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CMMB 403 > Tutorial Quiz 1 > Flashcards

Tutorial Quiz 1 Flashcards

1
Q

What does the blue stain tell us in a B-galactosidase reporter assay?

A
  • When and where an enhancer is active
  • When and where a gene of interest is normally expressed
  • When and where a reporter gene is expressed
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2
Q

What is an example of an enzyme conjugated to an antibody in WISH?

A

Alkaline Phosphatase

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3
Q

What does the staining pattern in WISH tell us?

A

When and where mRNA of a specific gene is localized

When and where a gene of interest is expressed

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4
Q

What are ways we can use reporter genes to report gene expression?

A
  • Inserting the reporter gene into the coding sequence of a gene (nullify)
  • Insert the reporter gene inline with the coding sequence (using IRES)
  • Attach the reporter to the coding sequence of the gene of interest (fusion)

What is NOT a way
- Insert the reporter gene downstream of an enhancer of the gene of interest

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5
Q

Why might the sense control WISH embryos stain when no hybridization even has occurred?

A

The sense control probe gets stuck in some parts of the embryo

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6
Q

What kind of cells are used to do a standard or conditional gene knockout?

A

Embryonic stem cells

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7
Q

What are the 3 differences between making a transgenic mouse and doing a standard gene knockout in a mouse?

A
  • In standard gene knockout, you use electroporation to get the construct into the embryonic stem cells where as in a transgenic, you use pronuclear injection
  • In a knockout, a construct is targeted to a particular locus using regions of homology. The construct is randomly incorporated when making a transgenic
  • In a knockout, you are using embryonic stem cells but in a transgenic, you are using a fertilized egg (single cell embryo w/ 2 nuclei still)
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8
Q

What are conventional models (model organisms) typically used in developmental biology?

A

Mice, Zebrafish, Chicks (historic), Drosophila, C. elegans, Xenopus

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9
Q

What council governs the animal usage for teaching and learning in Canada?

A

CCAC - Canadian Council on Animal Care

- advance high standards and animal ethics

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10
Q

What are the 3 R’s ?

A

Replace- Alternatives been considered?
Reduce- Need to ensure the fewest number of animals is used
Refine- is the distress of the animals being reduced as much as possible?

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11
Q

Categories of Invasiveness

A

A- The use of tissues/blood
B - Experiments which cause little or no discomfort or stress
C - Experiments which cause minor stress or pain of short duration
D - Experiments which cause moderate to severe distress or discomfort
E - Cause severe pain - at or above the pain tolerance threshold - unanesthetized

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12
Q

How often is the U of C visited by CCAC?

A

Every 3 years
They identify strengths and weaknesses - make recommendations for change
U of C must then submit a response

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13
Q

How often must animal use protocols be re-reviewed?

A

Every four years if they are still in-use

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14
Q

What committee reviews and approves all U of C animal care and use?

A

Animal Care Committee (ACC)

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15
Q

What might gene expression tell us about a gene?

A
  • Where it functions
  • What it function may be
  • Its relationship with other genes
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16
Q

What does WISH stand for?

A

Whole-mount In Situ Hybridization

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17
Q

What can WISH tell you?

A
  • Stains whole embryos
  • A method to tell where in an embryo a given gene is transcribed (or expressed)
  • It utilizes a complementary RNA probe
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18
Q

What kind of probe does WISH utilize?

A

Complimentary RNA probe

- It will hybridize to transcribed sequences (mRNA) in the cells of an embryo

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19
Q

How to make WISH probe?

A

digoxigenin (DIG)-labelled RNA probe is made in a test tube
Need:
- nucleotides (A, G, C, and U with DIG label)
- template DNA for gene of interest
- RNA Polymerase

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20
Q

What may 2 genes having similar expression domains imply?

A

They could be related to one another or work together! For example, Shh ligand and Patched protein receptor will be expressed in similar regions.

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21
Q

What is up with the uracil in making the WISH probe?

A

It is a nucleotide added to form the mRNA probe complimentary to a gene of interest.
The uracil specifically is conjugated to digoxigenin (DIG), a big bulky steroid.
The bulky steroid allows the probe to be detected by an antibody

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22
Q

What happens in the WISH assay?

A
  1. The DIG-labelled RNA probe is added to fixed embryos (the probe will hybridize to the mRNA of the gene of interest)
  2. The probe is detected with anti-DIG antibody which is conjugated to an enzyme (Ab conj to enzyme an Ab binds to the hybridized probe)
  3. Substrate is added. The enzyme conjugate turns added colourless substrate purple - reveals when and where a gene is expressed (bc its binding to where the mRNA is)
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23
Q

What does fixing the embryos mean?

A

Killing the embryos and maintaining them at a particular stage in embryogenesis for studying

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24
Q

What and how is an enzyme used in the WISH Assay?

A

An enzyme, alkaline phosphatase, is conjugated to the antibody. The Ab is bound to the RNA probe that hybridized to the mRNA for the gene of interest.
- Alkaline phosphatase catalyzes the reaction of a colourless substrate into a purple one. So wherever you see purple is where the gene is expressed.

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25
Q

What are reporter genes?

A

Genes that are expressed along with or in place of a gene of interest.
They code for some product that we can detect
Reporter gene expression represents expression of our gene of interest (they report the expression!)

26
Q

How are reporter genes introduced into the genome?

A

Through homologous recombination!

27
Q

What are 3 different ways reporter genes can be used/introduced?

A
  • Inserted into the coding sequence of a gene, nullifying it. (This is only ok if the gene is haplosufficient)
  • Inserted in line with the coding sequence of a gene, w/o disrupting the reading frame (with an internal ribosome entry site). Reporter gene is after the gene of interest.
  • Attached to the coding sequence of a gene, creating a fusion protein. This adds something to the protein of interest that allows us to see where the protein is. EX. GFP

** The IRES is an RNA element that allows for translation to occur! to translate the reporter gene mRNA into a protein that can be used to identify location

28
Q

LacZ reporter gene

A

This reporter gene encodes the B-galactosidase enzyme. The enzyme than carries out a reaction to stain the embryo upon substrate addition.
The location of the staining indicates where the gene of interest id expressed.

29
Q

What are enhancers?

A
  • DNA sequences that communicate with the gene promoter to activate transcription of that gene
  • Contains binding sites for transcription factors - the transcription factors mediate the association between the enhancer and the promoter. The activity of the enhancer depends in the TFs present in the cell/tissue at the time
  • Their activity can be cell, tissue, or stage-specific
  • Eukaryotic enhancers have different ‘strengths’ (the ability to drive a certain amount of transcription)
30
Q

What are examples of reporter genes?

A

Luciferase, LacZ, GFP (Green Fluorescent Protein)

31
Q

What is the luciferase assay used for?

A

Can be used to quantify enhancer activity

32
Q

Strong enhancer?

A

Binds to RNA Pol more tightly. Ie. increased binding sites - this leads to more gene expression or more RNA produced

33
Q

What does a transgenic reporter assay tell you?

A

Uses a reporter gene to show when and where an enhancer is active

34
Q

How do you do a transgenic reporter assay?

A
  1. A DNA construct is made containing the enhancer sequence of interest, a promoter and the report gene,
  2. This construct is then introduced into a single cell embryo (with both pronuclei present) using pronuclear injection.
  3. The enhancer (when active) will cause the reporter gene to be expressed
  4. The reporter gene expression corresponds to when and where the enhancer is active
    - Location of staining is where the enhancer activated lacZ expression

** the incorporated DNA construct utilizes the cell machinery to drive its expression (of the reporter gene)

35
Q

What is in the DNA construct for a transgenic reporter assay?

A

The enhancer sequence of interest, a promoter, and the reporter gene

36
Q

What is a minimal promoter?

A

One that on its own is not capable of driving expression of the downstream gene

37
Q

How is the construct incorporated into the genome when creating a transgenic?

A

Construct is inserted into a single cell embryo with 2 pronuclei - the DNA construct is then RANDOMLY INCORPORATED into genome housed within the pronucleus
- Generally will insert into junk DNA and not affect the expression of other genes.
Multiple copies of the transcript may be incorporated.
The construct will then be present in ALL of the cells

38
Q

What is luciferase?

A

It is a reporter gene that codes for the luciferase enzyme. It catalyzes the reaction of luciferin into a bioluminescent product that can be detected/assayed for.
- Activity can be monitored using a luminometer.

39
Q

What does the vector contain in the luciferase assay?

A

Vector contains:

  • enhancer of interest
  • minimal promoter
  • luciferase reporter gene
40
Q

What is transfection and when is it used?

A

The vector containing the enhancer interest is transfected into cells. Transfection is just covering it in nonpolar lipids that allow it to diffuse through the plasma membrane.

41
Q

Why might the sense control WISH embryos stain when no hybridization event has occurred?

A

The sense control probe gets stuck in some parts of the embryo

42
Q

What does the luciferase assay tell you?

A

Can tell you if there is enhancer activity in a particular sequence or can determine the strength of a given enhancer.
- You can then manipulate the enhancer sequence or add/remove TF to see the effects it has on the
enhancer’s activity
- specific cells or tissues are used, not entire embryos
- the stronger the glow, the higher the activity of enhancer of interest.

THE LUCIFERASE ASSAY DOES NOT TELL YOU WHERE AN ENHANCER IS ACTIVE

43
Q

What does the blue stain tell us in a β-galactosidase reporter assay?

A
  • when and where an enhancer is active
  • when and where a gene of interest is normally expressed
  • when and where a reporter gene is expressed
44
Q

What enzyme is conjugated to an antibody in WISH?

A

Alkaline Phosphatase

45
Q

What is endogenous expression?

A

The normally occurring expression within a tissue/cell (NORMAL)

46
Q

What is ectopic expression?

A

The expression of a gene where it is not normally found (NOT NORMAL)

47
Q

What does a standard gene knockout do?

A

It disrupts the DNA sequence of gene, thereby preventing it from making a functional protein product

48
Q

Does standard gene knockout target endogenous or ectopic loci?

A

Targets endogenous loci using homologous recombination

49
Q

What is included in the construct used for standard gene knockouts?

A
  • Regions of homology (sequences bordering gene of interest) to target endogenous locus
  • A marker (antibiotic resistance marker)
  • Upon homologous recombination, the targeted gene will be replaced with the Ab resistance gene - ES cells that have undergone this switch will survive in the presence of antibiotics!
    Target gene will be knocked out
50
Q

Why might one want to use conditional knockouts vs a standard knockout?

A

Often the function of particular developmental genes are necessary for survival of the embryo.

A conditional knockout allows you to study gene expression that has only been knockout out in particular targeted tissues

  • avoiding embryonic lethality
51
Q

How does the DNA construct enter the cell in standard gene knockouts?

A

Electroporation is used to increase the permeability of the ES cell membrane to the construct

52
Q

How is breeding to homozygosity achieved in a standard gene knockout?

A
  1. Inject cells with standard knockout completed into the inner cell mass of early blastula and insert into surrogate
  2. Developed embryo will have mixed cells. Select a germ cell - breed it with white phenotype (if the germ cell is of the mutant (brown) phenotype, than all of the resultant offspring will be brown because brown is dominant.
  3. Breed the F1 progeny (heterozyg) together - at least some of the resultant progeny will be fully mutant.
53
Q

How are loxP sites introduced into the genome?

A

Through homologous recombination!

54
Q

What is a floxed gene?

A

A gene that is bordered by loxP sites. Cre recognizes loxP sites.

55
Q

What is required for a conditional knockout?

A

Cre viral enzyme, cell- or tissue- specific promoter, and loxP sites to flox genes.

56
Q

How is a tissue specific Cre activated and what does it do?

A

It is activated if the tissue-specific promoter it is attached to is activated. The promoter will be active when this gene is normally active at the time the Cre is inserted. This specific Cre that is now active will excise the floxed gene targeted for knockout.

** Cre is present in all cells but will only be active when the promoter of the gene of interest is active. Bc the tissue-specific Cre has that same promoter.

57
Q

What promoter is necessary for overexpressing a gene?

A

Rosa26 - Constitutive promoter
The Rosa26 promoter can be used to drive expression of an extra copy of a gene of interest.
- We use Cre/loxP recombination to limit the overexpression to particular tissues.

-

58
Q

What is a constitutive promoter?

A

A promoter that drives expression of all genes during development constantly. ex Rosa 26

59
Q

How is Cre/loxP used to conditionally overexpress a gene?

A
  1. Flox STOP cassettes with homologous recombination and loxP sites
  2. When Cre is active (when cell- or tissue- specific promoter is active) it will excise out the STOP cassettes, causing gene expression to go ahead (as well as the normal gene expression) (no stop signal)
60
Q

How is Cre activity characterized?

A
  1. Use a Cre-inducible reporter allele
    This reporter gene express will tell the viewer where the Cre is active in the embryo (where the tissue-specific promoter is active and driving Cre expression)

CMMB 403 (7 decks)

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