Floxing

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This figure depicts how Floxing is used in scientific research for spatial and temporal control of gene expression. Floxing Flow Chart.svg
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 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".

Contents

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]

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. CreLoxP experiment.png
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 -myosin heavy chain promoter (-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 -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]

Related Research Articles

<span class="mw-page-title-main">Chromosomal crossover</span> Cellular process

Chromosomal crossover, or crossing over, is the exchange of genetic material during sexual reproduction between two homologous chromosomes' non-sister chromatids that results in recombinant chromosomes. It is one of the final phases of genetic recombination, which occurs in the pachytene stage of prophase I of meiosis during a process called synapsis. Synapsis begins before the synaptonemal complex develops and is not completed until near the end of prophase I. Crossover usually occurs when matching regions on matching chromosomes break and then reconnect to the other chromosome.

Gene knockouts are a widely used genetic engineering technique that involves the targeted removal or inactivation of a specific gene within an organism's genome. This can be done through a variety of methods, including homologous recombination, CRISPR-Cas9, and TALENs.

Lox is a fillet of cured salmon.

A transgene is a gene that has been transferred naturally, or by any of a number of genetic engineering techniques, from one organism to another. The introduction of a transgene, in a process known as transgenesis, has the potential to change the phenotype of an organism. Transgene describes a segment of DNA containing a gene sequence that has been isolated from one organism and is introduced into a different organism. This non-native segment of DNA may either retain the ability to produce RNA or protein in the transgenic organism or alter the normal function of the transgenic organism's genetic code. In general, the DNA is incorporated into the organism's germ line. For example, in higher vertebrates this can be accomplished by injecting the foreign DNA into the nucleus of a fertilized ovum. This technique is routinely used to introduce human disease genes or other genes of interest into strains of laboratory mice to study the function or pathology involved with that particular gene.

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.

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.

<span class="mw-page-title-main">FLP-FRT recombination</span> Site-directed recombination technology

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.

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.

Conditional gene knockout is a technique used to eliminate a specific gene in a certain tissue, such as the liver. This technique is useful to study the role of individual genes in living organisms. It differs from traditional gene knockout because it targets specific genes at specific times rather than being deleted from beginning of life. Using the conditional gene knockout technique eliminates many of the side effects from traditional gene knockout. In traditional gene knockout, embryonic death from a gene mutation can occur, and this prevents scientists from studying the gene in adults. Some tissues cannot be studied properly in isolation, so the gene must be inactive in a certain tissue while remaining active in others. With this technology, scientists are able to knockout genes at a specific stage in development and study how the knockout of a gene in one tissue affects the same gene in other tissues.

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).

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A P1-derived artificial chromosome, or PAC, is a DNA construct derived from the DNA of P1 bacteriophages and Bacterial artificial chromosome. It can carry large amounts of other sequences for a variety of bioengineering purposes in bacteria. It is one type of the efficient cloning vector used to clone DNA fragments in Escherichia coli cells.

In molecular cloning and biology, a gene knock-in refers to a genetic engineering method that involves the one-for-one substitution of DNA sequence information in a genetic locus or the insertion of sequence information not found within the locus. Typically, this is done in mice since the technology for this process is more refined and there is a high degree of shared sequence complexity between mice and humans. The difference between knock-in technology and traditional transgenic techniques is that a knock-in involves a gene inserted into a specific locus, and is thus a "targeted" insertion. It is the opposite of gene knockout.

A knockout mouse, or knock-out mouse, is a genetically modified mouse in which researchers have inactivated, or "knocked out", an existing gene by replacing it or disrupting it with an artificial piece of DNA. They are important animal models for studying the role of genes which have been sequenced but whose functions have not been determined. By causing a specific gene to be inactive in the mouse, and observing any differences from normal behaviour or physiology, researchers can infer its probable function.

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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.

References

  1. ↑ Nagy A (February 2000). "Cre recombinase: the universal reagent for genome tailoring". Genesis. 26 (2): 99–109. doi: 10.1002/(SICI)1526-968X(200002)26:2<99::AID-GENE1>3.0.CO;2-B . PMID   10686599. S2CID   2916710.
  2. 1 2 Hayashi S, McMahon AP (April 2002). "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. 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   9781493912155. OCLC   885338722.{{cite book}}: CS1 maint: location missing publisher (link) CS1 maint: others (link)
  4. ↑ Green JE, Ried T (2012). Genetically Engineered Mice for Cancer Research. doi:10.1007/978-0-387-69805-2. ISBN   978-0-387-69803-8. S2CID   40599715.
  5. ↑ Friedel RH, Wurst W, Wefers B, Kühn R (2011). "Generating Conditional Knockout Mice". Transgenic Mouse Methods and Protocols. Methods in Molecular Biology. Vol. 693. pp. 205–31. doi:10.1007/978-1-60761-974-1_12. ISBN   978-1-60761-973-4. PMID   21080282.
  6. ↑ Sakamoto K, Gurumurthy CB, Wagner KU (2014), Singh SR, Coppola V (eds.), "Generation of Conditional Knockout Mice", Mouse Genetics, Methods in Molecular Biology, vol. 1194, Springer New York, pp. 21–35, doi:10.1007/978-1-4939-1215-5_2, ISBN   9781493912148, PMID   25064096
  7. ↑ Imuta Y, Kiyonari H, Jang CW, Behringer RR, Sasaki H (March 2013). "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. doi:10.1002/dvg.22376. PMC   3632256 . PMID   23359409.
  8. ↑ Hall B, Limaye A, Kulkarni AB (September 2009). "Overview: generation of gene knockout mice". Current Protocols in Cell Biology. Chapter 19: Unit 19.12 19.12.1–17. doi:10.1002/0471143030.cb1912s44. PMC   2782548 . PMID   19731224.
  9. ↑ Rodrigues JV, Shakhnovich EI (2019-08-01). "Adaptation to mutational inactivation of an essential E. coli gene converges to an accessible suboptimal fitness peak". bioRxiv: 552240. arXiv: 1902.06630 . doi: 10.1101/552240 .
  10. 1 2 Schwenk F, Baron U, Rajewsky K (December 1995). "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. doi:10.1093/nar/23.24.5080. PMC   307516 . PMID   8559668.
  11. ↑ Griffiths AJ, Miller JH, Suzuki DT, Lewontin RC, Gelbart WM (2000). "Inversions". An Introduction to Genetic Analysis. 7th Edition.
  12. 1 2 Xu J, Zhu Y (August 2018). "A rapid in vitro method to flip back the double-floxed inverted open reading frame in a plasmid". BMC Biotechnology. 18 (1): 52. doi: 10.1186/s12896-018-0462-x . PMC   6119287 . PMID   30170595.
  13. ↑ Oberdoerffer P, Otipoby KL, Maruyama M, Rajewsky K (November 2003). "Unidirectional Cre-mediated genetic inversion in mice using the mutant loxP pair lox66/lox71". Nucleic Acids Research. 31 (22): 140e–140. doi:10.1093/nar/gng140. PMC   275577 . PMID   14602933.
  14. ↑ Griffiths AJ, Miller JH, Suzuki DT, Lewontin RC, Gelbart WM (2000). "Translocations". An Introduction to Genetic Analysis (7th ed.).
  15. ↑ Pugach EK, Richmond PA, Azofeifa JG, Dowell RD, Leinwand LA (September 2015). "Prolonged Cre expression driven by the α-myosin heavy chain promoter can be cardiotoxic". Journal of Molecular and Cellular Cardiology. 86: 54–61. doi:10.1016/j.yjmcc.2015.06.019. PMC   4558343 . PMID   26141530.
  16. ↑ Danielian PS, Muccino D, Rowitch DH, Michael SK, McMahon AP (December 1998). "Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase". Current Biology. 8 (24): 1323–6. Bibcode:1998CBio....8.1323D. doi: 10.1016/s0960-9822(07)00562-3 . PMID   9843687.
  17. ↑ Transgenesis techniques : principles and protocols. Clarke, Alan R. (2nd ed.). Totowa, NJ: Humana Press. 2002. ISBN   9781592591787. OCLC   50175106.{{cite book}}: CS1 maint: others (link)
  18. ↑ Cancer and zebrafish : mechanisms, techniques, and models. Langenau, David M. Switzerland. 10 May 2016. ISBN   9783319306544. OCLC   949668674.{{cite book}}: CS1 maint: location missing publisher (link) CS1 maint: others (link)
  19. ↑ Kobayashi K, Kamei Y, Kinoshita M, Czerny T, Tanaka M (January 2013). "A heat-inducible CRE/LOXP gene induction system in medaka". Genesis. 51 (1): 59–67. doi:10.1002/dvg.22348. PMID   23019184. S2CID   25211137.
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