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Today we'll be discussing FLEx, or "flip excision" switches. FLEx switches are a powerful tool for genetic manipulation, especially in vivo. Before we can talk about FLEx switches, however, we need to explain the basics of Cre-Lox recombination. The Cre-Lox system relies on two components to function: a site-specific recombinase called Cre, and a DNA sequence called loxP. A loxP site is a 34 bp sequence made of two palindromic recognition sites, separated by an 8 bp spacer that gives directionality to the sequence. Cre recombinase recognizes loxP sites and initiates site-specific recombination between those sites. The outcome of the recombination depends on two factors: the position of the lox sites, and their orientation. If loxP sites flank a gene in the same orientation, recombination will result in the gene being excised. This is an irreversible process. If loxP sites flank a gene in opposing orientations, recombination results in gene inversion. Since the loxP sites remain unchanged, this process is reversible and the gene can flip back and forth between both orientations. The continual flipping of the insert during Cre-loxP inversion means it's not very useful for genetic manipulation. Scientists, however, have found a clever way to get around this problem. They discovered that Cre will only recombine two lox sites if they have the same spacer sequence. So, they developed different lox sites that have different spacer regions that are not cross-compatible. This property has been exploited to make constructs with FLEx switches. A FLEx switch has the gene of interest flanked by two sets of different lox sites. Two recombination steps will occur. The first will be an inversion of the flanked sequence using one set of lox sites. This will leave two identical sites with the same orientation on one side of the gene. The second recombination will cause the excision of the intervening sequence. The final product will be the inverted gene of interest flanked by two different lox sites. Because these sites aren't cross-compatible, any further Cre-based recombination events will not occur. The inverted version of a gene will not be expressed, so a simple FLEx switch can be used to irreversibly turn gene expression on or off when Cre is present. Cre-On vectors are often called "D-I-O" vectors because the gene of interest is "Double-floxed with Inverted Orientation". Cre-Off vectors are often called "D-O" vectors because the gene of interest is "Double-floxed in Orientation". FLEx switches are an extremely flexible research tool that can be used for many purposes. In the next section, we'll discuss some of the novel ways FLEx switches can be used. These include: tissue-specific gene expression, inducible gene expression, control of multiple genes simultaneously, and gene knock-ins. FLEx switches are commonly used during in vivo experiments to achieve tissue-specific expression. If a mouse expresses Cre under the control of a tissue-specific promoter, the FLEx switch will only be triggered in the desired tissue type. This limits ubiquitous gene expression that can confound experimental results. Researchers can use this FLEx reporter...