Quiz 3

Question 1

What are the steps to preparing a transgene?

Answer 1

1. In a plasmid add a foreign gene
   from another organism
2. Add the plasmid to a plant cell (it is now transformed)
3. Culture the cells on antibiotic containing plates
4. Grow the plantlet
5. You now have a NEW transgenic plant

Question 2

Why does every cell in the transformed plant express the fluorescent protein?

Answer 2

Because the foreign gene is integrated into the genome

Question 3

What does plant cell plasticity allow?

Answer 3

Regeneration of a whole plant form a single cell differentiation of the cell and then redifferentiation

Question 4

Can seed sets also be transformed?

Answer 4

Yes

Question 5

What are the benefits of arbidopsis cell lines?

Answer 5

• Quick to grow
• Diploid -> easy to engineer just knockout once
• Related to several food crops
• Self-pollinate
• Large number of seeds
• Small genome

Question 6

What is the difference between the onion epidermal cell expression and arabidopsis?

Answer 6

The plasmid is integrated into the genome for arabidopsis

Question 7

What can you do to ensure there is little background in imaging arabidopsis?

Answer 7

Have the focal plane on the epidermal layer not below too many layers of tissue

Question 8

What are the drawbacks of using epifluorescence on intact plant tissue?

Answer 8

Autofluorescence

• Photosynthetic cells (mesophyll cells) have a high level of autofluorescence from chlorophyll
• The cell wall and fluroescent chemicals add to autofluorescence

Question 9

What does immuno mean?

Answer 9

Antibodies

Question 10

What is the antibody conjugated to?

Answer 10

A fluorescent protein to label a specific endogenous protein

Question 11

What is the process for immunofluorescence?

Answer 11

1. Fix the tissue with formaldehyde or glutaraldehyde
2. Conjugate the antibody
3. Probe with the antibody

Question 12

Where should primary and secondary antibodies be made?

Answer 12

In different animal species

Question 13

What is direct immunofluorescence?

Answer 13

• When the primary antibody is conjugated with the fluorescent molecule
• There is less artifactual signal
• Good for high abundance proteins

Question 14

What is indirect immunofluorescence?

Answer 14

• When the secondary antibody is conjugated with the fluorescent molecule
• Multiple secondary antibodies can interact with the primary antibody = signal amplification

Question 15

What is callose made of?

Answer 15

B-glucan

Question 16

What is callose to the plasmodesmata?

Answer 16

The proteins on the plasmodesmata

Question 17

What is the benefit of using a secondary antibody with a fluorescent tag?

Answer 17

• Amplify the signal
• Useful for targets with low abundance