- [Narrator] Site-specific recombination, abbreviated SSR,
is a method utilized in molecular biology
that exchanges DNA segments
possessing at least a certain degree of sequence homology.
This form of genetic engineering is specific,
fast, and efficient.
It has been implemented in simple prokaryotic
and more complex eukaryotic systems.
In this module, we will discuss SSR
in the context of transgenic mice.
To be able to understand the use of
site-specific recombination in the mouse animal model,
we will discuss four main topics.
First, we will present a brief history of transgenic mice.
Then we will describe the need for site-specific
recombination in these genetically modified animals.
Following, we will discuss the process of
Cre-Lox recombination.
And finally, we will show how the Cre-Lox system
of site-specific recombination is used
developing knockout mice.
Transgenic mice were first developed
by Mario Capecchi in the 1980s.
Dr. Capecchi invented a method to change or remove a gene
within a mouse genome by hijacking the cells
on homologous recombination.
This repurposing of the host cells' processes
earned him a Nobel Prize in 2007.
In order to perform this method, embryonic stem cells
are harvested from the inner cell mass of a blastocyst
of a white mouse.
A blastocyst is a preimplantation embryo,
a few days after fertilization.
The inner cell mass lies within the blastocyst,
surrounded by a cellular layer called the trophoblast.
After harvesting the embryonic stem cells,
you plate them in a culture dish and grow them in vitro.
A DNA sequence is developed that is very similar
to the original gene, but sufficiently different enough
to cause the new gene to be inactive.
This gene also carries one or two markers
that do not exist in the mouse genome,
such as a fluorescent gene like GFP,
or a drug resistance gene.
The new genetic sequence is introduced
to the stem cells by electroporation.
Electroporation is a method that applies
an electrical current to cells that increases
the cell membrane permeability, thus allowing molecules
such as DNA to be introduced into the cell.
A small portion of the cells will undergo
homologous recombination, and incorporate
the new DNA sequence into their genome.
The cells that underwent homologous recombination
will be identified by screening for fluorescence
or drug resistance.
These cells will then be expanded in culture
and injected into the recipient blastocyst,
usually a different coat color
than the donor mouse, such as gray.
The resulting embryo will contain stem cells
from the original gene from the gray mouse,
and new gene from the white mouse.
This embryo will then be implanted into the foster mother.
In this example, it is a gray mouse.
The embryos will develop, and the resulting offspring
will be chimeras, containing both the original and new gene,
or a normal gray mouse without an altered genome.
The chimeric mouse will be crossed with another gray mouse.
Some of the offspring will be heterozygous
for the altered gene, and will be white,
or will contain the original gene, and be gray.
The resulting heterozygous mouse can be bred
with one another to produce mice that are homozygous
for the new gene to develop a transgenic knockout strain.
Although the method developed by Dr. Capecchi
was revolutionary, there were also
major drawbacks of this method.
For example, some genetic knockouts can cause
abnormal development of the animal.
It can also lead to early embryonic death
or neonatal lethality.
Another downside is the resulting animal
will have an inherited mutated state that can limit the user
if the desired model is an acquired disease.
Therefore, the need arose for a method that could result in
transgenic animals with mutations
that were site and time-specific.
Fortunately, researchers were able to
introduce bacteriophage and yeast derived site-specific
recombination systems into mammalian cells.
As described at the beginning of this module,
site-specific recombination is a type of
genetic recombination in which a genetic exchange
takes place between homologous segments of DNA
using enzymes called recombinases.
These enzymes are derived from bacteria and fungi
that catalyze directionally sensitive
DNA exchange reactions, between 30 to 40
nucleotide short target site sequences
that are all specific to each recombinase.
Most site-specific recombinases are grouped
into one of two families, serine or tyrosine.
Some examples of the serine recombinase family
are gamma-delta resolvase from the Tn1000 transposon,
Tn3 resolvase from the Tn3 transposon,
and phiC31 integrase from the phiC31 phage.
Some examples of the tyrosine recombinase family
are cyclization recombinase and flippase recombinase.
The two recombinase families perform reactions
with similar outcomes, however the two families
are unrelated to each other, having different
protein structures and reaction mechanisms.
Cre recombinase, also known as cyclization recombination,
is a tyrosine recombinase enzyme derived from the
P1 bacteriophage, and was developed by Dr. Brian Sauer
initially for use in activating gene expression
in mammalian cell lines in the late 80s.
This recombinase was first used in mice in the mid-90s
to manipulate T lymphocytes.
Here you can see the use of four tyrosines working together
to nick two strands of DNA for recombination.
Cre recombinase interacts with recognition sites
with DNA called LoxP sites.
LoxP stands for locus of crossing over of bacteriophage P1.
This is a 34-base pair sequence consisting of two
13-base pair palindromic repeated sequences
with an eight-base pair core region,
which determines the orientation of the target site.
The result of Cre-mediated recombination
is dependent upon the location and relative orientation
of the LoxP sites within the DNA strand.
The result of Cre-mediated recombination
is dependent upon the location and relative orientation
of the LoxP sites, giving three main types of recombination.
Here, the LoxP sites are on the same DNA strand,
and are in opposite orientations.
Recombination results in an inversion,
and the region of DNA between the LoxP sites is reversed.
If the sites face in the same direction,
the sequence between the LoxP sites is excised
as a circular piece of DNA which is not maintained.
This is therefore named a deletion.
If the sites are on separate DNA strands,
a translocation event is generated at the LoxP sites
where the gene of interest is now located
on a separate DNA strand.
The Cre recombinase method of site-specific recombination
is one of the most commonly used methods
to form transgenic mice with acquired mutations,
versus the inherited mutations
that were discussed at the beginning of this module.
The method to attain this type of mouse
is similar to the traditional method,
with some modifications that allow both spatial
and temporal control of Cre recombinase activity.
The spatial control of genetic alteration
involves the selection of a tissue-specific promoter
to drive Cre expression, for example,
using a kidney-specific cadherin promoter
for renal-specific activity.
Temporal control takes advantage of various
ligand-binding domains that have been developed,
such as fusing the Cre enzyme with a mutated
ligand-binding domain for the human estrogen receptor, ERT.
This imparts temporal control, because when mice
are introduced to the drug tamoxifen,
an estrogen receptor antagonist, the CreERT construct
is able to penetrate the nucleus
and induce targeted mutation.