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 system to map specific neurons in the brain, for example.
FLEx switches are also often used for inducible gene expression or repression.
If Cre expression is controlled by an inducible system, the FLEx switch will flip only when
the inducer is present.
Commonly used Cre-inducible systems include tamoxifen and tetracycline, which can both
be used in vivo.
Multiple genes can be controlled at once using a FLEx switch.
If two genes are inside the switch, one inverted and the other not, you can 'turn on' one
gene while 'turning off' another.
Or, by changing the position of the lox sites,  you can even cause the activation of one
gene and the simultaneous excision of another!
For example, you can use FLEx to easily replace the wildtype version of a gene with a mutated version.
This is an excellent way to study essential
genes, since traditional knockout studies
would be lethal.
Gene knock-ins can become much easier by using the FLEx system for Recombinase Mediated Cassette
Exchange.
In this approach, you would begin with a Cre-expressing mouse that has a genomic region flanked by
different lox sites.
By introducing a vector carrying a gene of
interest flanked by those same lox sites,
you can easily swap in the gene of interest.
This is a powerful tool for in vivo gene knock-in, since the same mouse line can be used to generate
many different knock-in strains; you simply have to change the gene in your vector!
Other than their utility, FLEx switches have some unique advantages over other techniques.
FLEx switches are ideal for working with AAV, which is very popular for in vivo gene therapy,
but has a limited packaging capacity.
By controlling expression of the gene of interest indirectly via Cre, you can use large inducible
or tissue-specific promoters that aren't
able to be packaged inside AAV.
The FLEx switch itself is also very compact, taking up only 5% of an AAV's packaging capacity.
Another advantage of using FLEx switches is that one Cre-expressing mouse line can be
used in many different experiments, cutting down on costs, time, and labour.
Because Cre-lox recombination is a well-established research tool, there are many Cre-expressing
vectors, cell lines, and mice readily available.
I hope you've found this video helpful and
now better understand the advantages of using
the FLEx system for in vivo genetic manipulation.
For more information, check out our knowledge base article on Cre-Lox recombination, or
order a custom "D-I-O" vector for Cre-dependent expression of your gene of choice.
