cre-lox Flashcards
Cre-lox system
powerful method used to target and manipulate gene expression in specific cells/tissues.
relies on 2 components:
> cre recombinase (enzyme only found in P1 bacteriophages)
> loxP (34 base pair SNA sequences)
cre recombinase
- Cre recombinase catalyses the recombination of DNA
- Recognises loxP sites
loxP
- Long directional sequences that only occur in P1 bacteriophages
- loxP sites can be inserted to ‘flank’ a gene of interest
crelox excision (gene knockout)
Gene excision occurs when the loxP sites are in the SAME orientation
1) Two Cre proteins recognise and bind to loxP sites that are in the same orientation flanking a gene sequence
2) Recombination occurs between the loxP sites
3) Results in excision of gene sequence from its original site
This excision of the gene via recombination is
IRREVERSIBLE
This type of gene excision is useful to study the effect of gene knockouts
But it can also be adapted to study gene expression….
crelox excision (gene expression)
The Cre-lox system can also be used to activate gene expression.
Insertion of ‘stop’ sequence that is flanked by loxP sites between the promoter and gene of interest prevents the translation/expression of the gene.
* Without Cre, the stop cassette prevents gene translation
* With Cre present, the stop cassette is deleted and gene expression proceeds
1) Cre recombinase recognises the loxP sites
that are in the same orientation and flanking a stop cassette.
2) Stop sequence is excised and the gene is expressed
cre-lox inversion
The Cre-lox system can also be designed to be reversible (i.e., turn the same gene on/off).
Inversion of a gene occurs when loxP sites are in the OPPOSITE orientation.
* Recombination results in the inversion of that sequence’
* LoxP sites remains unchanged and therefore this process is REVERSIBLE
* Gene can “flip” back and forth indefinitely as long as the loxP sites are in opposite
orientations
Cre-lox inversion alone is not stable…
to be useful, we need something that can switch the process on/off
DIO and DO systems (FLEx switches)
Discovered that the 8bp spacer region of loxP can be altered
* Create mutant lox sites that doesn’t undergo Cre recombination with wild type loxP sites
* But the same mutant lox can recombine with each other Combining 2 different loxP sequences is called ‘double flox’ and there are two ways in which they can be used to control gene expression:
- Double floxed Inverse Orientation (DIO) = “Cre-On”
* Double floxed Orientation (DO) = “Cre-Off”
DIO
cre on
FLEx switches work by having
the gene of interest flanked by 2 different lox sites
Two recombination steps occur:
1) Inversion between 1 set of lox sites (in opposite directions)
2) Excision of the middle lox sequence (in the same direction)
Remaining 2 lox sites are not cross compatible so no further recombination will occur. IRREVERSIBLE
DO cre off
For DO, the gene of interest begins in the correct orientation.
Undergoes the 2 recombination steps to move the gene to the inverted orientation, therefore turning the gene off
hoe does cre get into cells
Cre recombinase is only found naturally in P1 bacteriophages.
* Use viruses or transgenic animals to express the Cre protein
Viruses
* Viral vectors, like adeno-associated viruses (AAVs), expressing lox sequences or the Cre gene sequence can be stereotaxically injected into specific brain regions.
* Genes can be manipulated in non-genetically modified animals
Transgenic organisms
* Organisms can be modified to express the Cre gene in specific cell types or tissues
* Organisms can be modified to have a gene of interest flanked by lox sites
* Most commonly used in mice
promoters
Promoters are regions of DNA where transcription of a gene is initiated
The Cre-lox system can target cells/tissues using promoters.
* Insert the gene for Cre next to a cell/tissue-specific promoter allows for Cre to be made in that areas that have that promoter
crelox viruses
One strategy of using the Cre-lox system in experiments is through injecting wildtype mice with:
Virus containing the Cre gene + Virus with the gene floxed with lox sites
transgenic cre lines
Other, more common, strategies of using the Cre-lox system is through transgenic animals
* Cre lines are available in many different organisms:
Mouse, Zebrafish, drosophila, C-elegans
Different ways that transgenic animals can be used:
* Cre-animal + virus injection of the floxed gene of interest
* Animal modified to have floxed gene of interest + virus containing Cre gene
* Cre-animal x Flox-animal (breed pairs of transgenic organisms)
CreERT
In some experiments, you may want to only activate the Cre-lox system at a specific time.
Tamoxifen-inducible Cre-loxP system
* Fusing Cre to the estrogen receptor (Cre-ERT) and heat shock protein 90 (HSP90) prevents Cre from entering the nucleus/binding to lox
* Synthetic steroids (e.g., tamoxifen) breaks the interaction between Cre-ERT and HSP90.
* The Cre-ERT is able to translocate to the nucleus and start the Cre-lox mechanism
temporal control over cre recombination
Using the CreERT system, recombination is only possible when a steroid is given (e.g., tamoxifen)
Can be used to:
* Target cells that may be present at different developmental stages (e.g., newborn vs. adult)
* Identify cells born at different stages of life
* Look at the effects of gene knock-ins/outs at different life stages
neuroscience and its application cell labelling
Cre-lox can be used to induce expression of a fluorescent protein into a specific cell type
* Label specific brain regions & map their projections
Bareyre et al. 2005
* Cre expression driven by Emx1 promoter (gene marker for the embryonic forebrain)
* Excision of the STOP cassette through Cre-lox recombination allows for
expression of the fluorescent protein YFP in cells with the Thy1 promoter
The amount of Cre can be controlled to limit the number of cells that undergo Cre recombination
* Tested multiple dilutions of Cre virus injections
* Potential to visualise individual cells
neuroscience and its application gene knockout
Target recombination in a specific population of cells to knock out genes
* Used to identify the role of genes in behaviors, neurological processes, and disease
Antunes et al., 2022
* Looked at effect of deleting Tet3 from Camk2a-expressing neurons on anxiety
neuroscience and its application gene knockin
Target recombination in a specific population of cells to knock in genes and look at cellular function and
behavior
Feng et al., 2016
* Tested if overexpressing BDNF can delay vision loss following ocular
hypertension (OHT)
* Used Thy-1-CreERT(T2) mice – express yellow fluorescent protein in retina and brain
* Used BDNFstopmice – contains a stop cassette in front of BDNF gene
caveats of cre lox
The Cre-lox system is not 100% full proof
Off-target Cre expression can occur in cells that don’t have the promoter for Cre
* Papthanou et al. (2019) bred DAT-Cre mice x floxed tdTomato reporter mice = tdTomDAT-Cre mice
* Vglut2 = glutamatergic neurons; Gad1 = GABAergic neurons
* Found Cre recombinase activity also occurred in non-dopaminergic neurons
Unintentional Cre recombination in germline cells can also occur → results in Cre-mediated manipulation in ALL cells instead of targeted cells
