Floxing, or flanking by loxP, is a process used by geneticists by which a specifically targeted gene is flanked by two loxP sites. Although seemingly simple, this process is relatively new and quite complex. In most cases, gene targeting with the use of floxing is enhanced and catalyzed through a process known as Cre/lox recombination. This can lead to the production of appropriate genetic models for the study of various genetic diseases, as well as diseases that can be triggered by the mutation or inactivation of a certain gene. Many studies have shown the effectiveness of using floxed animal models – especially mouse models – in order to obtain conditional, tissue-specific modifications as well as the alteration of various gene expressions.
In essence, through the use of floxing and Cre recombinase, it is possible to splice a gene entirely out of the genome, invert the orientation of the gene by removing it and splicing it back in with the opposite orientation, and replace the gene with a floxed DNA sequence that originated elsewhere. All of these options are available once Cre recombinase is expressed and becomes active within a cell with a floxed gene.
One of the greatest advantages of floxing is that it allows for detailed in vivo study of abnormalities and diseases associated with genetic modifications. During the development of floxed mouse models, gene knockouts and other alterations can be triggered by certain processes at specific times. This action is made possible by the use of floxing together with the Cre/lox recombination system, allowing for the generation of better, more precise animal models. The development of drugs and genetic treatments can be highly dependent on new breakthroughs and results that are found during the in vivo study of floxed mouse models. Better models can accelerate this process, as they allow researchers to obtain more accurate and practical results.
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Tissue-specific knockouts using Cre/lox recombination and floxed mice can prevent many of the problems that occur during conventional knockout mutations. These can include serious concerns such as increased embryonic lethality and unwanted inconsistencies in the gene knockout process. As a result, whenever greater accuracy or a tissue-specific recombination is needed, most experts will recommend the use of floxed mouse models that have the gene in question inactivated only in a specific type of tissue, such as the kidneys or liver.
Other uses for the site-specific recombinase system using gene floxing can also include conditional gene trapping, the creation of a multipurpose conditional allele and the ability to facilitate more accurate methods for gene silencing. All these uses come with their own advantages, including the reduction of embryo lethality in the case of gene trapping studies and the possibility to induce gene silencing in less time, compared to conventional methods.
In summary, gene floxing is powerful in today’s research laboratories as it allows precise and discrete analysis of gene function. Its use in animal biology will better allow us to delineate the exact role of multiple cell types and at varying times throughout the lifespan of an animal. For example, using gene floxing with the alpha-MyHC promoter causes the floxed gene to be inactivated in the heart alone. Further, these knockouts can be inducible. In several mouse studies, tamoxifen is used to induce the Cre recombinase. In this case, Cre recombinase is fused to a portion of the mouse estrogen receptor which contains a mutation within its ligand binding domain. The mutation renders the receptor inactive, which leads to incorrect localization through its interactions with chaperone proteins such as heat shock protein. 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. Thus, this genetic resource is available to continue the remarkable progress made recently in understanding how certain genes function to regulate development and signaling activities.
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