L16 - Non-genetic Analysis of Gene Function

Question 1

What do you use to tell where a protein is in an organism?

Answer 1

Antibodies

Question 2

What do you use to tell where a gene is transcribed in an organism?

Answer 2

RNA in situ

Question 3

What do you use to visualise gene expression and protein localisation in living cells?

Answer 3

GFP

Question 4

How to make a protein specific antibody overview

Answer 4

Use protein to immunize animal (rabbit)

Generates immune response - antibodies

Question 5

Making a protein specific antibody full method

Answer 5

1. Need to make a lot of the protein
2. Expression vectors use a bacteriophage promoter to drive high levels of RNA synthesis
   a. Promoters have to be inducible (can turn them off and on) otherwise bacteria die rapidly
3. Chemicals or temperature shifts are used to induce protein expression
4. Bacteria harvested in a centrifuge and lysed to make a crude extract
5. Expression plasmids often include an epitope tagging system
   a. Allows for rapid and efficient purification of protein
6. Tag is fused in-frame to the cDNA during the cloning – helps purify the protein

Question 6

What are epitope tags?

Answer 6

Peptides for which antibodies are already available

Antibodies bind to small regions of proteins with very high specificity - epitope

Question 7

Antibody-affinity purification method

Answer 7

1. Bacterial cells are lysed and the resulting solution (crude extract) is poured onto a column
2. Column contains small beads coated with antibody - recognise the epitope tag which contains protein of interest
3. Tagged antibodies have dyes or enzymes attached so we can determine their location
4. Protein eluted for column using a pH 3 buffer

Question 8

What are the 4 detection methods?

Answer 8

Fluorescent dyes
Antibodies
Enzyme conjugates
Two antibody sandwhich

Question 9

Detection - fluorescent dyes

Answer 9

Allow us to detect location using specific wavelenghs of light

Question 10

Detection - antibodies

Answer 10

Examine the sub-cellular localization of the protein

Testing where the protein is in the organism

Question 11

Detection - enzyme conjugates

Answer 11

Alkaline phosphatase - substrate turns blue
Horseradish peroxidase - substrate turns brown
Enzyme detection can enhance sensitivity

Question 12

Detection - two antibody sandwhich

Answer 12

Amplifies the signal
Many 2º antibodies bind to each 1º antibody
1º antibody made in rabbit
2º antibody made in mouse - binds rabbit antibodies and carries the tag

Question 13

How to use antibodies method

Answer 13

1. Chemically fix (formaldehyde) animal or tissue to stabilize the structure (whole structure or slice)
2. Incubate with tagged antibody
3. Wash of excess antibody
4. Whole or slice mount

Question 14

RNA in situ analysis method

Answer 14

1. Purified vector containing the cDNA of interest (template)
2. Synthesise RNA antisense probe - incorporate epitope tagged nucleotides
3. Incubate embryo with antisense probe
4. Antisense probe hybridises with the endogenous mRNA
5. Wash off excess probe
6. RNA detection systems use a blue substrate

Question 15

Bicoid in situ vs antibody

Answer 15

Whole mount analysis of Drosophila embryo

mRNA highly concentrated at anterior end

Question 16

GFP excitation

Answer 16

475 nM

Question 17

GFP emission

Answer 17

510 nM

Question 18

Generating a GFP transgenic line – transgenesis method

Answer 18

1. Clone the entire gene into a plasmid
2. Genetically engineer GFP onto the end of the last exon or replace the gene (reporter construct)
3. Integrate the GFP fusion gene back into organism’s genome
   o Involves microinjecting a solution of the DNA into the one-cell zygote
   o DNA then randomly integrates into genome
4. Integrated gene - transgene

Question 19

What are the 3 uses of GFP transgenic lines?

Answer 19

To follow expression of a gene in the animal
To follow subcellular localisation of a protein
To follow the behaviour of cells in vivo

Question 20

GFP transgenic lines - follow expression of gene in animal

Answer 20

To see which tissues gene is expressed in

Question 21

GFP transgenic lines - follow subcellular localisation of a protein

Answer 21

Some transcription factors become nuclear upon activation
This happens after the cell receives a signal - use a GFP fusion
GFP fusion does not usually alter the activity of the protein

Question 22

GFP transgenic lines - follow the behaviour of cells in vivo

Answer 22

GFP line used to mark specific cells so they are distinguishable - use a GFP reporter construct