Lecture 14 - genetic manipulation of animals

Question 1

3 reasons why trasngenic animals are effective bioreactors for pharmeceutical proteins

Answer 1

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

Question 2

What is a good place to express proteins in mammals and why

Answer 2

Milk
ease of production (already make lots)
mammary gland specific promoters available so protein not expressed in entire animal
high level production possible

Question 3

Name some other sources of protein expression in animals

Answer 3

blood, urine, saliva, semen, eggs

Question 4

List the disadvantages of useing milk as location to express desired proteins

Answer 4

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

Question 5

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

Answer 5

Express in sheep’s milk instead

costs $40,000 a year and 60mg/kg required once a week per patient

Question 6

How was AAT expressed successfully in good old tracy the sheep

Answer 6

Ovine beta lactoglobulin gene promotor fused to human AAT gene.
Sheep created by pronucelar injection
40g/l conc in milk was the result

Question 7

What is Antithrombin III (AT III)

Answer 7

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

Question 8

What is ATT used for and what is the world wide requirement

Answer 8

Used for patients with antithrombin deficiency undergoing high risk surgical procedures
100kg

Question 9

Compare mammalian cell culture as a way of expressing AAT with transgentic goat

Answer 9

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

Question 10

What is xenotransplantation

Answer 10

the transplantation of living cells, tissues, and or organs between one species and another

Question 11

Potential benefits of xenotransplanation

Answer 11

animals could potentially provide an unlimited supply of cells tissues and organs for transplanting into humans
Human organs are in short supply

Question 12

Why are pigs favoured as organ donors over e.g. baboons

Answer 12

fewer viruses
,more offspring
fewer moral objections
pig heart valves already a success

Question 13

What are the essential prerequisites for successful xenotransplantation

Answer 13

immunological barriers overcome
transmission of pathogens prevented
anatomy and physiology compatible

Question 14

What is the main immunological issue facing xenotransplantation

Answer 14

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

Question 15

What is a potential solution to HAR

Answer 15

create transgenic pigs with the carbohydrate knocked out

Question 16

What are the agricultural uses of GM animals

Answer 16

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

Question 17

What is the official name of mad cow disease

Answer 17

Bovine Spongiform Encephalopathy

Question 18

What is BSE caused by

Answer 18

propogation of misfolded forms of normal cellular prion protein PrPc

Question 19

How have cows been genetically modified to get resistance to mad cow disease

Answer 19

knocked out PRPN gene in fibroblasts, produced clones from these cells

Question 20

How is carcass composition in pigs altered, and how genetically?

Answer 20

more unsaturated linoleic acid in meat

FAD2 demnaturase from spinach introduced via the microinjection of pronuclear embryos

Question 21

Is the introduction of FAD2 to pigs efficient? If not state why

Answer 21

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.

Question 22

How might one want to alter milk composition and why. O,h and how

Answer 22

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)

Question 23

What do enviropigs genetically contain and how was it introduced

Answer 23

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

Question 24

What is the problem with pigs

Answer 24

Can’t digest phylate, so lots in their manure

leads to eutrophication

Question 25

What changes have been made to pigs to make them greener

Answer 25

phytase enzyme in salivary gland so less phosphorous products in manure