Lecture 14 - genetic manipulation of animals Flashcards
3 reasons why trasngenic animals are effective bioreactors for pharmeceutical proteins
many proteins require complex modifications only possible in eukaryotes
proteins can be produced in large amounts - >2g per litre
increased safety compared with extraction from human tissues
What is a good place to express proteins in mammals and why
Milk
ease of production (already make lots)
mammary gland specific promoters available so protein not expressed in entire animal
high level production possible
Name some other sources of protein expression in animals
blood, urine, saliva, semen, eggs
List the disadvantages of useing milk as location to express desired proteins
purification can be hard:
-Milk is biocolloid ie. a suspension of micelles which are made up of casein and calcium (precipitate to remove casein)
-Fat must centrifuged out
The gene has the potential to negatively affect milk supply
Covelant modifications may not be conserved between different tissue types
What would be an alternative to human alpha 1-antitrypsin being extracted fromnn blood plasma, why would we look for an alternative and what is it for
Express in sheep’s milk instead
costs $40,000 a year and 60mg/kg required once a week per patient
How was AAT expressed successfully in good old tracy the sheep
Ovine beta lactoglobulin gene promotor fused to human AAT gene.
Sheep created by pronucelar injection
40g/l conc in milk was the result
What is Antithrombin III (AT III)
plasma protein with anticoagullant and anti inflammatory properties.
A glycoprotein usually synthesised in the liver
The first transgenically produced protein to be approved for human therapeutic use
What is ATT used for and what is the world wide requirement
Used for patients with antithrombin deficiency undergoing high risk surgical procedures
100kg
Compare mammalian cell culture as a way of expressing AAT with transgentic goat
MCC can produce 100mg/l
has 50% recovery
would need 2,000,000l of mammalian culture to fulfil world wide need
Transgenic goat producing AAT in milk produced by pronuclear injection, pro nuclear transfer from transgenic fetal fibroblasts expressing AAT
2g/l goats milk achieved , only need 150 goats
What is xenotransplantation
the transplantation of living cells, tissues, and or organs between one species and another
Potential benefits of xenotransplanation
animals could potentially provide an unlimited supply of cells tissues and organs for transplanting into humans
Human organs are in short supply
Why are pigs favoured as organ donors over e.g. baboons
fewer viruses
,more offspring
fewer moral objections
pig heart valves already a success
What are the essential prerequisites for successful xenotransplantation
immunological barriers overcome
transmission of pathogens prevented
anatomy and physiology compatible
What is the main immunological issue facing xenotransplantation
Hyper Acute Rejection
immediate and catastrophic because the recipient already has antibodies against donor tissue, specifically against the small carbohydrate Gal alpha (1,3)-gal
What is a potential solution to HAR
create transgenic pigs with the carbohydrate knocked out
What are the agricultural uses of GM animals
Disease resistance - introduce genes that confer animal Resistance to parasites and pathogen
Carcass composition - make leaner meat
Lactation - modify milk components or make hypoallergenic
Wool production - incr yield
Environmentally friendly farm animals - address intense farming issues
What is the official name of mad cow disease
Bovine Spongiform Encephalopathy
What is BSE caused by
propogation of misfolded forms of normal cellular prion protein PrPc
How have cows been genetically modified to get resistance to mad cow disease
knocked out PRPN gene in fibroblasts, produced clones from these cells
How is carcass composition in pigs altered, and how genetically?
more unsaturated linoleic acid in meat
FAD2 demnaturase from spinach introduced via the microinjection of pronuclear embryos
Is the introduction of FAD2 to pigs efficient? If not state why
11 out of 16 recipient pigs (of injected embryos) became pregnant, 70 piglets
Of these only 15% injected embryos developed to full term
Only 6 of the piglets were transgenic-2 still born, 1 died day after birth, 2 males 1 female survived
FAD2 mRNA detected in 1 male and 1 female founder pig
male mated with 2 WT females, 32 piglets produced, 21 alive. 8 carried transgene
female mated with WT male, 12 piglets, 3 live.
How might one want to alter milk composition and why. O,h and how
Increase levels of human lysozyme
It is a:
Non specific immune factor
anti inflammitory ]
protects against gram positve bacteria
will confer disease resistance to infants if consumed
How: expressed in transgenic cloned cattle
Remove B-lactoglobulin because it’s a major milk allergen
How: use RNA interference to target BLG gene
Introduce RNAi construct in calf fibroblast perform somatic cell nuclear transfer (like dolly the sheep)
What do enviropigs genetically contain and how was it introduced
a transgene construct containing the murine (mouse) parotid secretory protein promoter gene sequnce and the Ecoli phytase gene.
Introduced via pro nuclear microinjection of fertilized embryos
What is the problem with pigs
Can’t digest phylate, so lots in their manure
leads to eutrophication
