Transgenic Animals and Plants

8/8/2019 Transgenic Animals and Plants

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Transgenic Products of rDNA technology are produced by

genetic modification in which DNA coding for therequired product is introduced, usually by meansof a plasmid or a viral vector, into a suitable

microorganism or cell line or plant cell or animalcell, in which that DNA is expressed andtranslated into protein.

The desired product is then recovered byextraction and purification.


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Transgenic plants as factoriesfor biopharmaceuticals


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Molecular farming is the use ofcrops for the large-scale production

of valuable recombinant proteins.

Molecular farming in plants has arelatively short history, and the

commercial adoption of thistechnology occurred very recently.


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Glycosylation of proteins is a highly complex post-translational modification process taking place in

the endoplasmic reticulum and Golgi apparatusand involving more than a hundred differentproteins (and genes).

This glycosylation machinery is absent in E. coli,present but different from mammalian cells in the

yeast Saccharomyces cerevisiae, but highly

conserved among mammalian cells (e.g., human,Chinese hamster).


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Transgenic plants can also produce a variety ofproteins used in diagnostics for detecting humandiseases and therapeutics for curing human andanimal diseases in large-scale with low cost.

,proteins, peptide hormones and cytokinins arebeing produced in transgenic plants and theirparts such as tobacco (in leaves), potato (in

tubers), sugarcane (in stems) and maize (in seedendosperm).


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Plants are amazing and cheap chemical factoriesthat need only water, minerals, sun light andcarbon dioxide to produce thousands ofsophisticated chemical molecules with different

structures.

Given the ri ht enes lants can serve as

bioreactors to modified or new compounds suchas amino acids, proteins, vitamins, plastics,pharmaceuticals (peptides and proteins), drugs,

and enzymes for food industry and so on


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Producing therapeutic proteins in plants hasmany economic and qualitative benefits,

including reduced health risks from pathogencontamination,

comparatively high yields, and in seeds or otherstorage organs


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The cultivation, harvesting,storage,and processing of transgenic crops

would also use an existinginfrastructure and require relatively

e cap a nves men ma ng ecommercial production ofbiopharmaceuticals an exciting

prospect.


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Plants are potentially a cheapsource of recombinant products.

Estimated that the cost of

producing recombinant proteins inplants could be 10- to 50-fold lowerthan producing the same protein byEscherichia coli fermentation.


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The cultivation, harvesting, storage, andprocessing of transgenic crops would also use anexisting infrastructure and require relatively little

capital investment making the commercialproduction of biopharmaceuticals an excitingprospect.

In addition, purification may not always benecessary, for example, in the case of edible

vaccines. Plant-derived products, whetherpurified or not, are less likely to be contaminated

with human pathogenic microorganisms thanthose derived from animal cells, because plantsdont act as hosts for human infectious agents


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because they are easily transformed and provide a

cheap source of protein.

Several biotechnology companies are now actively

developing, field testing, and patenting plantexpression systems, while clinical trials are

proceeding on the first biopharmaceuticals derived

from them.

One transgenic plant-derived biopharmaceutical,

hirudin, is now being commercially produced inCanada for the first time.


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Techniques Used for Generating

Transgenic Plants

As with bacteria, the ability to geneticallymodify plants depends on obtaininggenetically identical populations and readily

manipulating DNA. How do you clone aplant?

It is also possible to grow plants in culture

from small explants.


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Another method is to culture plants fromtotipotent cells found in plant meristems.

These plant cells can divide and differentiateinto the various types of specialized cells. In atest tube, plant cells will divide and form an

.

When hormones in the culture medium areadjusted, the callus will sprout shoots androots and eventually develop into a plantletthat can be transplanted to soil.


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To clone a plant perhaps a plant withnew genes the growing callus issimply subdivided. Thousands of

genetically identical plants can begenerated in this way.


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Transformation transformed transgenic plants are produced using

Agrobacteriummediated transformation,

particle bombardment, or

other standard transformation techniques.

expression system, but other plants have been used,including

Nicotiana bethamiana, Arabidopsis thaliana,

tomato, banana, turnip, black-eyed bean, oilseedrape, Ethiopian mustard, potato, rice, wheat, andmaize.


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The second strategy is to infect nontransgenicplants with recombinant viruses that expresstransgenes during their replication in thehost.

used are

tobacco with tobacco mosaic virus (TMV) or

cowpeas with cowpea mosaic virus (CPMV)


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Expression of pharmaceutical proteinsin transgenic plants is mostly byconstitutive CaMV 35S promoters.

Promoter


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Seed expression has been achieved by placingtransgenes downstream of the glutelin (Gt3)signal peptide sequence and transcribing from

Gt3 promoters2.

Transcription of immunoglobulin transgenes intobacco has also been controlled b the seed-

specific, developmentally regulated, legumin B4promoter11.

As immunoglobulins in the cytoplasm often

interfere with cell function, it is desirable to havethem secreted into the apoplasm, or trafficked


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Trafficked to the endoplasmic reticulum,where they generally accumulate to a muchhigher proportion of TSP (15%)40. Traffickingcan e rec e y e egum n 4 s gnasequence


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Full-scale commercial production willprobably involve grain and oilseedcrops, such as maize, rice, wheat,soybeans, and oilseed rape.

Proteins stored in seeds become

esiccate , an cou remain intact orlong periods, making seeds a convenientmethod of storing, distributing, and

administering biopharmaceuticals suchas vaccines.


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Genetic Modification of Animals Dolly thelamb stole the headlines as the first example oflivestock cloned from DNA of an adult animal.

But the real breakthrough came with Polly, thefirst transgenic lamb. Born the year after Dolly,

P ll w iv n h m n n th t n

blood-clotting factor IX, the protein missing inpeople with one form of hemophilia.

Harvesting such proteins from transgenic

livestock is one goal of this research.


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The road to Polly and subsequenttransgenic animals began with researchusing genetically altered mice. Along the

way, technologies for cloning animals,modifying DNA, and targetingexpression of proteins to specific tissues

were developed.


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The production of pharmaceutical humanproteins in transgenic animals is still a minorbusiness, in which only a few companies are

.

has yet reached the market but the stewards ofthis technology hope to achieve this within thenext couple of years. Currently, most research is

directed towards products for industrializedcountries


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Transgenic animal

A transgenic animal is by definition an animalwhose genetic composition has been altered toinclude selected genes from other animals or

breeding. In other words, an animal altered by the

introduction of recombinant DNA through

human intervention.


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The first transgenic animal was a mouse, createdin 1981, carrying a gene which made the animalsusceptible to cancer.

-

created in 1985.

Biotechnology firms and research institutesinvolved in pharmaceutical development and

production use transgenic animals for threedifferent ends.


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As apharmaceutical production unit. Viatransgenic technology such as micro-injection, genes that code for the productiono a uman pro e n are nser e n o egenome of an animal, which in turn producesthe human protein.


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Pharming

This new branch of transgenic animalproduction which has emerged in recent yearshas a new name:pharming. Pharming is thepro uc on o p armaceu ca umanproteins in transgenic farm animals.

In most cases of pharming, changing thecomposition of milk is the main strategy.


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Mammary glands of cows produce largevolumes of milk, a protein-rich solution whichcan be collected non-invasively. In October1997, a ure o ec no ogy repor e on eachievement of producing a biologically activehuman protein in the milk of a transgenic pig.This demonstrated the feasibility of

producing large and complex proteins in thisway.


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However, it remains difficult to generate animalswhich produce these medical proteins of aconsistent quality and in sufficient quantities. If a

successfully engineered transgenic animal can becloned, and then bred successfully, it will bepossible to create herds of these animals for

quality.Research also extends to areas other than milk.TheAgricultural Research Service of the United

States Department of Agriculture (USDA) hasdeveloped mice which produce stable amounts ofhuman growth hormone in their urine.


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Disadvantage

The researchers see some clear disadvantages of milkbased pharmaceutical protein production: firstly,lactation occurs only in females and is non-

,

months before lactation starts. Finally, milk is acomplex substance usually containing 3 to 6 per centtotal protein and therefore needs extensive

purification to obtain the pharmaceutical protein.Purification of urine seems to be easier, according tothe USDA research.


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Microinjection


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Why transgenic livestock The use of transgenic livestock in protein

production aims at overcoming several majorbarriers presented by cell-based systems.

o en a y, s approac cou prov e argequantities of complex proteins in a cost-effective way. Compared to the facilities andchemicals required for cell culture

production, capital investment required foranimal production facilities is relatively low.


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The British companyPPL Therapeutics (PPL)estimates that the basic costs for productsdeveloped by transgenic animals are four to ve mes ower an ce cu ure pro uc on.

However, this does not take into accountdevelopment costs. To date, the use oftransgenic animals is developing fast for a

range of pharmaceutical products.


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Drug Disease/Target Animal Company

alpha-lactalbumin anti-infection cow PPL

alpha1 anti trypsin(AAT)

deficiency leads toemphysema

sheep PPL

CFTR cystic fibrosis sheep, mouse PPL

R&D of medicine production by

transgenic animals

uman pro e n rom os s p g, s eep

tissueplasminogenactivator (tPA)

thrombosis mouse, goat PPL

human calcitonin osteoporosis rabbit PPL

factor VIII hemophilia pigsheep

PharmingPPL

factor IX hemophilia pig, cowsheep

PharmingPPL

Drug Disease/Target Animal Company


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g / g p y

fibrinogen wound healing cowsheep

PharmingPPL

alpha-glucosidase

Pompe disease rabbit Pharming

collagen I

collagen II

tissue repairrheumatoid

arthritis

cow

cow

Pharming

lactoferrinGI tractinfection,

cow Pharming

arthritisantithrombin 3(ATIII)

thrombosis goat GTC

glutamic aciddecarboxylase

type 1 diabetes mouse, goat GTC

human serumalbumin (HSA)

maintains bloodvolume

mouse, cow GTC

msp-1 malaria mouse GTC

Pro542 HIV mouse, goat GTC

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Transgenic Animals and Plants