Techniques Used in Cellular and Molecular Neuroscience Flashcards
This method uses light to directly excite or inhibit specific neurons in living animals, can use to turn cells off and on
Optogenetics
What are pluripotent cells?
Cells that can become anything
What happens if a sample is continuously put under an EM?
The electron beam will eventually destroy the sample
Examples of this are mutations that remove activation domain or dimerization domain to allow the protein to compete for the target without performing the function
Dominant negative protein expression
Allows us to see several figures during course of synapses in great detail
Electron microscopy
These two can be used in cells in culture or in the brain of living animals to destroy specific RNA targets
siRNA and shRNA
Stem cells that generate cells from patents with neurological disorders
Induced pluripotent stem cells (iPSC)
What does a full KO do in cre-lox systems?
KOs the gene everywhere
This method aims to answer questions regarding how exciting or inhibiting neurons effects complex processes like addiction, learning, reward, fear conditioning, neuromodulation of disease symptoms, sleep, and depression
Optogenetics
These are good to use bc they allow for proteins to be expressed 2-4 days after injection
Oocytes
These light channels are used to excite (optogenetics)
Channelrhodopsins
These are self organizing, small (5mm), can be used to test drugs, study development, and have therapeutic potential
Organoids
What do halorhodopsins do?
Let in Cl- when a certain wavelength of light hits it
Methods used to alter endogenous genes and proteins at the mRNA level
RNA interference, morpholinos
These are identified by function and molecular markers
Stem cells
What type of rhodopsin is used to modulate intracellular signaling in optogenetics
G-protein coupled rhodopsins
Breeding strategy for producing homozygous KO mice
Mosaic x Wild Type —> Heterozygote
Heterozygote x Heterozygote —> 25% wild type and 25% with both copies of KO (homozygous KO)
What does a conditional KO do in cre-lox systems?
KOs the gene in specific places only. Tissue specific promoters are used to mark cells where it is to be KOd
3D tissue cultures derived from stem cells
Organoids
What do channelrhodopsins do?
Let in Na+ when a certain wavelength of light hits it
These are present in bacteria to defend against viruses and plasmids (provide memory or adaptive immunity to organisms previously exposed to)
CRISPRs
These are pluripotent cells with the capacity to self renew
Stem cells
These can be used to deliver genes to cultured cells, brain slices, or brain regions in vivo
Viruses
What cant be controlled in transgenic mice?
Where the new DNA integrates in the genome
These are classified by source (embryonic, adult, induced) and tissue they generate
Stem cells
What type of stem cells are multipotent and can self renew?
Multipotent
Neural cell culture that can be used to maintain connectivity (acute for short term electrophysiology, organotypic for extended studies of synapse formation, migration)
Slice cultures (250-400 um)
Can be used to introduce genes for inducing or inhibiting neural activity such as channelrhodopsins, halorhodopsins, and DREADDs
Transgenic mice
4 types of stem cell cultures
- Embryonic stem cells
- adult stem cells
- neural stem cells
- induced pluripotent stem cells (iPSC)
Where are the sites of recombination in the cre-lox system?
After the gene has been KOd, the recombination site is combining the two parts that were on either side of the KOd gene with the lox sites
New gene added, native gene still present
Transgenic mice
Can be used to mark specific organelles
Transgenic mice
This is used to distinguish specific cells, trace processes and connections, highlight cell structures, and to visualize fixed and living tissues
Brainbow
This method is used to measure and compare small amounts of RNA or DNA
Polymerase chain reaction (PCR)
Type of EM that allows for viewing over the surface
SEM (scanning)
Light that allows for receptor mediated intracellular signaling
Green light
These can be used to deliver genes when transgenic/KO or knockin animals can’t or don’t need to be used
Viruses
This is used to compare expression of genes in different neurons, different stages of development, mutant animals or tissue
Polymerase chain reaction (PCR)
Measure in microscopes used to tell how far apart two things have to be before you can tell theyre apart
Resolving power
Homology directed repair leads to this in CRISPR
The KOd gene gets replaced with a new gene. A new sequence is inserted
Is optogenetics limited to mice?
No it can be applied to many animals
What are oocytes
Frog eggs
Can be used to inactivate a gene at a specific time in development if inactivating it too early is lethal (or to see what inactivating it at different stages does)
Knockout
Cells that are more like real neurons, theyre more relevant but they produce heterogeneous populations with high variability
Primary cell culture
This is the ultimate form of ablation, completely getting rid of a gene
Knockout
Does confocal or epifluorescence produce a better image?
Confocal
Stem cells that are self renewing, produce neurons, astrocytes, oligodendrocytes, more present in embryonic than in adult brain
Neural stem cells
Microscopy in which optics are used to exaggerate differences in light scattering properties
Nomarski (light)
Are neurons and glia non-dividing or dividing cells?
Neurons = non-dividing Glia = dividing
Can be used to ablate cells by expressing toxin or toxin receptors specifically
Transgenic mice
What are the 3 parts to the Core-Lox system?
Tissue specific promoter used to help cre recombinase find the correct tissue
Cre Recombinase enzyme used to cut out the gene at the lox sites
Lox sites used to flank a gene and mark it for KO
Microscopy that is deeper, has less photobleaching, and less background
Two photon (fluorescence)
These methods operate on DNA by altering endogenous genes (genes that are already in the organism) and proteins
Knockout, Knockin, and CRISPR-Cas9
Pluripotent stem cells that can be induced to form neural progenitors
Embryonic stem cells
How can the role of a specific protein be tested?
Blocking function of the protein
Do the majority of offspring wind up being transgenic or not?
It depends, lots of DNA will be destroyed by the mice but some of the offspring will wind up being transgenic
Rhodopsin activated by blue light
Channelrhodopsin (excite)
Type of EM that allows for viewing through a thin section
TEM (transmission)
Cells that are easier to grow, more stable, and fairly well characterized but don’t have all the properties of neurons
Immortalized cell lines
Used to express gene in specific cells, determine effects on cell or circuit formation
Transgenic mice
These allow for multiple electrodes to be used to record ion channel activity
Oocytes
Where must the transgene (new gene being put in) go to create transgenic mice?
It has to go in front of the correct promoter you want it to go in front of
What does it mean to be floxed
When the target gene is flanked by lox sites
When foreign DNA is injected into mouse germ line by injection of one-cell egg or injection of ES (embryonic stem) cells into blastocyst
Transgenic mice
This is used to look at structures, not how things change
Electron microscopy
How does CRISPR work?
RNA generated from spacers and CRISPRs targets Cas9 nuclease to invading DNA and CRISPR loci are transcribed
Rhodopsin activated by yellow light
Halorhodopsin (inhibit)
Can be used to inactivate a gene to look at the function of it
Knockout
Type of microscopy that collects light from the entire depth of tissue and illuminates the entire sample
Epifluorescent (fluorescence)
Type of culture that extends on explant culture and further makes it easier to see the neurons
Dissociated cells
Microscopy in which there is illumination from the side, only highly refractive parts such as organelles are seen
Darkfield (light)
Can be used to overexposes a gene to make a product
Transgenic mice
Can be used to insert genes to measure neural activity (Ca2+, voltage, synaptic vesicle fusion)
Transgenic mice
Type of culture that is taking out a chunk of the brain
Explant culture
What are the 5 steps for creating knockout/knockin mice?
- Create targeting construct
- Place in embryonic stem cells (ES), select for proper incorporation
- Place ES cells into blastocyst
- Insert blastocyst into female mouse
- Breeding of the mice
These are an array of identical repeats intercalated with DNA-targeting spacers derived from bacteria and plasmids
CRISPRs
What do Cre-Lox systems do?
Knockout a gene in a specific tissue or at at specific time
What does in situ mean?
In place
These are just starting off and far from being able to be applied to use in people for therapeutic purposes
Organoids
Activators, repressors, and GFPs lead to being able to do these 3 things in CRISPR
Turn on gene, turn off gene, and be able to see gene not in replication
Type of microscopy that collects light from thin section, 1 um, tissues or whole animals like fruit fly, zebrafish, photobleaching, long exposure to intense light
Confocal (fluorescence)
This method requires tiny amounts of tissue, can be done in situ (in place), and is used for genomic sequencing and mapping
Polymerase chain reaction (PCR)
This method uses transgenic and cre-lox technology to generate mice (or other animals) expressing unique combinations of fluorescent markers in individual cells
Brainbow
Can be used to inactivate a gene in a specific group of cells
Knockout
What is photobleaching?
Exposing samples to so much light they lose their fluorescence
These method allows the use of methods (promoters, virus injection) to express rhodopsins in specific cells at specific times
Optogenetics
What happens once the transgene is placed in front of the proper promoter in making transgenic mice?
The new DNA created will be injected into fertilized eggs and implanted in a mouse. The offspring will be transgenic (but a lot of them still won’t be)
Microscopy in which light is transmitted through the sample, usually only see pigmented or stained structures
Brightfield (light)
SLIDE 48
SLIDE 48
These are usually used from African clawed frog and 1 mm in diameter
Frog eggs (oocytes)
Methods used to alter endogenous genes and proteins at the protein level
Dominant negatives
The idea of these stem cells is to take any cell from someone, reprogram it to make it a stem cell, and use it in the needed area and avoid the immune response bc its the persons cells
Induced pluripotent stem cells (iPSC)
Non-homologous end joining tries to repair KO and leads to this in CRISPR
Bad repair leading to the gene being KOd
This allows for the creation of receptors that bind a ligand but don’t transmit the signal
Dominant negative protein expression
These stem cells have a more limited capacity to self renew and may be unipotent
Progenitor cells (ex NPC)
Can be used to trace axons and dendrites with membrane-bound fluorescent reporters (trace connections to see what connects to what)
Transgenic mice
These types of cells can only become one certain thing
Unipotent
Microscopy in which light scatters due to differences in density, amplified here
Phase contrast (light)
This method allows for specific parts of neurons to be turned off and on at the flip of a switch
Optogenetics
Microscopy that visualizes molecules only at surface in great detail. Can be used to track what is on the surface and see what is in the membrane
Total internal reflectance (TIRF) (fluorescence)
These can be used to test the function of a gene or effect of mutation in gene
Viruses
These two things already found in our bodies are used in combination with siRNA and shRNA to target and degrade specific RNAs
Dicer and RISC
These can be used to examine how the animal nucleus is effected bc it appears black or brown
Oocytes
What does an inducible conditional KO do in cre-lox systems?
KOs the gene at a specific time. Add something like tamoxifen to do this
What are DREADDs?
Designer receptors exclusively activated by designer drugs. A custom receptor is designed by humans so crosstalk is not a factor and a custom drug is also made that only reacts at that receptor
How did electron microscopy improve resolution
Light resolution = 200 nm
EM = 0.2 nm (radius of a glutamate molecule)
This works by adding different fluorescent proteins between different lox sites and having the cre recombinase enzyme cut them to produce different colored cells
Brainbow
This method can produce cells expressing rhodopsins constitutively (all the time) or inducibly (turn on and off)
Optogenetics
Different versions of these are used in optogenetics for different times/patterns of stimulation
Rhodopsins
What is a blastocyst?
A developing embryo with a few hundred cells
What does multipotent mean?
Cant become every cell, but can become certain kinds
Neural cell cultures that can be grown under the right conditions
Cell lines with neural properties and primary cells
To use this, samples must be fixed, dried, and chemically treated. They cant be alive
Electron microscopy
What do more mature organoids start to develop?
More neurons, other cells like glia, and vasculature
What type of microscope is needed to see cells don’t touch at synapses?
Electron microscope (Cajall and Golgi didn’t have this)
This is useful to visualize what cells are communicating with what cells
Brainbow
What two things can result from CRISPR?
Gene KO or gene being replaced by new sequence
Microscopy that images sections one at a time to get a good 3D image
Confocal (fluorescence)
These can be used to inject RNA or cDNA
Oocytes
Can be used to replace a gene with a modified version of the gene to determine effect of mutations in disease genes
Knockin
Methods used to alter endogenous genes and proteins at the DNA level
Gene targeting (KOs, knockins, conditional KOs)
Acting at these two times allows the altering of endogenous genes and proteins by blocking RNA expression to remove function without destroying genes
Transcription and translation
Can be used to mark specific cells with GFP (green fluorescent protein or other colors), luciferase, Lac Z
Transgenic mice
These light channels are used to inhibit (optogenetics)
Halorhodopsins
Used to identify neural structure and function, protein relationships, locations of specific proteins, structural changes due to neural activity, visualize transport down axons, different fluorophores can be used at the same time (multiple labels at once)
Fluorescence microscopy
