Non-genetic gene analysis of function (L2)

Question 1

What is meant by non-genetic analysis of gene function?

Answer 1

Includes fining out about where a gene is isolated in the cell and organisms. This may give an idea to its function. E.g. if its solely in the eye it may be used for phototransduction. To do this, you can use immunohistochemistry, in situ hybridisation or both, to find out where the gene is being transcribed, but not translated. You can also visualise gene expression and protein localisation in living cells using things like GFP

Question 2

Explain how you make protein-specific antibodies.

Answer 2

First, you need to make a lot of the protein. Do this by inserting cDNA into a plasmid using reverse transcriptase. This makes an expression vector of the gene. Expression vectors use a bacteriophage promoter to drive high levels of RNA synthesis (they have an increased expression compared to a normal vector due to having more promoters). The promoters have to be inducible, otherwise, the bacteria would die rapidly (because all their energy goes to making that and not their own stuff) Chemicals or temp shifts induce the protein expression. After expression, they are lysed and centrifuged to make a crude extract. Expression plasmids often include an epitope to allow for easy purification. Once the protein has been purified you can inject animals (usually rabbits) with it over a long period of time. The rabbit’s immune system will start making antibodies that are complementary to the target protein. You can then use tagged antibodies that are complementary to the rabbit ones (have des or enzymes attached) to see where the protein is.

Question 3

What are the different tags commonly used on antibodies?

Answer 3

Some conjugates are dyes that are fluorescent, allowing us to detect the location using specific wavelengths of light. Sometimes, we use antibodies to examine the subcellular localisation of the protein, and other times we are looking at the expression pattern of the whole organism. Commonly used enzyme conjugates include Alkaline phosphatase (substrate turns blue) or horseradish peroxidase (substrate turns brown). Enzyme detection can enhance sensitivity. Often, we make a 2-antibody sandwich, this amplifies the signal because multiple secondary antibodies can bind to the primary one- so when there’s only a small amount of protein you can see it more clearly. The antibodies have to be made in different organisms otherwise they won’t recognise each other

Question 4

How would you use the antibodies to find proteins now you’ve produced them?

Answer 4

First, you chemically fix the animal tissue to stabilise it - formaldehyde is the most commonly used - it forms cross-links between all the proteins, so stops them moving in vitro. Then you incubate the tissue with the tagged antibody then after a time (enough to let them anneal) you wash off the excess. In a whole mount- you use the whole organism and in a section system, you cut the organism into section (microtome/cryosectioning). This enabled you to see in more detail where expression is, e.g. axon pathways and new axonal guidance protein expression.

Question 5

Explain how you would conduct an in situ hybridisation

Answer 5

Easier and quicker than immunohistochemical staining.
1. The purified vector containing cDNA (from a library) - synthesise the RNA antisense and incorporate epitope-tagged nucleotides into the cDNA
2. incubate the antisense strand with the embryo
3. The antisense strand hybridises with the endogenous mRNA
4. Wash off the excess.
5. add the substrate which will bind to the epitope and turn blue when it binds
You can use this to see the distribution of expression of certain mRNA like bicoid in a whole mount analysis of a Drosophila embryo (before cellularisation)

Question 6

Explain how you can use GFP tagging to analyse proteins.

Answer 6

GFP was first isolated from jellyfish. When you excite it with blue light (at 475nm) the electrons rise energy levels and then drop, when they drop they release energy in the form of green light. There are now dozens of new fluorescent proteins that have been cloned and characterised.
To generate a GFP transgenic line you
1. Clone the entire gene (from a genomic library) with all of the regulatory elements in a plasmid.
2. genetically engineer GFP onto the end of the last exon (gene fusion) or replace the gene (receptor construct)
3. integrate the GFP fusion gene back into the genome of the organism - this usually involves microinjecting a solution of the DNA into the one-cell zygote, DNA then randomly integrates into the genome (done in ES cells in mice)
You can use GFP transgenes for things like tracking microtubules during cell division or how proteins become localised after different divisions. Allows you to follow in vivo behaviour of cells.