Brainscape's Knowledge GenomeTM


Top Flashcards (Certified)
All Flashcards

A BMS336 > Techniques > Flashcards

Techniques Flashcards

(85 cards)

Start Studying
1
Q

How can you discover what TF is responsible for the specification of a cell?

A
  • Cell specific promoter driving RFP
  • FACS
  • Microarray - look for TF that are upregulated with no known function in comparison to other cells
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

How can you determine if something is required for an event?

A
  • Right place at the right time (ISH)
  • Necessary (KO, knockdown)
  • Sufficient (ectopic over expression)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What are LOF/GOF studies used to determine?

A

The function of a cell/tissue/gene product

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

At what levels can LOF/GOF occur?

A

1) Tissue/cell level (ablation/graft)
2) Gene level (KO, over expression)
3) Protein level

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

What are the different ways the KO of a gene function can occur?

A

1) Pre mRNA processing
2) Nuclear transport –> cytoplasm
3) Translation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What are ways that KOs can occur?

A

1) Homologous recombination
2) Cre/lox
3) CreERT2/lox
4) CRISPR/Cas9
5) Morpholino

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Homologous recombination?

A

Removes the gene in EVERY CELL of the body:

1st way:

  • Genomic clone of the gene to be knocked out with NEO in the middle - KO
  • TK off to one side of the gene

Into bacteria –> double selection to select for HOMOLOGOUS recombination:
- Grow on NEOMYCIN - taken up NEO –> Survive

  • Grow on GANC media - taken up TK –> Die (not-homologous)

2nd way:
- Gene for ANTIBIOTIC RESISTANCE into the gene –> disrupting it

INTO MICE:

  • Into ES cells –> into pseudopregnant mouse
  • Level of integration in the mouse is determined by the level of chimerism of the mouse
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Cre/lox?

A

Removes the gene in SPECIFIC CELLS of the body:

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

CreERT2/lox

A

Removes the gene in SPECIFIC CELLS at a SPECIFIC TIME in the body

  • ERT2 = triple mutant form of the human estrogen receptor
  • Requires the addition of TAMOXIFEN to get a response
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

CRISPR/Cas9?

A
  • Cas9 cuts DNA - is guided to the DNA cut site of interest by the complimentary RNA sequence

–> can remove the gene

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Morpholino?

A

Blocks PROTEIN SYNTHESIS (translation):
- RNA chain that is ANTISENSE to the DNA of the gene wanting to block

  • Base pairs with the target DNA and blocks progression of the transcription initiation machinery
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What is the difference between KO of a signal and KO of a specific cell type receptor?

A

Signal - Effects the whole body

Receptor - Effects that particular cell

Can compare if they are the same and conclude the signal is acting on the specific cell to get the body response

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What can be done after a KO of a signal?

A

Can READMINISTER the signal –> see if restore the phenotype

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What can a hs promoter be used for?

A

1) INDUCIBLE promoter for a floxed transgenic gene (eg. GFP)
2) Cause overexpression

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What techniques can be used to cause over expression?

A

1) GAL4/UAS
2) hs promoter

BOTH CONDITIONAL??

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

GAL4/UAS?

When can this system be used?

A
  • GAL4 gene under the regulation of a TISSUE SPECIFIC promoter (only active in a cell where specific transcriptional activators are present)
  • UAS (upstream activating sequence promoter) –> fused upstream to a coding sequence that want to upregulate
  • Cross transgenes –> up regulation in SPECIFIC cells

Can be used:

  • To up regulate a GENE
  • Up regulate a TOXIN - which can then have drugs applied to -> see effects
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

How can the Cre system be used other than to KO genes?

A

1) Lineage tracing

2) Labelling of 2 transgenic genes (eg. GFP and RFP)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

What are the techniques that show WHERE a cell/tissue is present?

A

1) Transgenic reporter lines

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

What are the techniques that show WHERE mRNA is present?

A

1) ISH

2) GFP-transgenic lines

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

What are the techniques that show WHERE protein is present?

A

1) Immunohistochemistry

2) GFP transgenic lines - gene fusion

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

Describe the the theory of transgenic lines?

A

Genes are differentially transcribed in different cells/tissues as a function of interaction at the promoters and enhancers

Promoters are TISSUE SPECIFIC (only on in specific tissues) - need the right activators present

Transcriptional activators/repressors are cell specific

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

How can transgenic reporter lines be used to follow:
- A specific cell/tissue

  • A specific gene
A

Cell/tissue: Use a tissue/cell specific promoter

Gene: Replace the gene of interest with GFP

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

What can transgenic reporter lines be used for?

A

Can follow the reporter OVER TIME

Can distinguish cells from neighbouring cells

Can see the effects of: Health, disease, insult, drug administration, different conditions on the cell/tissue OR gene

Can follow cellular proliferation/migration (real-time cells)
Can follow cell function
Can identify if these are normal/aberrant

Can COUNT cell number and monitor/drug screen

Can label a signal/hormone - gets brighter/duller in certain situations (amount of hormone produced)

Can see what tissues/cells a certain gene is expressed

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

How is GFP inserted into the genome?

A

Using restriction enzymes

IRREVERSIBLY –> stable (can follow over lifetime)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
How can GFP gene fusion be used to follow the protein product of a gene? How is this different to a GFP reporter construct?
Gene fusion: - Contains BOTH mRNA for GFP AND mRNA for the gene of interest - Gene of interest can be transcribed and translated - fused to GFP Reporter: - mRNA of interest is replaced by GFP (under the control of the endogenous gene but cannot make a protein product)
26
What is FACS? What is it used for?
Fluorescence activated cellular sorting Used to ISOLATE labelled cells/tissues
27
What can be done after FACS?
1) Drug screening | 2) Find a gene responsible for the specification of the cel (eg. using microarrays)
28
How can cell death be monitored?
1) By the loss of fluorescence from a transgenic reporter line 2) TUNEL staining 3) Using labelled antibodies for caspases (up regulated during apoptosis)
29
What are other uses of GFP in the genome?
1) Fuse GFP to nls (nuclear localisation signal) (promoter:nlsGFP) 2) Track cell divisions over time (by labelling the nuclei using histone2a-mCherry) 3) Can label 2 transgenes using GFP and cre-lox
30
In what situation is it advantageous to have GFP in the nucleus?
When following cells/cell number: | - Easier to count than when GFP is in the cytoplasm
31
What other ways are there of tracking cell division/proliferation other than using mCherry fused to histone2a?
1) EDU/BrdU labelling
32
How can cre-lox and GFP be used to label 2 transgenes?
Transgenic 1: - Tissue specific promoter driving the expression of CreERT2 Transgenic 2: - CA promoter (present everywhere) driving the expression of a floxed GFP gene and a RFP gene Cross transgenics (and apply tamoxifen) --> loxp sites cleaved, GFP removed (in certain tissues) --> RFP END UP WITH DIFFERENT CELLS - DIFFERENT COLOURS
33
Describe the technique of monitoring cell proliferation with EDU and BrdU labelling
EDU and BrdU are analogues of thymidine (a component of DNA) that can be visualised using labelled antibodies - Before a cell proliferates - must replicate the DNA - EDU/BrdU incorporated into the DNA in the place of T - Non-dividing cells --> not incoporated
34
When can cell proliferation/death be altered?
In response to CUES
35
What is the disadvantage of EDU/BrdU to monitor cell proliferation?
It cannot be done in LIVE cells
36
How can specific cells be ablated (conditionally)?
Using SPECIFIC expression of NITROREDUCTASE NTR on its own is NOT harmful, but the addition of metronidazole (MTZ) converts NTR --> toxic product
37
How can cell ablation using nitroreductase be shown to be effective?
Nitroreductase is fused to a genetic reporter (eg. GFP) under the same tissue specific promoter Tissue/cellular specific promoter When Metronidazole (MTZ) is added --> see that cells have been deleted because the fluorescence disappears
38
What is TUNEL staining used for? Describe this technique
To see if cells go through apoptosis - Fix tissues and add Tdt (a DNA polymerase that adds nucleotides to the end of DNA) - In cells going through apoptosis - lots of free DNA --> lots of DNA synthesis by Tdt - Use BrdU DNA - can be recognised with an antibody labelled with a reporter/flurophore
39
How can different populations of cells be identified over time?
Different stages of differentiation of a cell express different markers - Express some markers - Repress other markers - -> specific combination
40
How can if be seen what cells derive from a particular cell? How?
Using lineage tracing Cells are permanently marked, all the cells from this cell and all the cells from that cell etc are marked and look the same
41
Describe the lineage tracing technique (eg. for tanycytes)
- Use a specific cell promoter in the adult/stem cell that is not present in the daughters? (eg. tanycytes --> GLAST1) to drive creERT2 - In the other transgene, have ROSA26 driving the expression of loxP sites flanking a stop site before GFP (no gene - blank casette) - Cross transgenes and add tamoxifen - cells will fluoresce (ROSA now driving GFP (no stop site in between) - PERMANENT genetic change - IRREVERSIBLY expresses GFP even if GLAST no longer expressed
42
What is a ubiquitous promoter?
Expressed in MANY cell types/tissues
43
What is the disadvantage of using lacZ as reporter?
It cannot be used in live animals
44
How can stem cells be studied?
Can KO key genes that induce/maintain each cell type --> differentiation?? Can over express these genes --> extra cells?? ectopically?
45
What are 3 ways that fate mapping can occur?
1) Transplantation of cells and tissues (eg. quail --> chick, antibodies recognise surface of the quail nucleus but not the chick) 2) Tissue/cell labelling with dye 3) Genetic labelling (transgenic lines, brainbow, GFP)
46
Describe the brainbow technique of fate mapping
- Introduce MANY genes each encoding a DIFFERENT FLUOROPHORE - Each construct framed by a DIFFERENT pair of loxP sites that are each slightly different to each other and recognised by different cre recombinases - Only have ONE TYPE of excision --> determines what genes are left - Get wide range of combinations of colours that are all distinct in each cell type where recombination took place
47
What animals is the brainbow technique used in?
Zebrafish and mice
48
When is the brainbow technique particularly useful?
In the brain and cortex (eg. around the neural tube) can see the neural progenitors and their daughter cells
49
What does fgf encode?
Cell proliferation
50
What does shh encode?
Differentiation | Proliferation
51
What does wnt encode?
Self-renewal - regulate stem cells in the niche
52
What does 'activating' smoothed mutations cause?
Increase in smoothened activation, which is normally repressed by patched More GliA in the nucleus More cell proliferation --> Basal cell carcinoma
53
What are xenotransplants? Example?
Transplantation organs/tissues from one species to another Eg. Can transplant human cell lines --> NUDE MICE and grow human tumours in these mice
54
What is the advantage of nude mice?
Can transplant human cells into them - won't be rejected
55
What is the disadvantage of nude mice?
They are immunocompromised
56
What is epistasis analysis? Example
Experiments to determine what genes are upstream of others by interfering with 2 pathways Block shh --> block proliferation/regeneration If Wnt is DOWSTREAM, then activation of wnt will reactivate/restore regeneration
57
What is forward genetics?
- RANDOMLY mutating the genome - Select for phenotype of interest (eg. tumor formation) - Find gene responsible (microarray??)
58
What are chemical mutagens?
ENU OR Electromagnetic radiation
59
What is complementation testing?
Testing to see if 2 mutations that cause the same phenotype are due to mutations in 2 different alleles of the same gene OR mutations in DIFFERENT genes
60
What is the process of complementation testing?
Cross m1/+ and m2/+: 1/4 of offspring with phenotype --> Fail to compliment --> alleles of the sam gene No phenotype --> compliment each other --> different genes
61
How can you demonstrate the instructive function of a tissue? What would be the next steps once identified?
Tissue manipulation: Graft ectopically or in the same place/ablation Next steps: - To identify the signalling molecule in the tissue responsible - Necessary?? - KO - Sufficient?? - Bead soaked in the signalling molecule
62
How can CHANGES in gene expression be determined?
Using MICROARRAYS: - Detect the difference in gene expression between 2 cell types - Eg. treated vs untreated, WT vs mutant
63
What is the process of using a microarray?
- Take the mRNA from each cell --> cDNA and label with fluorescent marker - Each grid has mRNA - If cDNA is present --> will hybridise to the mRNA - One cell labelled with red, one with green - If in BOTH cells --> yellow
64
In tumours, what kinds of genes are UPREGULATED?
POTENTIAL oncogenes
65
In tumours, what kinds of gene are DOWNREGULATED?
POTENTIAL tumour supressor genes
66
What can stem cells be used for?
Modelling of a disease in vitro
67
Where are ES stem cells from?
The ICM of an embryo
68
How can stem cells be defined?
1) DESCRIPTIVE - express PLURIPOTENCY MARKERS | 2) FUNCTIONAL - transplant into the kidney of a mouse embryo --> should form a teritocarcinoma
69
What are the pluripotency markers?
Oct4, nanog, sox2
70
What is the 3D approach to disease modelling with stem cells?
- REMOVE signals that keep stem cells in undifferentiated state - Let them differentiate/organise on their own --> Makes aggregates (embryoid bodies) - if left for longer --> CEREBRAL ORGANOIDS -> can apply drugs
71
What is the 2D approach to disease modelling with stem cells?
- Remove signals | - Add signals that push the cells to differentiate to a particular cell type (known differentiation signals)
72
What are the advantages/disadvantages of the 3D approach?
A: - Mimics the environment in vivo (cell interactions etc) D: - Hard to dissect out individual signals
73
What are the advantages/disadvantages of the 2D approach?
A: - Can test specific signals - Can use LIVE IMAGING D: - Cannot see the cell interactions
74
How can signalling be studied?
1) Visualisation with FRET 2) Use of chemical inhibitors 3) Miss expression/over expression 4) Genetics - forwards, reverse, transgenics, mutation analysis
75
What can fluorescently labelled proteins show?
Can FOLLOW the movement of a protein when signalling is ACTIVE compared to when inactive
76
What is FRET analysis used for?
1) To monitor the interaction between 2 proteins 2) To see any confirmation changes in a single protein - 2 fluorophores in different parts of the protein 3) To see when a protein is CLEAVED - Activity = close together, cleaved = drift apart - REAL TIME - LIVING cells
77
What is the idea behind FRET analysis?
Blue - one protein Green - other protein - Blue is excited by violet light - Green is excited by blue light If proteins interact - green light let out when excited by violet light If proteins don't interact - blue light let out when excited by violet light
78
What is advantageous about with chick and xenopus?
Both fertilised outside of the mother --> Easy access and easy to manipulate and move tissues around in the EARLY STAGES
79
What model is used for regeneration?
Salamander Zebrafish
80
What model organisms are used for reporter lines?
Mice and zebrafish
81
What are the advantages of the mouse animal model?
- Can follow into adulthood --> look @ disease progression over life - Look at gene x environment - Inbred populations - select for particular characteristics - Takes a long time to get transgenics (have to breed) - MAMMALS
82
What are the disadvantages of the mouse animal mode?
Expensive
83
What are the advantages of using zebrafish as a model?
- Transparent --> easy to score, can see under the microscope - High throughput screens - Fast genetics - Accessible - Cheap - Can inject --> mutants, transgenics - Small - 70% genes similar to humans - Model for regeneration and heart disease (same proteins/processes as make the blood vessels as in humans)
84
What are the disadvantages of using zebrafish as animal models?
- Size - limiting to some technologies - Rapid development --> moving baseline - Single circulation
85
Why use conditional KO?
- Can study potentially LETHAL genes that are important for survival - If the gene functions in another part of the animal - can KO in a specific tissue so know the phenotype is bc of a KO in that tissue

A BMS336 (1 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2025 Bold Learning Solutions. Terms and Conditions