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NEUR3310 Neuroscience > Lecture 5: Cre-lox System in Neuroscience > Flashcards

Lecture 5: Cre-lox System in Neuroscience Flashcards

1
Q

What is Cre-Lox system?

A

The Cre-lox system is a powerful METHOD USED TO TARGET AND MANIPULATE GENE EXPRESSION IN SPECIFIC/TISSUES.

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2
Q

Cre-Lox Relies on 2 components:

A
  1. Cre recombinase → enzyme only found in P1 bacteriophages
  2. loxP → 34 base pair DNA sequence
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3
Q

What is Cre recombinase?

A
  1. Cre recombinase CATALYSES the RECOMBINATION OF DNA
  2. Recognises loxP sites
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4
Q

What is LoxP?

A
  1. LONG DIRECTIONAL SEQUENCES that only occur in P1 BACTERIOPHAGES.
  2. loxP sites can be inserted to ‘flank’ A GENE OF INTEREST
  3. LoxP—GENE—LoxP
  4. 8 base pair spacer sequence = ATGTATGC

5’- ATAACTTCHTATA …TATACGAAGTTAT - 3’ = 13 base pair recognition sequence (PALINDROMIC REPEATS)

5’- ATAACTTCHTATA
ATGTATGC. TATACGAAGTTAT - 3’

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5
Q

Cre-Lox: excision (gene knockout) When does it occur?

A

Gene excision occurs when the loxP sites are in the SAME orientation

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6
Q

Gene excision occurs when the loxP sites are in
the SAME orientation…EXPLAIN = 3

A

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

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7
Q

Cre-Lox: excision (gene knockout) : FEATURES ?

REVERSIBLE? WHY STUDY?

A
  1. This excision of the gene via recombination is IRREVERSIBLE
  2. This type of gene excision is useful to study the effect of gene knockouts
  3. But it can also be adapted to study gene expression….
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8
Q

Cre-Lox: excision (gene expression) What is it?

A

The Cre-lox system can also be used to ACTIVATE gene expression.

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9
Q

Cre-Lox: excision (gene expression) EXPLAIN = 3

A
  1. 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.

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10
Q

Cre-Lox: excision (gene expression) PROCESS: 2

A

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

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11
Q

Cre-Lox: inversion WHAT IS IT?

A

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.

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12
Q

Cre-Lox: inversion: EXPLAIN PROCESS = 3

A

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

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13
Q

Cre-Lox: inversion STABLE?

A

Cre-lox INVERSION ALONE IS NOT ‘STABLE’.

to be useful, NEED SOMETHING THAT CAN SWITCH THE PROCESS ON/OFF

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14
Q

Discovered that the 8bp spacer region of loxP
can be altered …EXPLAIN = 2

A

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

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15
Q

Combining 2 different loxP sequences is called?

two ways in which
they can be used to control gene expression:

AKA:

A

Combining 2 different loxP sequences is called
‘DOUBLE FLOX’

there are two ways in which
they can be used to control gene expression:

  1. Double floxed Inverse Orientation (DIO) = “Cre-On”
  2. Double floxed Orientation (DO) = “Cre-Off”

AKA: Note: these can also sometimes be referred to as
FLEx or “flip-excision” switches

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16
Q

DIO: Cre-On…EXPLAIN

A
  1. FLEx switches work by having the GENE OF INTEREST FLANKED BY 2 DIFFERENT Lox SITES
  2. 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)

  1. REMAINING 2 lox sitesARE NOT CROSS COMPATIBLE so NO FURTHER RECOMBINATION will occur.
  2. IRREVERSIBLE
  3. Example of DIO as gene of interest starts INVERTED
17
Q

DO: Cre-OFF: START/BEGINING…

A
  1. For DO, the gene of interest begins in the
    CORRECT ORIENTATION.
  2. Undergoes the 2 RECOMBINATION steps to MOVE THR GENE TO the ‘INVERTED ORIENTATION’
    MOVE the gene to the
    therefore TURNING the GENE OFF.
18
Q

How does Cre get into cells? = 2

A
  1. Cre recombinase is only found naturally in P1 bacteriophages.

2 * Use viruses or transgenic animals to express the Cre protein

19
Q

WHAT ARE VIRUSES?

A

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’

20
Q

Transgenic organisms:

A
  1. 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
21
Q

What ARE PROMOTERS?

A

Promoters are regions of DNA where transcription of a gene is initiated

promoter sequence…transcription starts… gene

22
Q

Cre-lox system and Promoters…

A

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

23
Q

Understanding Cre-Lox Viruses:

A

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

24
Q

Transgenic Cre lines:

Cre-lox system and Transgenic animals

A

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
25
Q

Different ways that transgenic animals can be used: 3

A

1 * Cre-animal + virus injection of the floxed gene of interest

2 * Animal modified to have floxed gene of interest + virus containing Cre gene

3 * Cre-animal x Flox-animal (breed pairs of transgenic organisms)

26
Q

CreERT: activating Cre at a specific time

In some experiments, you may want to only activate the Cre-lox system
at a specific time.

‘Tamoxifen-inducible Cre-loxP system’ EXPLAIN = 3

A

1 * Fusing Cre to the ESTROGEN RECEPTOR (Cre-ERT) and HEAT SHOCK PROTEIN 90 (HSP90) prevents Cre from entering the nucleus/binding to lox

2 * SYNTHETIC STEROIDS (e.g., tamoxifen) BREAKS the INTERACTION between
Cre-ERT and HSP90.

3 * The Cre-ERT is able to TRANSLOCATE to the NUCLEUS AND START THE Cre-lox mechanism

27
Q

Temporal control over Cre recombination:

A

Using the CreERT system, RECOMBINATION is ONLY possible when aSTEROID IS GIVEN (e.g., tamoxifen)

28
Q

Using the CreERT system, recombination is only possible when a steroid is given (e.g., tamoxifen)

Can be used to: 3

A

1 * Target cells that may be present at DIFFERENT DEVELOPMENTAL STAGES (e.g., newborn vs. adult)

2 * Identify CELLS BORN AT DIFFERENT STAGES OF LIFE

3 * LOOK at the EFFECTS OF GENE KNOCK-INS/OUTS AT DIFFERENT LIFE STAGES
at different life stages

29
Q

Neuroscience applications: Cell labelling:

A

Cre-lox can be used to INDUCE EXPRESSION OF FLUORESCENT PROTEIN INTO A SPECIFIC CELL TYPE

  • Label specific brain regions & map their projections
30
Q

Neuroscience applications: Cell labelling

UNDERSTANDING : Bareyre et al. 2005 = 2

A
  • 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
31
Q

Neuroscience applications: Cell labelling:

The amount of Cre can be controlled to limit the number of cells that undergo Cre recombination = 2

A

The amount of Cre can be CONTROLLED TO LIMIT THE NUMBER OF CELLS THAT UNDERGO Cre RECOMBINATION

1 * Tested multiple dilutions of Cre virus injections

2 * Potential to visualise individual cells

32
Q

Neuroscience applications: Gene knock-out:

A

TARGET RECOMBINATION in a SPECIFIC POPULATION OF CELLS of cells to ‘KNOCK OUT’ genes

  • Used to identify the role of genes in behaviours, neurological processes, and disease
33
Q

Neuroscience applications: Gene knock-out:

Antunes et al., 2022

A
  • Looked at effect of deleting Tet3 from Camk2a expressing neurons on anxiety
34
Q

Neuroscience applications: Gene knock-in

A

Target recombination in a specific population of cells to KNOCK IN GENES AND LOOK AT A CELLULAR FUNCTION AND BEHAVIOUR

35
Q

Neuroscience applications: Gene knock-in: 3

Feng et al., 2016

A

1 * Tested if over-expressing BDNF can delay vision loss following ocular
hypertension (OHT)

2 * Used Thy-1-CreERT(T2) mice – express yellow fluorescent protein in retina and brain

3 * Used BDNFstopmice – contains a stop cassette in front of BDNF gene

36
Q

Caveats of Cre-lox: 2

A
  • The Cre-lox system is not 100% full proof
  1. Off-target Cre expression can occur in cells that don’t have the promoter for Cre
  2. Unintentional Cre recombination in germline cells can also occur
37
Q

Caveats of Cre-lox:

‘Off-target Cre expression can occur in cells that
don’t have the promoter for Cre’

A
    • Papthanou et al. (2019) bred DAT-Cre mice x
      floxed tdTomato reporter mice = tdTomDAT-Cre mice

2 * Vglut2 = glutamatergic neurons; Gad1 =
GABAergic neurons

3 * Found Cre recombinase activity also occurred in
non-dopaminergic neurons

38
Q

Caveats of Cre-lox:

‘Unintentional Cre recombination in germline cells can also occur/

A

→ results in Cre-mediated
manipulation in ALL cells instead of targeted cells

NEUR3310 Neuroscience (18 decks)

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