre recombinase with the α-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.[2] 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 (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.[16][17] Additionally, Cre recombinase can be induced by heat when under the control of specific heat shock elements (HSEs).[18][19]

## References

1. ↑ "Cre recombinase: the universal reagent for genome tailoring". Genesis 26 (2): 99–109. February 2000. doi:10.1002/(SICI)1526-968X(200002)26:2<99::AID-GENE1>3.0.CO;2-B. PMID 10686599.
2. ↑ 2.0 2.1 "Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse". Developmental Biology 244 (2): 305–18. April 2002. doi:10.1006/dbio.2002.0597. PMID 11944939.
3. ↑ Mouse genetics: methods and protocols. Singh, Shree Ram,, Coppola, Vincenzo. New York, NY. 26 July 2014. ISBN 978-1-4939-1215-5. OCLC 885338722.
4. ↑ Green, Jeffrey E.; Ried, Thomas (2012). Genetically Engineered Mice for Cancer Research. doi:10.1007/978-0-387-69805-2. ISBN 978-0-387-69803-8.
5. ↑ "Generating Conditional Knockout Mice". Transgenic Mouse Methods and Protocols. Methods in Molecular Biology. 693. 2011. pp. 205–31. doi:10.1007/978-1-60761-974-1_12. ISBN 978-1-60761-973-4.
6. ↑ Sakamoto, Kazuhito; Gurumurthy, Channabasavaiah B.; Wagner, Kay-Uwe (2014), Singh, Shree Ram; Coppola, Vincenzo, eds., "Generation of Conditional Knockout Mice", Mouse Genetics, Methods in Molecular Biology (Springer New York) 1194: pp. 21–35, doi:10.1007/978-1-4939-1215-5_2, ISBN 978-1-4939-1214-8, PMID 25064096
7. ↑ "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". Genesis 51 (3): 210–8. March 2013. doi:10.1002/dvg.22376. PMID 23359409.
8. ↑ "Overview: generation of gene knockout mice". Current Protocols in Cell Biology Chapter 19: Unit 19.12 19.12.1–17. September 2009. doi:10.1002/0471143030.cb1912s44. PMID 19731224.
9. ↑ Rodrigues, João V.; Shakhnovich, Eugene I. (2019-08-01). "Adaptation to mutational inactivation of an essential E. coli gene converges to an accessible suboptimal fitness peak" (in en). bioRxiv. doi:10.1101/552240.
10. ↑ 10.0 10.1 "A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells". Nucleic Acids Research 23 (24): 5080–1. December 1995. doi:10.1093/nar/23.24.5080. PMID 8559668.
11. ↑ Griffiths, Anthony JF; Miller, Jeffrey H.; Suzuki, David T.; Lewontin, Richard C.; Gelbart, William M. (2000). "Inversions". An Introduction to Genetic Analysis. 7th Edition. https://www.ncbi.nlm.nih.gov/books/NBK22042/.
12. ↑ 12.0 12.1 "A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid". BMC Biotechnology 18 (1): 52. August 2018. doi:10.1186/s12896-018-0462-x. PMID 30170595.
13. ↑ "Unidirectional Cre-mediated genetic inversion in mice using the mutant loxP pair lox66/lox71". Nucleic Acids Research 31 (22): 140e–140. November 2003. doi:10.1093/nar/gng140. PMID 14602933.
14. ↑ Griffiths, Anthony JF; Miller, Jeffrey H.; Suzuki, David T.; Lewontin, Richard C.; Gelbart, William M. (2000). "Translocations". An Introduction to Genetic Analysis (7th ed.). https://www.ncbi.nlm.nih.gov/books/NBK21947/.
15. ↑ "Prolonged Cre expression driven by the α-myosin heavy chain promoter can be cardiotoxic". Journal of Molecular and Cellular Cardiology 86: 54–61. September 2015. doi:10.1016/j.yjmcc.2015.06.019. PMID 26141530.
16. ↑ "Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase". Current Biology 8 (24): 1323–6. December 1998. doi:10.1016/s0960-9822(07)00562-3. PMID 9843687. Bibcode: 1998CBio....8.1323D.
17. ↑ Transgenesis techniques: principles and protocols. Clarke, Alan R. (2nd ed.). Totowa, NJ: Humana Press. 2002. ISBN 978-1-59259-178-7. OCLC 50175106.
18. ↑ Cancer and zebrafish: mechanisms, techniques, and models. Langenau, David M.. Switzerland. 10 May 2016. ISBN 978-3-319-30654-4. OCLC 949668674.
19. ↑ "A heat-inducible CRE/LOXP gene induction system in medaka". Genesis 51 (1): 59–67. January 2013. doi:10.1002/dvg.22348. PMID 23019184.

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