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30 Cards in this Set
- Front
- Back
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optogenetics |
- genetically encoded proteins which are either light-activated or fluoresce when excited (by light) |
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3 broad functions of optogenetics |
1. reporter 2. biosensor 3. control |
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fluorescence microscopy |
- light source is broad spectrum and passes through excitation filter which only allows desired wavelength to pass - light meets dichromic mirror which can reflect or transmit wavelength - an optical microscope that uses fluorescence and phosphorescence to study properties of organic and inorganic substances |
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electroporation |
high voltage pulses breakdown plasma membrane allowing entry of a plamid - ideal for cell cultures |
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stably expressing transgenic animals |
construct is introduced and incorperated in germ cells - so it is always present in their genome |
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viral injection |
- uses natures machinery to do the work - package virus with construct of interest, then transduce cells - the virus is injected into animal brain and will infect other cells and instead of spreading a virus it spreads the desired construct which is then processed and expressed |
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opsins |
- light sensitive proteins - when activated by a certain light wavelength they undergo some conformational change resulting in 'activation' of the protein |
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channelrhodospin (ChR2) |
the canonical opsin of optogenetic control - a 7-transmembrane protein that associates with all-trans-retinel |
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chlamydomona reinhardtii |
a single cell algae which expresses ChR2 to product the function of movement toward light, to facilitate photosynthesis |
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retinal |
- it is light sensitive, activated by blue light (470nm) which causes all-trans-retinal to change in to all-cis-retinal and in doing so, causes a conformational change in the molecule and the associated ChR2 protein - a mixed cation channel (predominantly Na+ with some Ca2+ and some K+ exit) |
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Halorhodopsin (HR) |
- an opsin that is a Cl- pump, that when activated by 590nm wavelength brings Cl- ions into cell causing membrane to hyperpolarise and reduce neuronal activity |
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CHeta |
a genetic variation of ChR2 which closes more quickly than wildtype therefore allowing faster repetitive stimulation of neurons |
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advtanges |
1. specificity 2. genetic modification 3. light is non-invasive 4. temporal resolution of manipulation of measurement 5. no artifact from photo-stimulation |
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1. specificity |
- gene experssion under specific cell-type promoter: target a single cell in a population of cells - light can be focused to a small, largely definable region - Different wavelengths of excitation or emission - light has no "off-target" effects |
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2. Genetic modifcation |
- customization of proteins to suit needs - can manipulate single amino acids - allows us to turn the excitation and emission of GFP to have a whole variety of colours |
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3. Light is non-invasive |
- although intense light can be damaging to tissue - although subject to photo-bleaching (for reporter and biosensor) |
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4. temporal resolution of manipulation of measurement |
- fast: don't need second messengers generally - biosoensor/measurement = slower |
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5. No artifact from photo-stimulation |
- unlike electrical stimulation |
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Green Fluorescent Protein (GFP) |
- isolated from jellyfish (Aequorea Victoria) - genetically modified: colour variations, customized excitation and emission wavelengths, improved fluorescence |
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reporter gene |
- GFP is tagged on to other proteins of interest - it permits examination of protein-protein interactions - used for targeted cell-type specific recording/observations |
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biosensors |
- genetically encoded proteins sensitive to: voltage, pH, calcium, protein phosphorylation - same advantages of GFP: cell-specific - often FRET-based mechanisms: closeness of the donor and acceptor chormophores is voltage-sensitive |
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FRET |
Fluorescence Resonance Energy Transfer - can be measured and used to observe current changes in cells |
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control |
- there is a need for precise neuronal control as electrical stimulation is not specific and pharmacological intervention is not specific and it is slow acting - optogenetics activates specific cells |
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generating animals for optogenetic investigations |
1. transgenic mouse line 2. 'on demand' animal approach |
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1. transgenic mouse line |
- stably expressing a construct - cheap to buy ($700pair) but expensive to important and maintain - inflexible - single promoter and single opsin - less time intensive |
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2. 'On demand' animal appraoch |
- intracerebral injection of viral vector - flexible - any target region, any promoter - cheaper - more time intensive |
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optogenetic inhibition |
- can silence a particular cell population - using a different opsin |
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temporal limitation |
- time it takes to return to baseline - affects how fast you can stimulate the cell - need 3x tau to return to baseline and the max freuqnecy is 1/3t = around 16Hz |
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limitation in vivo |
- blue light is strongly absorbed by blood and scattered by brain tissue resulting in small volume of photo-excited tissue |
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is red light better? |
is absorbed more and scattered less and therefore offers greater volume of photoexcited tissue |
