Week 6 Flashcards
Northern Blot/Hybridisation basics
ssRNA, electrophoresis, blotting membrane + probe insertion leads to hybridisation
What is RT-PCR?
RNA population converted to cDNA by reverse transcription. using reverse transcriptase.
What is the advantage of in situ hybridisation?
No assumption about region of gene
What are the 2 disadvantages of in situ hybridisation?
Difficult if low expression and is lengthy
What is in situ hybridisation? basics
DNA probe put in tissue slides. Blocked (prevent non-specific interaction), incubated and then visualised.
What is microarray and mRNA sequencing?
Hybridise oligonucleotides with cDNA sample and know if the cDNA is expressed in specific tissue.
What is microarray and mRNA sequencing used for? (3 points)
- Quantitative data
- Location and time of expression of gene
- Address change in gene expression.
How does capture microdissection work? basics
Tissue+paraffin on slide. Laser caps around cells and bind to transfer film. Individual cells can then make cDNA.
Promoter enhancer properties? (2)
- Greatly increase transcription rate from promoters on same DNA molecule
- Function in either orientation
What is the role of the binary system gene expression/ GAL4/UAS system?
Used to express transgenes in specific cells.
How does a binary system gene expression system works using GAL4/UAS as an example? basics
GAL4: An activator combined with promoter sequence specific to a tissue.
UAS: fused to target gene.
Cross: target gene expressed only in specific tissue. Can find where and when gene is expressed.
Role of activator proteins?
Bind to enhancer sequences in DNA and recruit RNA polymerase to bind to the promoter sequence, activating transcription.
What are the benefits of the GAL4/UAS system? basics
Easier to cross than build new constructs when multiple targets are to be analysed. In vivo expression + lethal gene study.
What is ideal in a genetic switch? (6)
- Low or no expression when off.
- High expression when on
- Fast response
- Reversible
- No toxicity or interference in general cell function
How does the tet-off system works? basics
No tetracycline: tetA binds to tet repressor, preventing it from binding to tet-o. expression.
How does the tet-on system work? basics
Tetracycline: conformational change in tetA, binds to tet-O, expression.
What are the uses of transgenic animals? (3)
- Medicine (e.g. model organisms)
- Agriculture (disease resistance, livestock change, reduce waste)
- Nutritional supplements and pharmaceuticals (insulin in milk, omega-3 fatty acids in transgenic pigs)
How does microinjection work?
Micro-inject sperm nucleus, fusion and implantation into female.
Problems with microinjection basics
Transgene can be inserted anywhere or multiple times in the genome, timing can make it a chimera.
How does embryonic stem cell transfer works?
Inner cell mass of blastocyst transformed with DNA and induced back into blastocyst. Result: chimera, must cross with wild-type.
What is illegitimate recombination?
Foreign DNA has an imperfect sequence recognition. Will cleave a gene halting its function, which may be a problem.
What is homologous recombination?
Perfect sequence recombination, when target gene is flanked by regiosn of homology with host DNA. Replaces native gene with target gene.
Positive selection marker
Selects for that which HAS the gene. Marker between homology 1 and homology 2.
Negative selection markers
Counter-select gene, so that it will only be inserted when separate form other markers (illegitimate) and will cause cell death. If legitimate, then negative marker not present and survival occurs.
