lecture 11 transgenic animals

Transgenic Transgenic AnimalsAnimals

Lum Mok Sam

RT 2014 Agriculture RT 2014 Agriculture BiotechnologyBiotechnology

Transgenic animalsTransgenic animals

v Mice

v Cows

v Pigs

v Sheeps

v Goats

v Birds

v Insects


Transgenic MiceTransgenic Micev produced by the transfer of embryonic stem cells

containing a gene of interest

v Used as a MODEL in the study of gene function for human diseases, e.g.

x Arthritis

x Hypertension

x Alzheimer’s disease

x Coronary heart disease

x Certain cancers

x Various neurodegenerative disorders


v If mice lack a particular human disease counterpart, they often can be genetically engineered, by knock-out (inactivation) technology or by replacement of the normal gene with a mutated counterpart, to acquire itx e.g. cystic fibrosis, β-thalassaemia, atherosclerosis,

retinoblastoma, & Duchenne muscular dystrophyx Transgenes introduced into the mouse or other

animals (e.g. cows & goats) are expressed & their products (e.g. interleukin-2, α1-antitrypsin, clotting factor IX) secreted in the milk for isolation

v The gene construct must includex mammary – specific promoters

ye.g. a promoter from the -casein genex Appropriate enhancers


Knock-out mouseKnock-out mouse


Cystic fibrosis transmembrane Cystic fibrosis transmembrane regulator (CFTR)regulator (CFTR)

v Act as a chloride channel allowing chloride ions to move in & out of cells

v CFTR mutated: the channel disrupteddisruption of ion flow

mucus to accumulate in organs

Mucus attract bacteria

Bacteria die

Release their DNA

Further inhibiting normal organ function


v CFTR protein secreted in milk can be used to study protein function effective therapies can be developed

Secretion into milk facilitates isolationSecretion into milk facilitates isolationv The mammary glands produce fat globules in milk

that are encapsulated by the plasma membranev Heterologous transmembrane protein could be

associated with the plasma membranes of the globule & isolated readily from milk during lactation


Transgenic cattleTransgenic cattle

v Produced by microinjection of eggs, BUTx Inefficientx Costlyx Time consuming (≈2 years)

v Future: x grow fasterx require less feedx produce more milkx leaner


v Genetic modificationsx Change the composition of milk products

yIncrease the protein -casein (a phosphoprotein in milk) in cows by over expressing the -casein gene in animals

Production of more from milkyRemove lactose from milk

x Increased resistance of animals to bacterial, viral & other pathogenic diseasesyMastitis

oA bacterial infection of the mammary glandsoCommon affliction of cows


Recombinant bovine Recombinant bovine somatotropin (rBST)somatotropin (rBST)

v Use in cow to milk productionv Somatotropin

x occur naturally in the milk & meat of certain animals

v BSTx A protein hormone produced in the cow’s pituitary

glandx Essential for milk productionx Stimulates increased milk productionx Facilitates the efficient conversion of feed into

milk rather than body fat


v rBST

x Produced in E. coli after transfer of the bst gene

x Not toxic to humans

x Cows injected with rBST do not have elevated levels of the protein in their bodies

x Remain controversial

v FDA

x Has approved rBST as an animal drug

x Has deemed products from cows treated with rBST safe for human consumption


Sheep & GoatsSheep & Goats

v Bioreactor to produce important human proteins/ pharmaceuticals


PigsPigs

v Recombinant porcine somatotropin

x Increase animal’s growth rate

x Feed efficiency

x Decreases fat deposition


Transgenic birdsTransgenic birdsv Produced by the transfer of transformed blastoderm

cells into the subgerminal space of the embryo

isolate blastoderm cells

transformation

blastoderm irradiated

yolk

blastoderm

yolk

subgerminal space

Inject transfected blastoderm cells into subgerminal space

Gene to be transferred


v Production of healthier birds used for food

v Chickens, ducks, geese & small birds – resistant to viral & bacterial diseases

v Improved poultry products

x Decreasing the fat content of meat

x Decreasing the cholesterol in chicken eggs

v Chicken/ duck eggs – serve as factories for producing valuable proteins


InsectsInsects

v Production of insecticide-resistant insect predatorsx Ladybugsx Honey beesx Preying mantisx Many species of antsx Arachnids (spiders)

v Disadvantages: potentially allow increased use of chemical pesticides

v A fruit fly gene for resistance to cyclodiene insecticides (e.g. Dieldrin)


Application of animal Application of animal biotechnologybiotechnology

v Medical & therapeutic purposes

v Artificial insemination & germ cell storage

v Ectogenesis

v Amniocentesis

v Transplantation

v Cloning technique

v Gene therapy


Medical & therapeutic Medical & therapeutic purposespurposes

v Recombinant protein productionSynthesizing & secreting recombinant protein by transgenic animals for the production of valuable pharmaceutical proteins

v The most advantages production system: The mammary gland-target transgenic animal bioreactorx A large amount of recombinant protein can be

obtainedx The running cost for rearing animals is much lower

than that for maintaining cell culture facilitiesx Down-stream processing – relatively easy to purify

the protein from the milk


v Produced recombinant proteins may cause little disturbance with the health of host animals (because recombinant proteins are immediately secreted in the lumen of mammary gland outside of the body)

v Production of pharmaceutical proteins by transgenic system

Genes encoding pharmaceutical proteins

+ milk gene promoters

Germ lines

Expressed specifically in the mammary gland


XenotransplantationXenotransplantationv Alternative organ sources from different animal species

as human donor organsv A suitable donor animal species: PIGS

x Their organs are almost the same size as those of humans with anatomical & physiological similarities (Cooper et. al., 1998)

x Produce many youngx Their diseases & rearing technology are well studied

v BUT, organs from pigs CANNOT easily transplanted due to severe & prompt rejection (Calne, 1970)x Immune response – hyperacute rejection (Sachs &

Bach, 1990)


v hyperacute rejection

x Not suppressed by the immune – suppressive agents

x Resolve by the use of inhibitory proteins


Artificial insemination & germ Artificial insemination & germ cell storagecell storage

v Semen

x diluted & stored at 40ºC

x Usable for 4 – 5 days

v After which period, unused semen will have to discarded

v Storage temperatures were gradually lowered

v At very low temperature, the sperm heads were disrupted

v Alteration: addition of glycerol

Prevented damage to the sperm head


v Deep-freezing techniquesx Used of liquid nitrogenx Ability to store spermatozoa for prolonged periods

of timesx Enable the transfer of valuable genetic material

between countriesx Provide a unique method for genetic conservation

v Greatest impact on animal breeding, particularly in cattle

v Sexing semenx Separation of spermatozoa into populations X & Yx Permitting the preselection of the sex of animals at

the time of fertilization


EctogenesisEctogenesis

v The development of the young one outside the body of female parent

v Fish & frog

v In mammals,

x Early culture may be successful within a glass vessel

x Later stage of development will need the presence of placenta to draw substances from the mother as well as to eliminate waste from the embryo


AmniocentesisAmniocentesisv A technique used mainly for diagnosing birth directs

in the fetus by means of needle be inserted into the amniotic fluid

v The needles can be used to inject substances or withdraw amniotic fluid

v Different biochemical disease & chromosomal defects can be detected

v Pregnant woman (≈90 days of pregnancy) through a transabdominal puncture to withdraws a small quantity of amniotic fluid for studyx The sex of the growing embryox Reveal any chromosomal abnormality


v Offers a monitoring procedure in cases where there is a prospect of producing an abnormal child

v Usually suggested for woman believed to be risk for transmission of genetic diseases

v Pregnant woman >35 years of age – advised to undergo amniocentesis because of the risk of transmitting chromosomal abnormalities

v Risk: injuring the infants

v Costly

v Latest modification: taking biopsy of the chorionic villi from the placental tissue


TransplantationTransplantation

v Determined the antigenic similarity between the recipient & the donor

v The closer the similarity, the greater the chance

v Rejection: antigens involved are of many types

v After transplantation, the recipient needs careful monitory to foretell graft rejection through the use of immune suppression drugs


Cloning techniqueCloning techniquev A number of genetically identical organisms are

derived from a single organism by vegetative propagation

The nucleus of mature unfertilized ovum surgery/ irradiation

Remove+

Nucleus (from the skin, intestine/ the respiratory belonging to the same species)

Egg cell develop

Yield an organism whose genetic constitution will be

similar to the parent


v Cloning successful in

x Plants

x Frogs

x Salamander

x Drosophila

All the cells of a clone are in identical with one another & with the parent cells


Gene therapyGene therapy

v >2000 genetic diseases of man could be cured by gene therapy

v Gene therapy should meet 5 conditions

v Not all genetic diseases will meet these conditions

v Those that will include

v Sickle cell anaemia

v Thalassmia

v Haemophilia – efficiencies in the factors responsible for the coagulation of blood

Cancer alterations in the haemoglobin protein molecule


5 conditions5 conditions

1. The gene that causes the genetic disorder should be responsible for a product that functions at the terminal stage of its formation

2. The etiology of the disease should be restricted to a single tissue so that the replacement is easy

3. The clinical symptoms of the disease should result from the expression of the single pair of recessive genes (e.g. sickle cell anaemia). Under this condition, it would necessary for replace in the target cell only one of alleles


4. The genetic disorder should be such that by altering the DNA of a few target cells, amelioration of the disease could be achieved

5. At the molecular level, the difference between normal & abnormal should be as small as possible

lecture 11 transgenic animals

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