Lab 6 - gene expression in zebrafish

Question 1

What are some advantages of IHC?

Answer 1

• look directly for protein of interest
• idenitfy: subcellular location, if its secreted and where
• proteins last longer than mRNA

Question 2

What are the general step of IHC?

Answer 2

1. fix tissue
2. permeabilze cells
3. add primary Ab
4. wash
5. add secondary Ab
6. wash
7. visualize

Question 3

Why do we fix tissue in IHC? What is a consequence of this?

Answer 3

freezes the molecules into place
- kills the organism

Question 4

How do we permeabalize cells in IHC, why do we do this?

Answer 4

how = poke holes into membrane using a detergent
why = allows Abs to enter cells

Question 5

How are primary Abs generated, what are some characteristics of them?

Answer 5

generation: add YFP into mouse –> immune response –> mouse anti-YFP IgG

characterisitcs:
- V region binds to protein
- C region specific to species
- Can have multiple primaries bind to the same protein

Question 6

What are some characterisitcs of secondary Abs?

Answer 6

• Has fluorescent tag
• Against the C region of the primary Ab
• Has to be made in another animal

Question 7

Why do we do wash steps in IHC and in situ hybridization?

Answer 7

removes non specific binding molecules

Question 8

Why isn’t the tag on primary Abs in IHC?

Answer 8

• increases amplification
• primary Abs are difficult to make and tend to be inefficient
• Gives more flexibility (Can be used against any mouse primary Abs)

Question 9

What are some drawbacks to IHC?

Answer 9

• Can’t watch in real time
• Animals where Ab is made have to be sacrificed
• Because proteins have a greater half life, can’t tell if gene expression has changed

Question 10

What is a control treatment for IHC?

Answer 10

add secondary and not primary Abs to provide a background

Question 11

What proteins are involved in the RA pathway that we can make transgenes for?

Answer 11

RAR and RARE

Question 12

What is the advantage of in situ hybridization?

Answer 12

detect gene expression

Question 13

What are the steps for in situ hybridization?

Answer 13

1. Fix the embryos
2. Permeabilize the embryos
3. Incubate with DIG labeled RNA probe (Probe is complemetatory to mRNA of interest)
4. Wash to remove non specific binding
5. Incubate with Ab conjugated to alkaline phosphotase that recognizes DIG
6. Wash to remove nonspecific binding
7. Detection of gene expression by adding a colourless compound that becomes coloured when it’s phosphate is removed

Question 14

Where is Krox20 expressed?

Answer 14

rhomdameres 3 and 5

Question 15

How is RA gradient in the brain generated?

Answer 15

• of RA synthesizing enzymes in spinal cord
• Presence of RA metabolizing enzymes in anterior hindbrain (Also in tail!)