The Cre-LoxP system is a technique used in a variety of research studies that allows for
spatiotemporal control over gene deletion, inversion and translocation.
The technique utilizes the Cre recombinase enzyme to catalyze site specific recombinations.
Due to it’s efficiency in manipulating genes and chromosomes in a wide range of organisms,
it is a powerful tool that is widely used in modern day biochemical and molecular biological
research.
Previous genetic knockout experiments were restricted if the genes of interest were necessary
for embryonic survival or if the gene in question functioned in multiple tissues or cell types.
Cre/Lox made it possible to conditionally knockout genes solely in subsets of cells
where Cre recombinase is expressed.
Cre recombinase is named because it creates recombination. It is an enzyme derived from
the P1 bacteriophage, and is designed to cut out genetic sequences between two LoxP sites.
The Cre recombinase enzyme contains an active site, consisting of a catalytic triad with
residues Arginine 173, Histidine 289, and Arginine 292, and nucleophilic residues Tyrosine
324 and Tryptophan 315. This allows for Cre recombinase to bind to its substrate, DNA.
The name LoxP comes from the locus of crossover in P1 bacteriophage. LoxP is a site on the
P1 bacteriophage consisting of 34 base pairs. This includes two symmetric 13 base pair sequences,
and an asymmetric 8 base pair sequence.
Because of the asymmetry of the LoxP site, Cre recombinase can either catalyze a deletion,
inversion, or translocation of the genetic sequence between the sites, depending on the
orientation of these LoxP sites. LoxP sites going in the same direction results in deletions.
LoxP sites going in opposite directions can result in inversions. Interchromosomal recombination
can also result in translocations.
There are two main ways to get Cre recombinase to be expressed in a cell. The first is to
use a virus to introduce the gene for Cre recombinase into the cell. The other method
is to generate transgenic mice that insert the gene for Cre recombinase after a specific
promoter. Because this promoter can be expressed in certain cell types, the expression of Cre
recombinase can also be limited to these cells.
There are many applications of Cre, one of which is lineage analysis, which is the determination
of the origin or fate of cells. One example of lineage analysis was used by the Melton
lab in determining the origin of adult pancreatic beta cells. They used an inducible version
of Cre to determine whether new beta cells were derived from stem cells or preexisting
beta cells. In their experiment, transgenic mice were generated in which the insulin promoter
drove the expression of tamoxifen-dependent Cre recombinase. The enzyme then existed in
the cytosol as a complex with the estrogen receptor and heat shock protein. When tamoxifen
was injected in the cell, the CreER complex separated, and the Cre recombinase entered
the nucleus to perform recombination. Once in the nucleus, the Cre recombinase cut out
the lacZ stop sequence, and thus allowed the expression of the HPAP label, short for human
placental alkaline phosphatase. Through this process, cells that normally produce insulin,
namely the pancreatic beta cells, were tagged with the HPAP label.
Using these mice, the Melton lab performed a “Pulse-Chase experiment, the “Pulse”
being the set of cells labeled by HPAP using Cre recombinase in the pancreas, and the “Chase”
being the set of cells that had the label after a given amount of time. Starting with
a population of fully labeled beta cells, they hypothesized that if new beta cells arose
from preexisting ones, the percentage of stained cells wouldn’t change. However, if they
developed from unlabeled stem cells, the new population would have a much smaller percentage
of labeled cells. Their data showed that the percentage of labeled cells remained constant,
which suggests that new beta cells were derived from pre-existing ones rather than from stem cells.
Thus, the Cre- Lox system can be used for numerous types of modifications. Because it
can be designed in many different ways, it has been used in a variety of different research
studies that involve many different tissue types and organ systems. Perhaps in the future,
this technology can also be used for cures and treatments in humans. It has already been
demonstrated that Cre-loxP functions in human embryonic stem cells . There is definitely great potential
for this system not only in research but also in clinical settings.