week 7 Flashcards
(29 cards)
what is glycosylation
- glycosylation - 50% of eukaryotic proteins
- carbohydrates are added to proteins as co- and post-translational modifications. N-linked and O-linked
- glycosylation can affect protein function and protein-protein interactions, protein uptake, secretion, half-life, solubility and antigenicity
- N-linked glycosylation is common in eukaryotes but not in prokaryotes. bacteria are not capable of proper glycosylation of recombinant mammalian proteins -> may need to use other systems
example of GM organisms
- bacteria
- mammalian cells
- yeasts
- algae
- crop plants
- livestock
examples of delivery methods
- electroporation
- chemical treatment
- biolistics
- microinjection
other expression considerations
random insertion into genome:
- unpredictable levels of expression
- insertional mutagenesis - The insertion can disrupt important host genes, potentially causing malfunction or diseases (like cancer if tumor suppressors are hit).
gene does not normally confer a selective advantage to host (usually opposite):
- point mutations
- rearrangements
- DNA methylation
describe promoters
- Promoters are DNA sequences that control the start of gene transcription—they’re like “on switches” for genes.
- It provides a binding site for RNA polymerase
- It also contains binding sites for transcription factors, proteins that help turn genes on or off.
- promoters are usually organism-specific (human does not work in bacteria or yeast)
- often need spatial or temporal control:
- inducible promoters (time)
- tissue-specific promoters (place)
describe plant production
- all cells are totipotent - can de-differentiate to recreate a new plant
- possible to alter just one cell and recreate an altered plant
- if we can deliver DNA to plant cells, we will generate a whole GM plant
describe the biolistic method (gene gun)
directly ‘fire’ DNA into cells
- originally a CO2-powered Crosman air pistol
- now helium-propelled tungsten/gold particles
describe agrobacterium biology
- uses horizontal gene transfer (T-plasmid) to cause tumours in plants - transfers ‘T-DNA’ to cause dysregulated growth:
- auxins - uncontrolled growth
- opines for selective growth advantage
- integrates into plant’s genome
describe the agrobacterium method
- agrobacterium contains a DNA sequence that allows integration into plant genome
- can be altered to include specific genes - antibiotic resistance, increased growth, plant promoters
describe transgenic rapeseed
- herbicide-resistant rapeseed (also soybean, cotton and maize) - resistant to glyphosate (roundup)
- more pesticides can be used
- gene transfer to weeds = resistant weeds?
describe GM corn
- corn is genetically modified to express BT proteins. these are delta-endotoxins which are pore-forming (natural insecticide)
- less insecticide needed
- meant to be inactive on mammals, possibly off-target effects… butterflies, bees
describe GM rice
- Vitamin A deficiency kills 700,000 children under 5 a year due to poor diet
- Golden Rice 2:
rice engineered to produce 23x beta-carotene (vitamin A) in the grain
2 years of ‘successful’ field testing
describe GM tomatoes
senescence (biological aging):
- accompanied by cell wall degradation
- accompanied by increase in n-glycoproteins
increased shelf life:
- inhibition of N-glycoprotein modifying enzymes, a-Man and B-Hex
- longer lasting tomatoes, bananas, strawberries
describe GM animals
- food production/quality (increased yield, parasite and pest resistance)
- therapeutic applications (growth hormone, organ transplant)
- humanised products (breast milk)
- research: knockout/knock-in models
- bacteria are clonal and plant cells are totipotent
- most cells in adult animals have limited potency - alter developing embryo
- we want altered germline - can breed/cross offspring
describe the development stage of genetic mutation
- cells lose potential as development progress
- zygote is the first stage in development
- pronuclear phase:
- first sign of fertilisation
- both nuclear membranes dissolve
- haploid genomes combine
- permissive to foreign DNA incorporation
describe pronuclear microinjection
- glass micropipette with 0.5um diameter
- 200x magnification
- negative pressure micromanipulators
describe how transgenic animals are made
- stem cells isolated from blastocyst stage (very early embryo) - these cells are pluripotent
- A targeting vector (engineered DNA) is introduced into the cells, containing the desired gene modification and a resistance marker. introduced DNA recombines in place of original gene in rare cases
- select for successful recombination via resistance marker e.g. antibiotics (heterozygous)
- transfer into an anucleated oocyte (egg cell)
- transfer to surrogate mouse
- breed to create homozygous mutant
describe homologous recombination
- gene targeting constructs can be used to:
- add selection/reporter gene - important as correct incorporation is a rare event
- Modify the Genome in Targeted Ways - disrupt/alter/insert gene e.g. frameshift, deletions, insertions, gene additions, reporters etc
describe transgenic animals models
Cre/Lox recombination system:
Cre recombinase recombines Lox sequences
- Generate mouse line with gene of interest altered by adding flanking LoxP sites (Floxed mouse)
- Breed with mouse that expresses Cre recombinase (in next generation the gene will be gone -floxed out!-)
- Can provide deletions, insertions, translocations and inversions
- Cre recombinase can be expressed in particular tissues to provide selective knockouts. Incredibly powerful system
describe enviropig
- pig that produces more environmentally-friendly faeces - phytase production in saliva helps digest phytate (indigestible part of phosphorus feed) - mouse secretory promoter, e.coli phytase gene
- less phosphorous run-off
- reduces algae overgrowth and subsequent anoxia of rivers/lakes (eutrophication)
describe humanised cow’s milk
- humanised milk production for breast milk replacement
- pronuclear injection of human genes that improve immune function:
- lysozyme
- lactroferrin
- 20-30% increased fat content
- tastes ‘stronger’
describe viral vectors
common features:
- multiple cloning site
- selection gene(s)
- reporter gene(s)
- promoters - ubiquitous, tissue specific
- enhancers to increase expression
make replication incompetent:
- deletion of essential viral components
- use of separate vectors for packaging and viral production
make host specific:
- production of viruses/viral vectors with foreign viral envelope proteins. generates a pseudotyped virus particle
describe promoters
- sometimes want expression in any cell - reporters/markers, ubiquitous promoters, preferences for different cell types
- tissue specific promoters - relies on expression of tissue-specific transcription factors, aimed to be unique to particular tissue
describe adenoviral vectors
- non-integrating DNA virus - not integrated into genome and not replicated during cell division
- hijacks cellular transcription/ translation to make viral proteins
- hgihly immunogenic as inolved in human respiratory diseases
- usually used for in vitro protein production experiments
