Techniques Used in Cellular and Molecular Neuroscience Flashcards
1
Q
This method uses light to directly excite or inhibit specific neurons in living animals, can use to turn cells off and on
A
Optogenetics
2
Q
What are pluripotent cells?
A
Cells that can become anything
3
Q
What happens if a sample is continuously put under an EM?
A
The electron beam will eventually destroy the sample
4
Q
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
A
Dominant negative protein expression
5
Q
Allows us to see several figures during course of synapses in great detail
A
Electron microscopy
6
Q
These two can be used in cells in culture or in the brain of living animals to destroy specific RNA targets
A
siRNA and shRNA
7
Q
Stem cells that generate cells from patents with neurological disorders
A
Induced pluripotent stem cells (iPSC)
8
Q
What does a full KO do in cre-lox systems?
A
KOs the gene everywhere
9
Q
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
A
Optogenetics
10
Q
These are good to use bc they allow for proteins to be expressed 2-4 days after injection
A
Oocytes
11
Q
These light channels are used to excite (optogenetics)
A
Channelrhodopsins
12
Q
These are self organizing, small (5mm), can be used to test drugs, study development, and have therapeutic potential
A
Organoids
13
Q
What do halorhodopsins do?
A
Let in Cl- when a certain wavelength of light hits it
14
Q
Methods used to alter endogenous genes and proteins at the mRNA level
A
RNA interference, morpholinos
15
Q
These are identified by function and molecular markers
A
Stem cells
16
Q
What type of rhodopsin is used to modulate intracellular signaling in optogenetics
A
G-protein coupled rhodopsins
17
Q
Breeding strategy for producing homozygous KO mice
A
Mosaic x Wild Type —> Heterozygote
Heterozygote x Heterozygote —> 25% wild type and 25% with both copies of KO (homozygous KO)
18
Q
What does a conditional KO do in cre-lox systems?
A
KOs the gene in specific places only. Tissue specific promoters are used to mark cells where it is to be KOd
19
Q
3D tissue cultures derived from stem cells
A
Organoids
20
Q
What do channelrhodopsins do?
A
Let in Na+ when a certain wavelength of light hits it
21
Q
These are present in bacteria to defend against viruses and plasmids (provide memory or adaptive immunity to organisms previously exposed to)
A
CRISPRs
22
Q
These are pluripotent cells with the capacity to self renew
A
Stem cells
23
Q
These can be used to deliver genes to cultured cells, brain slices, or brain regions in vivo
A
Viruses
24
Q
What cant be controlled in transgenic mice?
A
Where the new DNA integrates in the genome
25
These are classified by source (embryonic, adult, induced) and tissue they generate
Stem cells
26
What type of stem cells are multipotent and can self renew?
Multipotent
27
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)
28
Can be used to introduce genes for inducing or inhibiting neural activity such as channelrhodopsins, halorhodopsins, and DREADDs
Transgenic mice
29
4 types of stem cell cultures
1. Embryonic stem cells
2. adult stem cells
3. neural stem cells
4. induced pluripotent stem cells (iPSC)
30
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
31
New gene added, native gene still present
Transgenic mice
32
Can be used to mark specific organelles
Transgenic mice
33
This is used to distinguish specific cells, trace processes and connections, highlight cell structures, and to visualize fixed and living tissues
Brainbow
34
This method is used to measure and compare small amounts of RNA or DNA
Polymerase chain reaction (PCR)
35
Type of EM that allows for viewing over the surface
SEM (scanning)
36
Light that allows for receptor mediated intracellular signaling
Green light
37
These can be used to deliver genes when transgenic/KO or knockin animals can't or don't need to be used
Viruses
38
This is used to compare expression of genes in different neurons, different stages of development, mutant animals or tissue
Polymerase chain reaction (PCR)
39
Measure in microscopes used to tell how far apart two things have to be before you can tell theyre apart
Resolving power
40
Homology directed repair leads to this in CRISPR
The KOd gene gets replaced with a new gene. A new sequence is inserted
41
Is optogenetics limited to mice?
No it can be applied to many animals
42
What are oocytes
Frog eggs
43
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
44
Cells that are more like real neurons, theyre more relevant but they produce heterogeneous populations with high variability
Primary cell culture
45
This is the ultimate form of ablation, completely getting rid of a gene
Knockout
46
Does confocal or epifluorescence produce a better image?
Confocal
47
Stem cells that are self renewing, produce neurons, astrocytes, oligodendrocytes, more present in embryonic than in adult brain
Neural stem cells
48
Microscopy in which optics are used to exaggerate differences in light scattering properties
Nomarski (light)
49
Are neurons and glia non-dividing or dividing cells?
```
Neurons = non-dividing
Glia = dividing
```
50
Can be used to ablate cells by expressing toxin or toxin receptors specifically
Transgenic mice
51
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
52
Microscopy that is deeper, has less photobleaching, and less background
Two photon (fluorescence)
53
These methods operate on DNA by altering endogenous genes (genes that are already in the organism) and proteins
Knockout, Knockin, and CRISPR-Cas9
54
Pluripotent stem cells that can be induced to form neural progenitors
Embryonic stem cells
55
How can the role of a specific protein be tested?
Blocking function of the protein
56
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
57
Rhodopsin activated by blue light
Channelrhodopsin (excite)
58
Type of EM that allows for viewing through a thin section
TEM (transmission)
59
Cells that are easier to grow, more stable, and fairly well characterized but don't have all the properties of neurons
Immortalized cell lines
60
Used to express gene in specific cells, determine effects on cell or circuit formation
Transgenic mice
61
These allow for multiple electrodes to be used to record ion channel activity
Oocytes
62
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
63
What does it mean to be floxed
When the target gene is flanked by lox sites
64
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
65
This is used to look at structures, not how things change
Electron microscopy
66
How does CRISPR work?
RNA generated from spacers and CRISPRs targets Cas9 nuclease to invading DNA and CRISPR loci are transcribed
67
Rhodopsin activated by yellow light
Halorhodopsin (inhibit)
68
Can be used to inactivate a gene to look at the function of it
Knockout
69
Type of microscopy that collects light from the entire depth of tissue and illuminates the entire sample
Epifluorescent (fluorescence)
70
Type of culture that extends on explant culture and further makes it easier to see the neurons
Dissociated cells
71
Microscopy in which there is illumination from the side, only highly refractive parts such as organelles are seen
Darkfield (light)
72
Can be used to overexposes a gene to make a product
Transgenic mice
73
Can be used to insert genes to measure neural activity (Ca2+, voltage, synaptic vesicle fusion)
Transgenic mice
74
Type of culture that is taking out a chunk of the brain
Explant culture
75
What are the 5 steps for creating knockout/knockin mice?
1. Create targeting construct
2. Place in embryonic stem cells (ES), select for proper incorporation
3. Place ES cells into blastocyst
4. Insert blastocyst into female mouse
5. Breeding of the mice
76
These are an array of identical repeats intercalated with DNA-targeting spacers derived from bacteria and plasmids
CRISPRs
77
What do Cre-Lox systems do?
Knockout a gene in a specific tissue or at at specific time
78
What does in situ mean?
In place
79
These are just starting off and far from being able to be applied to use in people for therapeutic purposes
Organoids
80
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
81
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)
82
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)
83
This method uses transgenic and cre-lox technology to generate mice (or other animals) expressing unique combinations of fluorescent markers in individual cells
Brainbow
84
Can be used to inactivate a gene in a specific group of cells
Knockout
85
What is photobleaching?
Exposing samples to so much light they lose their fluorescence
86
These method allows the use of methods (promoters, virus injection) to express rhodopsins in specific cells at specific times
Optogenetics
87
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)
88
Microscopy in which light is transmitted through the sample, usually only see pigmented or stained structures
Brightfield (light)
89
SLIDE 48
SLIDE 48
90
These are usually used from African clawed frog and 1 mm in diameter
Frog eggs (oocytes)
91
Methods used to alter endogenous genes and proteins at the protein level
Dominant negatives
92
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)
93
Non-homologous end joining tries to repair KO and leads to this in CRISPR
Bad repair leading to the gene being KOd
94
This allows for the creation of receptors that bind a ligand but don't transmit the signal
Dominant negative protein expression
95
These stem cells have a more limited capacity to self renew and may be unipotent
Progenitor cells (ex NPC)
96
Can be used to trace axons and dendrites with membrane-bound fluorescent reporters (trace connections to see what connects to what)
Transgenic mice
97
These types of cells can only become one certain thing
Unipotent
98
Microscopy in which light scatters due to differences in density, amplified here
Phase contrast (light)
99
This method allows for specific parts of neurons to be turned off and on at the flip of a switch
Optogenetics
100
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)
101
These can be used to test the function of a gene or effect of mutation in gene
Viruses
102
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
103
These can be used to examine how the animal nucleus is effected bc it appears black or brown
Oocytes
104
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
105
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
106
How did electron microscopy improve resolution
Light resolution = 200 nm
| EM = 0.2 nm (radius of a glutamate molecule)
107
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
108
This method can produce cells expressing rhodopsins constitutively (all the time) or inducibly (turn on and off)
Optogenetics
109
Different versions of these are used in optogenetics for different times/patterns of stimulation
Rhodopsins
110
What is a blastocyst?
A developing embryo with a few hundred cells
111
What does multipotent mean?
Cant become every cell, but can become certain kinds
112
Neural cell cultures that can be grown under the right conditions
Cell lines with neural properties and primary cells
113
To use this, samples must be fixed, dried, and chemically treated. They cant be alive
Electron microscopy
114
What do more mature organoids start to develop?
More neurons, other cells like glia, and vasculature
115
What type of microscope is needed to see cells don't touch at synapses?
Electron microscope (Cajall and Golgi didn't have this)
116
This is useful to visualize what cells are communicating with what cells
Brainbow
117
What two things can result from CRISPR?
Gene KO or gene being replaced by new sequence
118
Microscopy that images sections one at a time to get a good 3D image
Confocal (fluorescence)
119
These can be used to inject RNA or cDNA
Oocytes
120
Can be used to replace a gene with a modified version of the gene to determine effect of mutations in disease genes
Knockin
121
Methods used to alter endogenous genes and proteins at the DNA level
Gene targeting (KOs, knockins, conditional KOs)
122
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
123
Can be used to mark specific cells with GFP (green fluorescent protein or other colors), luciferase, Lac Z
Transgenic mice
124
These light channels are used to inhibit (optogenetics)
Halorhodopsins
125
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
