Applications of genetic engineering 1 Flashcards
How can you find out what causes disease in tissues?
- Compare epigenetic changes of DNA (eg. methylation)
- Compare mRNA
- Compare proteins
- Compare post- translational modifications
What are the 2 steps of researching what causes a disease?
1) Target identification
- Microarrays
- Yeast- 2 - hybrid screens
2) Target validation
- Gene knockdown using siRNA
- Gene knockout using transgenic mice
What do RNA microarrays allow?
- Comparison of 1000s of TRANSCRIBED gene in 2 different tissues (heart vs liver) or 2 conditions of the same tissue (healthy vs diseased)
- Screening for differences
- Uses isolated mRNA
Which principle are RNA microarrays based upon?
The princle of hybridsation between nucleaic acid and probes
Why are RNA microarrays complicated?
Must have enough probes to represent ALL of the known genes
What is the process of RNA microarrays?
very long so write down step-by-step on paper
1) Have a chip with a grid of 45,000 fixed probe cells
- Probe cells contain MANY copies of 25bp ssDNA which match a particular gene
- Location in the genome of each 25bp sequence known
2) Label ssDNA from both samples with different fluroscent tags
3) Mix the control and experimental DNA EQUALLY
4) ssDNA from both samples (control and experiment) will hybridise with ssDNA in complementary probe cells - Only PERFECT (base-for-base) matches will hybridise
- Non-complimentary will wash off
5) Observe the colours of the probe cells
What does the colour of the chips in the RNA microarray show? (eg. if control is red and experimental is green)
- If grid appear RED: gene is downregulated in the experimental sample
- If grid appear GREEN: gene is upregulated in the experimental sample
- If grid appear YELLOW (the average colour of the probe cells) : there is no change in that DNA from the control to the experimental sample
How do you find out what regulates a certain protein?
Look for proteins which PHYSICALLY interact with it
What is a yeast-2-hybrid screen?
- Start with a known protein of interest (POI)
- Fish for proteins with interact with the POI
What might happen if a yeast-2-hybrid screen is performed in a prokaryote?
May not get the correct results as yeast is a eukaryote
What are the 2 domains of a regulatory protein and what do they do?
1) Binding domain (BD)
- Binds to gene in the promoter to be regulated
2) Activation domain (AD)
- Recruits other transcription factors
What is the process of a yeast-2-hybrid screen?
very long so write down step-by-step on paper
1) Seperate BD and AD of the regulatory protein
2) Create a yeast expression plasmid which contains a gene that codes the the BD of the regulatory protein
3) Fuse this BD to a known protein (known as the BAIT)
4) Create a second expression plasmid that contains a gene for the AD domain of the regulatory protein and fuse this with various different protein coding sequences from a cDNA libary
5) This will create a AD plasmid library
- With all the possible proteins bound to a single AD
6) Yeast strain cotransformed so that each transformant has the BD plasmid and one of the AD libary plasmids
7) BD with bait sequence will bind to the reporter gene (gene to be regulated)
8) One of the proteins attached to the AD region will bind to the bait sequence (known protein)
9) Reporter gene regulated when AD bound - used to identify which yeast cells had the correct combination of interactive protein
(will be expressed in the cell, eg. antibiotic R)
10) Isolate plasmid for the succesful yeast and sequence the plasmid to identify the gene that codes for the interacting protein
How can you find out the function of a certain protein? Or test its
Observe the effect of its ABSENCE
What is siRNA and what is it used for?
- Small interfering RNA
- Derived from double stranded RNA
- Used to reduce expression of a particular gene (gene knockdown, not complete knockout)
What is the interference pathway?
- Regulated gene expression using endogeneously expressed siRNA (called microRNA)
- Can be used by synthetic siRNA
Whatt is RISC?
RNA induced silencing complex
What is the process of gene knockdown using siRNA?
very long so write down step-by-step on paper
1) Double stranded precursor of siRNA bind to dicer (an endonuclease protein)
2) Dicer cleaves this into 21-22bp fragments
3) Short dsRNA fragments bind to argonaute protein (in RISC)
4) One of the strands remains bound to argonaute and becomes the ‘guide strand’
5) The other strand dissociates and is degraded
6) Argonaute-RNA with a ssRNA activates the RISC
7) siRNA bound to the RISC directs the complex to specific mRNAs
8) siRNA and mRNA has perfect complementary base pairing
9) Argonuate catalyses the cleavage of the mRNA strand, preventing it from being translated
What are the positives and negatives of gene kncokout?
+ Prefered expression is zero
- More money
- More time
Describe the pronuclear injection method of gene knockout
1) Forgein DNA (transgene) introduced into pro-nucleus of fertalised ova (1 cell stage)
2) Forgein DNA integrated at random sites in the genome (random integration)
3) Expression of transgene can be modulated by insertion site (ie. variable expression patterns)
- Each transgenic line is unique
What can pronuclear injection be used for?
To generate genetically modified crops or animals
Describe the gene targeting method of gene knockout
- Foreign DNA introduced into cultured mouse stem cells
- Used to generate knockins or knockouts at SPECIFIC sites
- Selection of correctly modified/ targeted mouse ES stem cells by southern blotting or PCR
Process gene knockout in transgenic mice???????
1) Targeting vector introduced into
