How are genes of interest found?
Through cDNA libraries, genomic libraries or microarrays
How is gene function analysed?
Through forwards and reverse genetics
Can also be found through NON-GENETIC ANALYSIS
What 3 questions do non-genetic analysis of gene function answer?
1) Where is the protein in the cell/organism?
2) Where is the gene transcribed in the tissue/organism?
3) Follow gene expression and protein localisation in LIVING cells?
How can you visualise gene expression and protein localisation in LIVING cells?
Visualising gene expression and protein localisation in living cells
How can you see where protein is in a cell?
How can you see where genes are transcribed in a tissue/organism?
RNA in situ
What is the first stage of making a protein specific antibody made and how is this done?
First, need to make lots of PROTEIN:
1) Insert cDNA for gene of interest into an EXPRESSION vector, with an inducible bacteriophage promoter
2) Introduce this into the bacteria
3) Grow a large colony of bacteria
4) Turn on the promoter
5) Harvest bacteria in a centrifuge
6) Lyse the bacteria to make a crude extract
6) Purify the protein from the crude extract
What is important in an expression vector?
Use a bacteriophage promoter to drive high levels of expression (RNA synthesis) in bacteria
What is a bacteriophage?
A virus that infects bacteria and forces the to make lots of the bacteriophage protein
Why does the bacteriophage promoter in an expression plasmid have to be inducible?
To avoid the bacteria from dying quickly
How can the bacteriophage promoter in an expression plasmid be induced?
Using temperature increase or a chemical to drive protein expression
How is the protein purified out of the crude extract?
Using an epitope tagging system and antibody-affinity purification:
1) Fuse on a small peptide (epitope tag) in frame to one end of the cDNA of interest in the plasmic vector during cloning
2) Pour the crude extract (from lysing bacteria) into the column - column filled with small beads that have been coated with the antibody that binds to the epitope tag
3) Collect fractions from the column until no more protein is found
4) Rinse the column with low pH (pH 3)
5) Test the fractions for the proteins of interest
What are epitope tags?
Peptides for which antibodies are already available
What is the column packed with in antibody-affinity purification?
Small beads that have been coated with the antibody that binds to the epitope tag
What does rinsing the column with a low pH do?
Break the antibody-protein interaction and release the epitope tag and protein of interest
What is the process of making a protein specific antibody?
1) Grow bacterial culture and purify protein
2) Inject into rabbit over 3-month period - rabbits will have immune response to the protein
3) Purify specific antibody from serum
Where do antibodies bind?
To an epitope on a protein with high specificity
How are antibodies detected?
They are tagged with dyes or enzymes
What are antibodies used to examine?
- The sub-cellular localisation of the protein
- See where the protein is in the organism (what tissues)
What are the 2 commonly used enzyme conjugates used in antibody detection?
What colours does the substrate turn?
1) Alkaline phosphatase -turns blue
2) Horseradish peroxidase - turns brown
How can enzyme detection enhance sensitivity?
Interact with many substrate to amplify many small signals
Using a two antibody sandwich to further amplify the signal:
- Many secondary antibodies bind to one primary antibody
Which antibody carries the tag in an antibody sandwich?
What is the process of using antibodies?
1) Collect animal or tissue and chemically fix using formaldehyde
2) Incubate with primary tagged antibody
3) Wash off excess antibody
Why fix tissue/animal with formaldehyde?
Stabilise the structure by cross-linking the proteins together
Also prevents the tissue from rotting
What is whole mount analysis?
Using the entire organism
What is section analysis?
Thinly slice through the organism and look at the proteins in each slice
How is a 3D picture of protein distribution in an organism formed?
Using both whole mount and section analysis
What is RNA in situ analysis used for?
To find out where the GENE is expressed
What doesn’t RNA in situ give information about?
Where the protein is localised
Is using antibodies or RNA in situ easier?
RNA in situ
What is the process of RNA in situ?
Start with a purified vector containing cDNA of interest
1) Synthesise RNA ANTISENSE probe (from sense strand DNA) whilst incorporating epitope tagged nucleotides
2) Incubate the embryo with antisense probe - antisense strand with hybridise with the endogenous mRNA (sense strand)
3) Wash off excess probe
4) RNA detection using alkaline phosphatase (blue substate)
How is the distribution of bicoid mRNA different to the distibution of the bicoid protein?
mRNA tightly regulated to the anterior pole
Protein diffuses from the anterior
Where is GFP isolated from?
What wavelength and colour of light is GFP excited with?
What wavelength and colour does it emmit?
Which has the higher energy?
475nm (blue) - higher energy
Describe what happens in the GFP when blue light is shone on an atokm
- Atom absorbs the light
- Electron is temporarily shifted out of its normal orbit to a HIGHER energy state
- When electron drops back down to normal orbit - loses energy in the form of light and emits a longer wavelength
How is a GFP transgenic line made?
Start with a GENOMIC CLONE of the gene of interest (in a plasmid)
1) Genetically insert GFP into the plasmid using restriction enzymes
2) Integrate the DNA with GFP fusion into the genome of the organism
Why start with a genomic clone when making a GFP transgenic line?
Contains the regulatory elements
What 2 ways is GFP genetically inserted into a plasmid?
Using restriction enzymes:
1) GENE FUSION - fuse onto the end of the last exon
- TAGS the gene of interest with GFP
2) REPORTER CONSTRUCT - replace the gene of interest
- GFP is transcriptionally regulated
Describe the process of the gene fusion technique of genetically inserting GFP into a plasmid
- Delete the normal stop codon of the gene of interest
- Fuse GFP onto the gene
- When transcribed/translater GFP is fused to the C-terminus of the protein
- Introduce a new stop codon
Describe the process of the reporter construct technique of genetically inserting GFP into a plasmid
- Replace the gene of interest with GFP (Insert GFP at the start site)
- GFP is then controlled by the regulatory sequences of the gene of interest
What does gene fusion show?
How is this different to reporter construct?
GF - Shows the subcellular localisation of the protein
RC - Doesn’t show subcellular localisation of the protein but shows where the GENE is expressed (no protein is made as the gene is removed)
How is the DNA with GFP fused inserted back into the genome of the organism?
What about in mice?
Microinjecting a solution of DNA into the one-cell zygote
In mice - introduce DNA into ES cells and introduce intot the embryo
How does the DNA integrate into the genome?
What is the integrated gene called?
What are 3 uses of GFP transgenic lines?
1) To FOLLOW the expression of a gene in the whole animal
2) To FOLLOW subcellular localisation of a protein
3) To FOLLOW the behaviours of cells in vitro (good for mobile cells)
Does GFP alter the activity of a protein?
What technique is used to mark cells to distinguish them from their neighbours?
When can subcellular localisation of a protein of change?
When signalling occurs - certain proteins (eg. transcription factors) are activated
Why is GFP especially useful in small organisms?
Can follow the expression of a gene throughout the whole lifetime of the organism, under a microscope