Cre-LoxP Recombination

Cre-LoxP Recombination

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.

30 thoughts on “Cre-LoxP Recombination”

  1. Hi, thanks for the video. It would help if you place the references in the video description, so we can research it easier.

  2. Can i have the accession number from ncbi or pdb to view its structure in more detail..?
    Thanks in advance.. πŸ™‚

  3. To how large of an area could you introduce the Cre-LoxP System in vivo given the tissue is easily accessible such as epidermal tissue?

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