Anda di halaman 1dari 43

Transgenic

• Products of rDNA technology are produced by


genetic modification in which DNA coding for the
required product is introduced, usually by means
of a plasmid or a viral vector, into a suitable
microorganism or cell line or plant cell or animal
cell, in which that DNA is expressed and
translated into protein.
• The desired product is then recovered by
extraction and purification.
• Transgenic plants as factories
for biopharmaceuticals
• Molecular farming is the use of
crops for the large-scale production
of valuable recombinant proteins.

• Molecular farming in plants has a


relatively short history, and the
commercial adoption of this
technology occurred very recently.
• Glycosylation of proteins is a highly complex post-
translational modification process taking place in
the endoplasmic reticulum and Golgi apparatus
and involving more than a hundred different
proteins (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).
• Transgenic plants can also produce a variety of
proteins used in diagnostics for detecting human
diseases and therapeutics for curing human and
animal diseases in large-scale with low cost.

• The monoclonal antibodies, blood plasma


proteins, peptide hormones and cytokinins are
being produced in transgenic plants and their
parts such as tobacco (in leaves), potato (in
tubers), sugarcane (in stems) and maize (in seed
endosperm).
• Plants are amazing and cheap chemical factories
that need only water, minerals, sun light and
carbon dioxide to produce thousands of
sophisticated chemical molecules with different
structures.

• Given the right genes, plants can serve as


bioreactors to modified or new compounds such
as amino acids, proteins, vitamins, plastics,
pharmaceuticals (peptides and proteins), drugs,
and enzymes for food industry and so on
• Producing therapeutic proteins in plants has
many economic and qualitative benefits,

• including reduced health risks from pathogen


contamination,

• comparatively high yields, and in seeds or other


storage organs
• The cultivation, harvesting,storage,
and processing of transgenic crops
would also use an existing
infrastructure and require relatively
little capital investment making the
commercial production of
biopharmaceuticals an exciting
prospect.
• Plants are potentially a cheap
source of recombinant products.

• Estimated that the cost of


producing recombinant proteins in
plants could be 10- to 50-fold lower
than producing the same protein by
Escherichia coli fermentation.
 The cultivation, harvesting, storage, and
processing of transgenic crops would also use an
existing infrastructure and require relatively little
capital investment making the commercial
production of biopharmaceuticals an exciting
prospect.
 In addition, purification may not always be
necessary, for example, in the case of edible
vaccines. Plant-derived products, whether
purified or not, are less likely to be contaminated
with human pathogenic microorganisms than
those derived from animal cells, because plants
don’t act as hosts for human infectious agents
• because they are easily transformed and provide a
cheap source of protein.

• Several biotechnology companies are now actively


developing, field testing, and patenting plant
expression systems, while clinical trials are
proceeding on the first biopharmaceuticals derived
from them.

• One transgenic plant-derived biopharmaceutical,


hirudin, is now being commercially produced in
Canada for the first time.
Techniques Used for Generating
Transgenic Plants

• As with bacteria, the ability to genetically


modify plants depends on obtaining
genetically identical populations and readily
manipulating DNA. How do you “clone” a
plant?
• It is also possible to grow plants in culture
from small explants.
• Another method is to culture plants from
totipotent cells found in plant meristems.
• These plant cells can divide and differentiate
into the various types of specialized cells. In a
test tube, plant cells will divide and form an
undifferentiated callus.
• When hormones in the culture medium are
adjusted, the callus will sprout shoots and
roots and eventually develop into a plantlet
that can be transplanted to soil.
To clone a plant — perhaps a plant with
new genes — the growing callus is
simply subdivided. Thousands of
genetically identical plants can be
generated in this way.
Transformation
• transformed transgenic plants are produced using
• Agrobacteriummediated transformation,
• particle bombardment, or
• other standard transformation techniques.
• Nicotiana tabacum is widely used as a model
expression system, but other plants have been used,
including
• Nicotiana bethamiana, Arabidopsis thaliana,
tomato, banana, turnip, black-eyed bean, oilseed
rape, Ethiopian mustard, potato, rice, wheat, and
maize.
• The second strategy is to infect nontransgenic
plants with recombinant viruses that express
transgenes during their replication in the
host.
• The two host–virus systems most frequently
used are
• tobacco with tobacco mosaic virus (TMV) or
• cowpeas with cowpea mosaic virus (CPMV)
Promoter
 Expression of pharmaceutical proteins
in transgenic plants is mostly by
constitutive CaMV 35S promoters.
• Seed expression has been achieved by placing
transgenes downstream of the glutelin (Gt3)
signal peptide sequence and transcribing from
Gt3 promoters2.
• Transcription of immunoglobulin transgenes in
tobacco has also been controlled by the seed-
specific, developmentally regulated, legumin B4
promoter11.
• As immunoglobulins in the cytoplasm often
interfere with cell function, it is desirable to have
them secreted into the apoplasm, or trafficked
 Trafficked to the endoplasmic reticulum,
where they generally accumulate to a much
higher proportion of TSP (1–5%)40. Trafficking
can be directed by the legumin B4 signal
sequence
• Full-scale commercial production will
probably involve grain and oilseed
crops, such as maize, rice, wheat,
soybeans, and oilseed rape.
• Proteins stored in seeds become
desiccated, and could remain intact for
long periods, making seeds a convenient
method of storing, distributing, and
administering biopharmaceuticals such
as vaccines.
Transgenic animals
for pharmaceutical
production
• Genetic Modification of Animals Dolly the
lamb stole the headlines as the first example of
livestock cloned from DNA of an adult animal.
• But the real breakthrough came with Polly, the
first transgenic lamb. Born the year after Dolly,
• Polly was given a human gene that encodes
blood-clotting factor IX, the protein missing in
people with one form of hemophilia.
• Harvesting such proteins from transgenic
livestock is one goal of this research.
 The road to Polly and subsequent
transgenic animals began with research
using genetically altered mice. Along the
way, technologies for cloning animals,
modifying DNA, and targeting
expression of proteins to specific tissues
were developed.
 The production of pharmaceutical human
proteins in transgenic animals is still a minor
business, in which only a few companies are
involved. After ten years of research, no product
has yet reached the market but the stewards of
this technology hope to achieve this within the
next couple of years. Currently, most research is
directed towards products for industrialized
countries
Transgenic animal
 A transgenic animal is by definition an animal
whose genetic composition has been altered to
include selected genes from other animals or
species by methods other than used in traditional
breeding.
 In other words, an animal altered by the
introduction of recombinant DNA through
human intervention.
 The first transgenic animal was a mouse, created
in 1981, carrying a gene which made the animal
susceptible to cancer.
 The first transgenic farm-animal was a sheep
created in 1985.
 Biotechnology firms and research institutes
involved in pharmaceutical development and
production use transgenic animals for three
different ends.
• As a pharmaceutical production unit. Via
transgenic technology such as micro-
injection, genes that code for the production
of a human protein are inserted into the
genome of an animal, which in turn produces
the human protein.
Pharming
 This new branch of transgenic animal
production which has emerged in recent years
has a new name: pharming. Pharming is the
production of pharmaceutical human
proteins in transgenic farm animals.

 In most cases of pharming, changing the


composition of milk is the main strategy.
 Mammary glands of cows produce large
volumes of milk, a protein-rich solution which
can be collected non-invasively. In October
1997, Nature Biotechnology reported on the
achievement of producing a biologically active
human protein in the milk of a transgenic pig.
This demonstrated the feasibility of
producing large and complex proteins in this
way.
 However, it remains difficult to generate animals
which produce these medical proteins of a
consistent quality and in sufficient quantities. If a
successfully engineered transgenic animal can be
cloned, and then bred successfully, it will be
possible to create herds of these animals for
pharmaceutical milk products of identical
quality.
Research also extends to areas other than milk.
The Agricultural Research Service of the United
States Department of Agriculture (USDA) has
developed mice which produce stable amounts of
human growth hormone in their urine.
Disadvantage
 The researchers see some clear disadvantages of milk
based pharmaceutical protein production: firstly,
lactation occurs only in females and is non-
continuous; secondly, for cows it takes at least 24
months before lactation starts. Finally, milk is a
complex substance usually containing 3 to 6 per cent
total protein and therefore needs extensive
purification to obtain the pharmaceutical protein.
Purification of urine seems to be easier, according to
the USDA research.
Microinjection
Why transgenic livestock
• The use of transgenic livestock in protein
production aims at overcoming several major
barriers presented by cell-based systems.
Potentially, this approach could provide large
quantities of complex proteins in a cost-
effective way. Compared to the facilities and
chemicals required for cell culture
production, capital investment required for
animal production facilities is relatively low.
 The British company PPL Therapeutics (PPL)
estimates that the basic costs for products
developed by transgenic animals are four to
five times lower than cell culture production.
However, this does not take into account
development costs. To date, the use of
transgenic animals is developing fast for a
range of pharmaceutical products.
R&D of medicine production by
transgenic animals
Drug Disease/Target Animal Company
alpha-lactalbumin anti-infection cow PPL
alpha1 anti trypsin deficiency leads to sheep PPL
(AAT) emphysema
CFTR cystic fibrosis sheep, mouse PPL
human protein C thrombosis pig, sheep PPL
tissue
plasminogen thrombosis mouse, goat PPL
activator (tPA)
human calcitonin osteoporosis rabbit PPL
factor VIII hemophilia pig Pharming
sheep PPL
factor IX hemophilia pig, cow Pharming
sheep PPL
Drug Disease/Target Animal Company
fibrinogen wound healing cow Pharming
sheep PPL
alpha-
Pompe disease rabbit Pharming
glucosidase
tissue repair Pharming
collagen I cow
rheumatoid
collagen II cow
arthritis
GI tract cow Pharming
lactoferrin infection,
infectious
arthritis
antithrombin 3
thrombosis goat GTC
(ATIII)
glutamic acid
type 1 diabetes mouse, goat GTC
decarboxylase
human serum maintains blood
mouse, cow GTC
albumin (HSA) volume
msp-1 malaria mouse GTC
Pro542 HIV mouse, goat GTC