Lecture 5: Cre-lox System in Neuroscience Flashcards
What is Cre-Lox system?
The Cre-lox system is a powerful METHOD USED TO TARGET AND MANIPULATE GENE EXPRESSION IN SPECIFIC/TISSUES.
Cre-Lox Relies on 2 components:
- Cre recombinase → enzyme only found in P1 bacteriophages
- loxP → 34 base pair DNA sequence
What is Cre recombinase?
- Cre recombinase CATALYSES the RECOMBINATION OF DNA
- Recognises loxP sites
What is LoxP?
- LONG DIRECTIONAL SEQUENCES that only occur in P1 BACTERIOPHAGES.
- loxP sites can be inserted to ‘flank’ A GENE OF INTEREST
- LoxP—GENE—LoxP
- 8 base pair spacer sequence = ATGTATGC
5’- ATAACTTCHTATA …TATACGAAGTTAT - 3’ = 13 base pair recognition sequence (PALINDROMIC REPEATS)
5’- ATAACTTCHTATA
ATGTATGC. TATACGAAGTTAT - 3’
Cre-Lox: excision (gene knockout) When does it occur?
Gene excision occurs when the loxP sites are in the SAME orientation
Gene excision occurs when the loxP sites are in
the SAME orientation…EXPLAIN = 3
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
Cre-Lox: excision (gene knockout) : FEATURES ?
REVERSIBLE? WHY STUDY?
- 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….
Cre-Lox: excision (gene expression) What is it?
The Cre-lox system can also be used to ACTIVATE gene expression.
Cre-Lox: excision (gene expression) EXPLAIN = 3
- Insertion of ‘stop’ sequence that is flanked by
loxP sites between the promoter and gene of
interest prevents the translation/expression of
the gene.
2 * WITHOUT Cre, the sSTP CASSETTE PREVENTS GENE TRANSLATION
3 * WITH Cre present, the STP CASSETTE IS ELETED AND GENE EXPRESSION PROCEEDS.
Cre-Lox: excision (gene expression) PROCESS: 2
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 WHAT IS IT?
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.
Cre-Lox: inversion: EXPLAIN PROCESS = 3
1 * RECOMBINATION results in the INVERSION OF THAT SEQUENCE
2 * LoxP SITES remains UNCHANGED and therefore
this process is REVERSIBLE
3 * Gene can “flip” BACK and FORTH INDEFINITELY as long as the ‘loxP sites’ are in OPPOSITE ORIENTATION
Cre-Lox: inversion STABLE?
Cre-lox INVERSION ALONE IS NOT ‘STABLE’.
to be useful, NEED SOMETHING THAT CAN SWITCH THE PROCESS ON/OFF
Discovered that the 8bp spacer region of loxP
can be altered …EXPLAIN = 2
1 * Create mutant lox sites that doesn’t undergo Cre recombination with wildtype
loxP sites
2 * But the same mutant lox can recombine with each other
Combining 2 different loxP sequences is called?
two ways in which
they can be used to control gene expression:
AKA:
Combining 2 different loxP sequences is called
‘DOUBLE FLOX’
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”
AKA: Note: these can also sometimes be referred to as
FLEx or “flip-excision” switches
DIO: Cre-On…EXPLAIN
- 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 (inOPPOSITE DIRECTIONS)
—– 2) EXCISION of the MIDDLE LOS SEQUENCE
(in the SAME DIRECTION)
- REMAINING 2 lox sitesARE NOT CROSS COMPATIBLE so NO FURTHER RECOMBINATION will occur.
- IRREVERSIBLE
- Example of DIO as gene of interest starts INVERTED
DO: Cre-OFF: START/BEGINING…
- For DO, the gene of interest begins in the
CORRECT ORIENTATION. - Undergoes the 2 RECOMBINATION steps to MOVE THR GENE TO the ‘INVERTED ORIENTATION’
MOVE the gene to the
therefore TURNING the GENE OFF.
How does Cre get into cells? = 2
- Cre recombinase is only found naturally in P1 bacteriophages.
2 * Use viruses or transgenic animals to express the Cre protein
WHAT ARE VIRUSES?
1 * Viral vectors, like adeno-associated viruses (AAVs), expressing lox sequences or
the Cre gene sequence can be stereotaxically injected into specific brain
regions.
2 * 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
What ARE PROMOTERS?
Promoters are regions of DNA where transcription of a gene is initiated
promoter sequence…transcription starts… gene
Cre-lox system and Promoters…
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 promote
- AAV expression of Cre under the control of CamKII promoter
- Cell-type/ tissue Promoter (s)
Neurons - hSynapsin
Excitatory neurons - CaMKiia, VGLUT2
Inhibitory neurons - Dlx1, Gad2, Pvalb
Dopaminergic neurons - Slc6a3
Astrocytes - Aldh1, GFAP
Oligodendrocytes PLP1
Understanding Cre-Lox 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:
Cre-lox system and Transgenic animals
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
