Tutorial Quiz 1 Flashcards
What does the blue stain tell us in a B-galactosidase reporter assay?
- 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
What is an example of an enzyme conjugated to an antibody in WISH?
Alkaline Phosphatase
What does the staining pattern in WISH tell us?
When and where mRNA of a specific gene is localized
When and where a gene of interest is expressed
What are ways we can use reporter genes to report gene expression?
- 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
Why might the sense control WISH embryos stain when no hybridization even has occurred?
The sense control probe gets stuck in some parts of the embryo
What kind of cells are used to do a standard or conditional gene knockout?
Embryonic stem cells
What are the 3 differences between making a transgenic mouse and doing a standard gene knockout in a mouse?
- 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)
What are conventional models (model organisms) typically used in developmental biology?
Mice, Zebrafish, Chicks (historic), Drosophila, C. elegans, Xenopus
What council governs the animal usage for teaching and learning in Canada?
CCAC - Canadian Council on Animal Care
- advance high standards and animal ethics
What are the 3 R’s ?
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?
Categories of Invasiveness
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
How often is the U of C visited by CCAC?
Every 3 years
They identify strengths and weaknesses - make recommendations for change
U of C must then submit a response
How often must animal use protocols be re-reviewed?
Every four years if they are still in-use
What committee reviews and approves all U of C animal care and use?
Animal Care Committee (ACC)
What might gene expression tell us about a gene?
- Where it functions
- What it function may be
- Its relationship with other genes
What does WISH stand for?
Whole-mount In Situ Hybridization
What can WISH tell you?
- Stains whole embryos
- A method to tell where in an embryo a given gene is transcribed (or expressed)
- It utilizes a complementary RNA probe
What kind of probe does WISH utilize?
Complimentary RNA probe
- It will hybridize to transcribed sequences (mRNA) in the cells of an embryo
How to make WISH probe?
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
What may 2 genes having similar expression domains imply?
They could be related to one another or work together! For example, Shh ligand and Patched protein receptor will be expressed in similar regions.
What is up with the uracil in making the WISH probe?
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
What happens in the WISH assay?
- The DIG-labelled RNA probe is added to fixed embryos (the probe will hybridize to the mRNA of the gene of interest)
- 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)
- 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)
What does fixing the embryos mean?
Killing the embryos and maintaining them at a particular stage in embryogenesis for studying
What and how is an enzyme used in the WISH Assay?
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.