Understanding the Cre-Lox System for Genome Manipulation in Mice

 
Recombinase-mediated toolbox
 
Cre-Lox
 
system
:
Cre recombinase recombines a pair of short target sequences
 
(
LoxP
 sites)
Depending on the location and orientation of the 
loxP
 sites, Cre recombinase can
initiate deletions, inversions, and translocations of the floxed locus
.
The
 
most
 
widely
 
used
 
site-specific
 
recombinase
 
system
 
for
 
genome
 
manipulation
 
in
mice.
 
Because
 
it
 
is
 
more
 
efficient
 
than
 
other
 
systems.
 
Flp-FRT system:
 
Flp
 
is analogous to cre
 
and
 
FRT
 
to loxP
.
Dre-rox
 
system:
 
Dre
 
is analogous to cre
 
and
 
rox
 
to loxP
.
Recombinase-mediated toolbox
reference
: 
Nagy
 
A 2000,
 
Genesis
image
 
from
 
https://www.jax.org/news-and-insights/2006/may/the-cre-lox-and-flp-frt-systems
Cre-Lox
 
system
Cre recombinase recombines a pair of short target sequences
 
(
LoxP
 sites)
 
Depending on the location and orientation of the 
loxP
 sites, Cre recombinase can
initiate deletions
 
(A)
, inversions
 
(B)
, and translocations
 
(C)
 of the floxed locus
.
 
Use
 
the
 
Cre-Lox
 
system
 
to
 
express
 
a
 
transgene
 
 
images
 
from
 
https://www.jax.org
 
Cre recombinase removes the stop sequence, so the transgene is expressed only in
the cells where Cre is active
 
 
Cre recombinase removes the stop sequence, so the transgene is expressed only in
the cells where Cre is active
 
Use
 
the
 
Cre-Lox
 
system
 
to
 
express
 
a
 
transgene
 
images
 
from
 
https://www.jax.org
 
Use
 
the
 
Cre-Lox
 
system
 
to
 
turn
 
off
 
a
 
transgene
 
~50% of the offspring will be heterozygous for the loxP allele and heterozygous for
the cre transgene.
 
images
 
from
 
https://www.jax.org
 
A
 transgene is designed with loxP sites flanking all or part of it.
 
So the transgene is
turned
 
off
 only in the cells where Cre is active
 
images
 
from
 
https://www.jax.org
 
A
 transgene is designed with loxP sites flanking all or part of it.
 
So the transgene is
turned
 
off
 only in the cells where Cre is active
 
Mate heterozygous mice from
 
the
 
1
st
 
generation
 
back to the homozygous loxP-flanked mice
.
 
Experimental
 
mice
 
(~25%)
:
 
homozygous for the loxP-flanked allele
and heterozygous for the cre transgene
 
Use
 
the
 
Cre-Lox
 
system
 
to
 
turn
 
off
 
a
 
transgene
 
Inducibility:
 
allows
 
temporal
 
control
 
of
 
gene
 
manipulation
 
The
 
tamoxifen-inducible cre alleles
:
 
t
he most common inducible Cre variety
.
 
Cre is
expressed as a fusion protein with a modified estrogen receptor (ER) ligand binding
domain. Binding to tamoxifen allows the Cre-ER fusion protein to translocate to the
nucleus where the recombinase takes
 
effect
.
 
The
 
RU-486
 
inducible
 
c
re alleles
:
 
:
 
cre
 
is
 
expressed
 
as
 
a
 
fusion
 
protein
 
with a modified
progesterone receptor, allowing induction with the progesterone analog RU-486.
 
The
 
TMP-inducible cre alleles
:
 
cre
 
is
 
expressed
 
as
 
a
 
fusion
 
protein
 
with
 
a
 
modified
 
E. coli
dihydrofolate reductase (DHFR) protein
,
 
which
 
can
 
be
 
induced
 
by
 
the common antibiotic
trimethoprim (TMP).
 
Use multiple recombinases
 
for
 
sophisticated
 
manipulation
 
As Cre, Flp, and Dre recognize different target sequences, the combined use of these
recombinases would facilitate more sophisticated genetic manipulation of restricted cell
populations.
 
Example1
: Intersectional control by either Cre/Flp or Cre/Dre recombinases, driven by
different promoters (P1 or P2), on a doubly regulated reporter line
.
 
Reference
:
 
Madisen
 
L
 
et
 
al.,
 
2015
 
Neuron;
 
Imayoshi
 
I
 
et
 
al.,
 
2011
 
Front.
 
Neurosci.
 
Use multiple recombinases
 
for
 
sophisticated
 
manipulation
 
Example2
:
 
express tdTomato for ‘‘Cre NOT Flp’’ subtraction and GFP for ‘‘Cre AND Flp’’
intersection.
 
Reference
:
 
He
 
M
 
et
 
al.,
 
2016
 
Neuron
 
Example
3
:
 
convert a
 
transient
 
Cre expression to a
 
permanent
 
Flp expression.
Slide Note
Embed
Share

The Cre-Lox system, a powerful tool in genetic engineering, employs Cre recombinase to recombine specific DNA sequences (LoxP sites), allowing for precise genetic modifications such as deletions, inversions, and translocations. This system enables conditional gene expression, turning on or off transgenes in a spatially and temporally controlled manner. By strategically incorporating LoxP sites flanking a transgene, researchers can regulate gene expression with high precision, offering valuable insights into gene function and disease modeling in mice.


Uploaded on Aug 04, 2024 | 5 Views


Download Presentation

Please find below an Image/Link to download the presentation.

The content on the website is provided AS IS for your information and personal use only. It may not be sold, licensed, or shared on other websites without obtaining consent from the author. Download presentation by click this link. If you encounter any issues during the download, it is possible that the publisher has removed the file from their server.

E N D

Presentation Transcript


  1. Recombinase-mediated toolbox Cre-Lox system: Cre recombinase recombines a pair of short target sequences (LoxP sites) Depending on the location and orientation of the loxP sites, Cre recombinase can initiate deletions, inversions, and translocations of the floxed locus. The most widely used site-specific recombinase system for genome manipulation in mice. Because it is more efficient than other systems. Flp-FRT system: Flp is analogous to cre and FRT to loxP. Dre-rox system: Dre is analogous to cre and rox to loxP.

  2. Recombinase-mediated toolbox Cre-Lox system Cre recombinase recombines a pair of short target sequences (LoxP sites) Depending on the location and orientation of the loxP sites, Cre recombinase can initiate deletions (A), inversions (B), and translocations (C) of the floxed locus. The most common reference: Nagy A 2000, Genesis image from https://www.jax.org/news-and-insights/2006/may/the-cre-lox-and-flp-frt-systems

  3. Use the Cre-Lox system to express a transgene Cre recombinase removes the stop sequence, so the transgene is expressed only in the cells where Cre is active images from https://www.jax.org

  4. Use the Cre-Lox system to express a transgene Cre recombinase removes the stop sequence, so the transgene is expressed only in the cells where Cre is active images from https://www.jax.org

  5. Use the Cre-Lox system to turn off a transgene A transgene is designed with loxP sites flanking all or part of it. So the transgene is turned off only in the cells where Cre is active ~50% of the offspring will be heterozygous for the loxP allele and heterozygous for the cre transgene. images from https://www.jax.org

  6. Use the Cre-Lox system to turn off a transgene A transgene is designed with loxP sites flanking all or part of it. So the transgene is turned off only in the cells where Cre is active Mate heterozygous mice from the 1stgeneration back to the homozygous loxP-flanked mice. Experimental mice (~25%): homozygous for the loxP-flanked allele and heterozygous for the cre transgene images from https://www.jax.org

  7. Inducibility: allows temporal control of gene manipulation The tamoxifen-inducible cre alleles: the most common inducible Cre variety. Cre is expressed as a fusion protein with a modified estrogen receptor (ER) ligand binding domain. Binding to tamoxifen allows the Cre-ER fusion protein to translocate to the nucleus where the recombinase takes effect. The RU-486 inducible cre alleles: : cre is expressed as a fusion protein with a modified progesterone receptor, allowing induction with the progesterone analog RU-486. The TMP-inducible cre alleles: cre is expressed as a fusion protein with a modified E. coli dihydrofolate reductase (DHFR) protein, which can be induced by the common antibiotic trimethoprim (TMP).

  8. Use multiple recombinases for sophisticated manipulation As Cre, Flp, and Dre recognize different target sequences, the combined use of these recombinases would facilitate more sophisticated genetic manipulation of restricted cell populations. Example1: Intersectional control by either Cre/Flp or Cre/Dre recombinases, driven by different promoters (P1 or P2), on a doubly regulated reporter line. Reference: Madisen L et al., 2015 Neuron; Imayoshi I et al., 2011 Front. Neurosci.

  9. Use multiple recombinases for sophisticated manipulation Example2: express tdTomato for Cre NOT Flp subtraction and GFP for Cre AND Flp intersection. FRT FRT LoxP LoxP Example3: convert a transient Cre expression to a permanent Flp expression. Reference: He M et al., 2016 Neuron

Related


More Related Content

giItT1WQy@!-/#giItT1WQy@!-/#giItT1WQy@!-/#giItT1WQy@!-/#