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Neuro 3000 > Techniques Used in Cellular and Molecular Neuroscience > Flashcards

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

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2
Q

What are pluripotent cells?

A

Cells that can become anything

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3
Q

What happens if a sample is continuously put under an EM?

A

The electron beam will eventually destroy the sample

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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

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5
Q

Allows us to see several figures during course of synapses in great detail

A

Electron microscopy

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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

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7
Q

Stem cells that generate cells from patents with neurological disorders

A

Induced pluripotent stem cells (iPSC)

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8
Q

What does a full KO do in cre-lox systems?

A

KOs the gene everywhere

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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

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10
Q

These are good to use bc they allow for proteins to be expressed 2-4 days after injection

A

Oocytes

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11
Q

These light channels are used to excite (optogenetics)

A

Channelrhodopsins

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12
Q

These are self organizing, small (5mm), can be used to test drugs, study development, and have therapeutic potential

A

Organoids

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13
Q

What do halorhodopsins do?

A

Let in Cl- when a certain wavelength of light hits it

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14
Q

Methods used to alter endogenous genes and proteins at the mRNA level

A

RNA interference, morpholinos

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15
Q

These are identified by function and molecular markers

A

Stem cells

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16
Q

What type of rhodopsin is used to modulate intracellular signaling in optogenetics

A

G-protein coupled rhodopsins

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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)

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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

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19
Q

3D tissue cultures derived from stem cells

A

Organoids

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20
Q

What do channelrhodopsins do?

A

Let in Na+ when a certain wavelength of light hits it

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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

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22
Q

These are pluripotent cells with the capacity to self renew

A

Stem cells

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23
Q

These can be used to deliver genes to cultured cells, brain slices, or brain regions in vivo

A

Viruses

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24
Q

What cant be controlled in transgenic mice?

A

Where the new DNA integrates in the genome

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25
Q

These are classified by source (embryonic, adult, induced) and tissue they generate

A

Stem cells

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26
Q

What type of stem cells are multipotent and can self renew?

A

Multipotent

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27
Q

Neural cell culture that can be used to maintain connectivity (acute for short term electrophysiology, organotypic for extended studies of synapse formation, migration)

A

Slice cultures (250-400 um)

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28
Q

Can be used to introduce genes for inducing or inhibiting neural activity such as channelrhodopsins, halorhodopsins, and DREADDs

A

Transgenic mice

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29
Q

4 types of stem cell cultures

A
  1. Embryonic stem cells
  2. adult stem cells
  3. neural stem cells
  4. induced pluripotent stem cells (iPSC)
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30
Q

Where are the sites of recombination in the cre-lox system?

A

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

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31
Q

New gene added, native gene still present

A

Transgenic mice

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32
Q

Can be used to mark specific organelles

A

Transgenic mice

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33
Q

This is used to distinguish specific cells, trace processes and connections, highlight cell structures, and to visualize fixed and living tissues

A

Brainbow

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34
Q

This method is used to measure and compare small amounts of RNA or DNA

A

Polymerase chain reaction (PCR)

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35
Q

Type of EM that allows for viewing over the surface

A

SEM (scanning)

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36
Q

Light that allows for receptor mediated intracellular signaling

A

Green light

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37
Q

These can be used to deliver genes when transgenic/KO or knockin animals can’t or don’t need to be used

A

Viruses

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38
Q

This is used to compare expression of genes in different neurons, different stages of development, mutant animals or tissue

A

Polymerase chain reaction (PCR)

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39
Q

Measure in microscopes used to tell how far apart two things have to be before you can tell theyre apart

A

Resolving power

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40
Q

Homology directed repair leads to this in CRISPR

A

The KOd gene gets replaced with a new gene. A new sequence is inserted

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41
Q

Is optogenetics limited to mice?

A

No it can be applied to many animals

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42
Q

What are oocytes

A

Frog eggs

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43
Q

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)

A

Knockout

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44
Q

Cells that are more like real neurons, theyre more relevant but they produce heterogeneous populations with high variability

A

Primary cell culture

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45
Q

This is the ultimate form of ablation, completely getting rid of a gene

A

Knockout

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46
Q

Does confocal or epifluorescence produce a better image?

A

Confocal

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47
Q

Stem cells that are self renewing, produce neurons, astrocytes, oligodendrocytes, more present in embryonic than in adult brain

A

Neural stem cells

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48
Q

Microscopy in which optics are used to exaggerate differences in light scattering properties

A

Nomarski (light)

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49
Q

Are neurons and glia non-dividing or dividing cells?

A 
Neurons = non-dividing
Glia = dividing
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50
Q

Can be used to ablate cells by expressing toxin or toxin receptors specifically

A

Transgenic mice

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51
Q

What are the 3 parts to the Core-Lox system?

A

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
Q

Microscopy that is deeper, has less photobleaching, and less background

A

Two photon (fluorescence)

53
Q

These methods operate on DNA by altering endogenous genes (genes that are already in the organism) and proteins

A

Knockout, Knockin, and CRISPR-Cas9

54
Q

Pluripotent stem cells that can be induced to form neural progenitors

A

Embryonic stem cells

55
Q

How can the role of a specific protein be tested?

A

Blocking function of the protein

56
Q

Do the majority of offspring wind up being transgenic or not?

A

It depends, lots of DNA will be destroyed by the mice but some of the offspring will wind up being transgenic

57
Q

Rhodopsin activated by blue light

A

Channelrhodopsin (excite)

58
Q

Type of EM that allows for viewing through a thin section

A

TEM (transmission)

59
Q

Cells that are easier to grow, more stable, and fairly well characterized but don’t have all the properties of neurons

A

Immortalized cell lines

60
Q

Used to express gene in specific cells, determine effects on cell or circuit formation

A

Transgenic mice

61
Q

These allow for multiple electrodes to be used to record ion channel activity

A

Oocytes

62
Q

Where must the transgene (new gene being put in) go to create transgenic mice?

A

It has to go in front of the correct promoter you want it to go in front of

63
Q

What does it mean to be floxed

A

When the target gene is flanked by lox sites

64
Q

When foreign DNA is injected into mouse germ line by injection of one-cell egg or injection of ES (embryonic stem) cells into blastocyst

A

Transgenic mice

65
Q

This is used to look at structures, not how things change

A

Electron microscopy

66
Q

How does CRISPR work?

A

RNA generated from spacers and CRISPRs targets Cas9 nuclease to invading DNA and CRISPR loci are transcribed

67
Q

Rhodopsin activated by yellow light

A

Halorhodopsin (inhibit)

68
Q

Can be used to inactivate a gene to look at the function of it

A

Knockout

69
Q

Type of microscopy that collects light from the entire depth of tissue and illuminates the entire sample

A

Epifluorescent (fluorescence)

70
Q

Type of culture that extends on explant culture and further makes it easier to see the neurons

A

Dissociated cells

71
Q

Microscopy in which there is illumination from the side, only highly refractive parts such as organelles are seen

A

Darkfield (light)

72
Q

Can be used to overexposes a gene to make a product

A

Transgenic mice

73
Q

Can be used to insert genes to measure neural activity (Ca2+, voltage, synaptic vesicle fusion)

A

Transgenic mice

74
Q

Type of culture that is taking out a chunk of the brain

A

Explant culture

75
Q

What are the 5 steps for creating knockout/knockin mice?

A
  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
Q

These are an array of identical repeats intercalated with DNA-targeting spacers derived from bacteria and plasmids

A

CRISPRs

77
Q

What do Cre-Lox systems do?

A

Knockout a gene in a specific tissue or at at specific time

78
Q

What does in situ mean?

A

In place

79
Q

These are just starting off and far from being able to be applied to use in people for therapeutic purposes

A

Organoids

80
Q

Activators, repressors, and GFPs lead to being able to do these 3 things in CRISPR

A

Turn on gene, turn off gene, and be able to see gene not in replication

81
Q

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

A

Confocal (fluorescence)

82
Q

This method requires tiny amounts of tissue, can be done in situ (in place), and is used for genomic sequencing and mapping

A

Polymerase chain reaction (PCR)

83
Q

This method uses transgenic and cre-lox technology to generate mice (or other animals) expressing unique combinations of fluorescent markers in individual cells

A

Brainbow

84
Q

Can be used to inactivate a gene in a specific group of cells

A

Knockout

85
Q

What is photobleaching?

A

Exposing samples to so much light they lose their fluorescence

86
Q

These method allows the use of methods (promoters, virus injection) to express rhodopsins in specific cells at specific times

A

Optogenetics

87
Q

What happens once the transgene is placed in front of the proper promoter in making transgenic mice?

A

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
Q

Microscopy in which light is transmitted through the sample, usually only see pigmented or stained structures

A

Brightfield (light)

89
Q

SLIDE 48

A

SLIDE 48

90
Q

These are usually used from African clawed frog and 1 mm in diameter

A

Frog eggs (oocytes)

91
Q

Methods used to alter endogenous genes and proteins at the protein level

A

Dominant negatives

92
Q

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

A

Induced pluripotent stem cells (iPSC)

93
Q

Non-homologous end joining tries to repair KO and leads to this in CRISPR

A

Bad repair leading to the gene being KOd

94
Q

This allows for the creation of receptors that bind a ligand but don’t transmit the signal

A

Dominant negative protein expression

95
Q

These stem cells have a more limited capacity to self renew and may be unipotent

A

Progenitor cells (ex NPC)

96
Q

Can be used to trace axons and dendrites with membrane-bound fluorescent reporters (trace connections to see what connects to what)

A

Transgenic mice

97
Q

These types of cells can only become one certain thing

A

Unipotent

98
Q

Microscopy in which light scatters due to differences in density, amplified here

A

Phase contrast (light)

99
Q

This method allows for specific parts of neurons to be turned off and on at the flip of a switch

A

Optogenetics

100
Q

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

A

Total internal reflectance (TIRF) (fluorescence)

101
Q

These can be used to test the function of a gene or effect of mutation in gene

A

Viruses

102
Q

These two things already found in our bodies are used in combination with siRNA and shRNA to target and degrade specific RNAs

A

Dicer and RISC

103
Q

These can be used to examine how the animal nucleus is effected bc it appears black or brown

A

Oocytes

104
Q

What does an inducible conditional KO do in cre-lox systems?

A

KOs the gene at a specific time. Add something like tamoxifen to do this

105
Q

What are DREADDs?

A

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
Q

How did electron microscopy improve resolution

A

Light resolution = 200 nm

EM = 0.2 nm (radius of a glutamate molecule)

107
Q

This works by adding different fluorescent proteins between different lox sites and having the cre recombinase enzyme cut them to produce different colored cells

A

Brainbow

108
Q

This method can produce cells expressing rhodopsins constitutively (all the time) or inducibly (turn on and off)

A

Optogenetics

109
Q

Different versions of these are used in optogenetics for different times/patterns of stimulation

A

Rhodopsins

110
Q

What is a blastocyst?

A

A developing embryo with a few hundred cells

111
Q

What does multipotent mean?

A

Cant become every cell, but can become certain kinds

112
Q

Neural cell cultures that can be grown under the right conditions

A

Cell lines with neural properties and primary cells

113
Q

To use this, samples must be fixed, dried, and chemically treated. They cant be alive

A

Electron microscopy

114
Q

What do more mature organoids start to develop?

A

More neurons, other cells like glia, and vasculature

115
Q

What type of microscope is needed to see cells don’t touch at synapses?

A

Electron microscope (Cajall and Golgi didn’t have this)

116
Q

This is useful to visualize what cells are communicating with what cells

A

Brainbow

117
Q

What two things can result from CRISPR?

A

Gene KO or gene being replaced by new sequence

118
Q

Microscopy that images sections one at a time to get a good 3D image

A

Confocal (fluorescence)

119
Q

These can be used to inject RNA or cDNA

A

Oocytes

120
Q

Can be used to replace a gene with a modified version of the gene to determine effect of mutations in disease genes

A

Knockin

121
Q

Methods used to alter endogenous genes and proteins at the DNA level

A

Gene targeting (KOs, knockins, conditional KOs)

122
Q

Acting at these two times allows the altering of endogenous genes and proteins by blocking RNA expression to remove function without destroying genes

A

Transcription and translation

123
Q

Can be used to mark specific cells with GFP (green fluorescent protein or other colors), luciferase, Lac Z

A

Transgenic mice

124
Q

These light channels are used to inhibit (optogenetics)

A

Halorhodopsins

125
Q

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)

A

Fluorescence microscopy

Neuro 3000 (15 decks)

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