1.

Biopharmaceuticals
 Introduction
• Biopharmaceuticals are medical drugs produced using
biotechnology.
• They are proteins (including antibodies), nucleic acids (DNA,RNA
or antisense oligonucleotides) used for therapeutic or in vivo
diagnostic purposes, and are produced by means other than direct
extraction from a native (non-engineered) biological source.
• The first such substance approved for therapeutic use was
biosynthetic 'human' insulin made via recombinant DNA
technology.
• Sometimes referred to as rHI, under the trade name Humulin, was
developed by Genentech, but licensed to Eli Lily and Company,
who manufactured and marketed the product starting in 1982.
 Biotherapeutic Agents
• Virtually all biotherapeutic agents in clinical use are
biotech pharmaceuticals
• A biotech pharmaceutical is simply any medically
useful drug whose manufacture involves
microorganisms or substances that living organisms
produce (e.g., enzymes).
• Most biotech pharmaceuticals are recombinant—that
is, produced by genetic engineering
 Biopharmaceutical Categories
• Biopharmaceuticals can be grouped into six categories.
– Cytokines
– Enzymes
– Hormones
– Clotting Factors
– Vaccines
– Monoclonal Antibodies
 Cytokines
• Cytokines - Cytokines are hormone-like molecules
that can control reactions between cells. They activate
immune-system cells such as lymphocytes and
macrophages.
– Interferon - potent glycoprotein cytokine that act
against viruses and uncontrolled cell proliferation
– Interleukins function as messengers for various
steps in the immune process. Ex. interleukin-2 (IL-
2) stimulates T lymphocytes, a recombinant variant
of IL-2, aldesleukin (Proleukin), for treating renal
cell carcinoma. IL-3 stimulates bone marrow stem
cells. IL-1 secreted by macrophages induces fever.
 Cytokines
• Cytokines
– Granulocyte-colony stimulating factor (G-CSF)
stimulates the bone marrow to produce neutrophils
(antibacterial leukocytes), used for cancer treatments
that are immunodepressants
– Granulocyte-macrophage colony-stimulating factor
(GM-CSF) stimulates the bone marrow to produce
neutrophils and macrophages. For chemo and radio
therapy that suppresses bone marrow function
 Enzymes
• Enzymes – are complex proteins that cause a
specific chemical change in other substances
without being changed themselves.
– Alteplase – dissolves blood clots
– Dornase alfa – a recombinant DNAse I that digests
DNA in the mucous secretions in lungs
– Imiglucerase. A recombinant glucocereborsidase for
Gaucher’s disease (bone destruction and
enlargement of the liver and spleen)
 Other Biopharmaceutical categories

• Hormones – chemicals that transfer information and

instructions between cells in animals and plants. Ex.
insulin, HGH.
• Clotting Factors – any factor in the blood that is essential
for the blood to coagulate.
• Vaccines – microorganisms that can be used to
stimulate resistance in a human to specific diseases.
Ex. – Hepatitis B virus, Ebola virus
• Monoclonal antibodies – immortal cells created by fusion
of a cancer cell with an antibiotic producing spleen cell.
Cloning Insulin
 Human Growth Hormone

• Facts
– Peak production during adolescence
– 60 year old secretes 25% of a 20 year old
– Anterior lobe of the pituitary gland
– Primarily released in pulses during beginning phases of sleep
– Converted in the liver into insulin-Like Growth Factor type I
– Incidence: 1 out of 4,000 to 10,000 births
 Human Growth Hormone

• Also known as
somatotropin
• Most abundant
hormone secreted by
the pituitary gland

The Pituitary gland is a small, pea-sized gland that is located in the middle
of your skull, just below the hypothalamus.
 Pituitary Gland
• The pituitary gland has two lobes, anterior
and posterior
• The anterior lobe also produces
– Prolactin – breast milk
– FSH, LS – sexual reproduction
– Thyrotropin – thyroid function
– Adrenocorticotropin - adrenal function
– Growth Hormone - growth
 Human Growth Hormone
• Usually the first hormone to be lost or
reduced
• Can be congenital
• Can be acquired
– Inflammation
– Surgery
– Radiation
– Autoimmune disease
– Tumors
 Human Growth Hormone
• 191 amino acid (approx. 22 kdal)
• Appears to be 5 variants of the gene on chromosome 17
• Takes 80 cadavers for one year’s worth of therapy
• Creutzfeldt -Jacob syndrome (odds – 1:500)
 Protein Pharmaceuticals
• Natural sources are often rare and expensive
– Difficult to keep up with demand
– Hard to isolate product
– Lead to immune reactions (diff. species)
– Viral & pathogen contamination

• Most protein pharmaceuticals today are produced recombinantly

– Cheaper, safer, abundant supply
 Recombinant Methods

• Developed in 1970’s &1980’s

• Paul Berg (1973) restriction enzymes
• Herbert Boyer (1978) cloning human insulin
into E. coli – Genentech
• Two general approaches
– Expression in isolated cells
– Expression in transgenic plants/animals
 Six step process
• Isolation of gene of interest
• Introduction of gene to expression vector
• Transformation into host cells
• Growth of cells through fermentation
• Isolation & purification of protein
• Formulation of protein product
 Cloning Process
• Gene of interest is cut
out with restriction
enzymes (RE)
• Host plasmid (circular
chromosome) is cut with
same REs
• Gene is inserted into
plasmid and ligated with
ligase
• New (engineered)
plasmid inserted into
bacterium (transform)
Cloning (Details)
Cloning (Details)

protein
Recombinant Protein Expression
Systems
• Escherichia coli
• Other bacteria Polyhedra
• Pichia pastoris
• Other yeast
• Baculovirus
• Animal cell culture
• Plants
• Sheep/cows/humans
 Expression System Selection
• Choice depends on size and character of protein
– Large proteins (>100 kD)? Choose eukaryote
– Small proteins (<30 kD)? Choose prokaryote
– Glycosylation essential? Choose baculovirus or
mammalian cell culture
– High yields, low cost? Choose E. coli
– Post-translational modifications essential? Choose
yeast, baculovirus or other eukaryote
 Which Vector?
• Must be compatible with host cell system (prokaryotic vectors for
prokaryotic cells, eukaryotic vectors for eukaryotic cells)
• Needs a good combination of
– strong promoters
– ribosome binding sites
– termination sequences
– affinity tag or solubilization sequences
– multi-enzyme restriction site
 Plasmids and Vectors
• Circular pieces of DNA ranging in size from
1000 to 10,000 bases
• Able to independently replicate and typically
code for 1-10 genes
• Often derived from bacterial “mini”
chromosomes (used in bacterial sex)
• May exist as single copies or dozens of copies
(often used to transfer antibiotic resistance)
 Key Parts to a Vector
• Origin of replication (ORI) – DNA sequence for DNA
polymerase to replicate the plasmid
• Selectable marker (Amp or Tet) – a gene, when
expressed on plasmid will allow host cells to survive
• Inducible promoter – Short DNA sequence which
enhances expression of adjacent gene
• Multi-cloning site (MCS) – Short DNA sequence that
contains many restriction enzyme sites
A Generic Vector
 Which Vector?
• Promoters
– arabinose systems (pBAD), phage T7 (pET), Trc/Tac
promoters, phage lambda PL or PR
• Tags
– His6 for metal affinity chromatography (Ni)
– FLAG epitope tage DYKDDDDK
– CBP- calmodulin binding peptide (26 residues)
– E-coil/K-coil tags (poly E35 or poly K35)
– c-myc epitope tag EQKLISEEDL
– Glutathione-S-transferase (GST) tags
– Celluluose binding domain (CBD) tags
Gene Introduction (Bacteria)
Bacterial Transformation
 Bacterial Transformation
• Moves the plasmid into bacterial host
• Essential to making the gene “actively” express the
protein inside the cell
• 2 routes of transformation
– CaCl2 + cold shock
– Electroporation
• Typical transformation rate is 1 in 10,000 cells (not very
efficient) for CaCl2, but 1 in 100 for electroporation
 Electroporator

25 microfarads = 2500 V
@ 200 ohms for 5 ms
 Electroporation
• Seems to cause disruption in
cell membrane
• Reconstitution of membrane
leads to large pores which
allow DNA molecules to
enter
• Works for bacteria, yeast and
animal cells
 Bacterial Systems
Advantages Disadvantages
• Grow quickly (8 hrs to • Difficulty expressing
produce protein) large proteins (>50 kD)
• High yields (50-500 • No glycosylation or
mg/L) signal peptide removal
• Low cost of media • Eukaryotic proteins are
(simple media sometimes toxic
constituents) • Can’t handle S-S rich
• Low fermentor costs proteins
Cloning & Transforming in
Yeast Cells

Pichia pastoris
 Pichia Pastoris
• Yeast are single celled eukaryotes
• Behave like bacteria, but have key advantages
of eukaryotes
• P. pastoris is a methylotrophic yeast that can
use methanol as its sole carbon source (using
alcohol oxidase)
• Has a very strong promoter for the alcohol
oxidase (AOX) gene (~30% of protein produced
when induced)
Pichia Cloning
 Pichia Pastoris Cloning
• Uses a special plasmid that works both in E. coli and
Yeast
• Once gene of interest is inserted into this plasmid, it
must be linearized (cut open so it isn’t circular)
• Double cross-over recombination event occurs to cause
the gene of interest to insert directly into P. pastoris
chromosome where the old AOX gene used to be
• Now gene of interest is under control of the powerful
AOX promoter
 Pichia Systems
Advantages More advantages
• Grow quickly (8 hrs to • Can express large
produce protein) proteins (>50 kD)
• Very high yields (50- • Glycosylation & signal
5000 mg/L) peptide removal
• Low cost of media • Has chaperonins to help
(simple media fold “tough” prtns
constituents) • Can handle S-S rich
• Low fermentor costs proteins
 Baculovirus Expression
• Baculoviruses are rod-shaped dsDNA viruses found mainly in insects. The most
common baculovirus used for expression studies is Autographa californica multiple
nuclear polyhedrosis virus, which relies on the lepidopteran species Spodoptera
frugiperda and Trichoplusia ni as host insects.
• AcMNPV particles surround themselves with a protective matrix consisting of the
protein polyhedrin, which permits survival in the environment and efficient spread
to new hosts.
• Under the control of the extremely strong promoter pPolh, polyhedrin is expressed
at extremely high levels (up to 50% of all cellular protein) at the end of the
baculovirus life cycle.
• The baculovirus expression system makes use of the fact that in cell culture a
polyhedrin coat is not essential for virus propagation and thus heterologous proteins
can be expressed under the control of the pPolh promoter.
• Autographica californica multiple nuclear polyhedrosis virus (Baculoviurs)
• Virus commonly infects insects cells of the alfalfa looper (small beetle) or
armyworms (and their larvae)
• Uses super-strong promoter from the polyhedron coat protein to enhance
expression of proteins while virus resides inside the insect cell
Baculovirus Expression
Baculovirus Expression

~12 days
 Baculovirus Successes
• Alpha and beta interferon
• Adenosine deaminase
• Erythropoietin
• Interleukin 2
• Poliovirus proteins
• Tissue plamsinogen activator (TPA)
 Baculovirus Systems
Disadvantages Advantages
• Grow very slowly (10-12 • Can express large proteins
days for set-up) (>50 kD)
• Cell culture is only • Correct glycosylation &
sustainable for 4-5 days signal peptide removal
• Set-up is time consuming, • Has chaperonins to help fold
not as simple as yeast “tough” prtns
• Very high yields, cheap
Mammalian Expression
Systems
 Mammalian Cell-line Expression
• Sometimes required for difficult-to-express proteins or
for “complete authenticity” (matching glycosylation and
sequence)
• Cells are typically derived from the Chinese Hamster
Ovary (CHO) cell line
• Vectors usually use SV-40 virus, vaccinia virus
promoters and DHFR (dihydrofolate reductase) as the
selectable marker gene
 Mammalian Expression
• Gene initially cloned and plasmid propagated in bacterial
cells
• Mammalian cells transformed by electroporation (with
linear plasmid) and gene integrates (1 or more times)
into random locations within different CHO chromosomes
• Multiple rounds of growth and selection using
methotrexate to select for those cells with highest
expression & integration of DHFR and the gene of
interest
 Methotrexate (MTX) Selection
Gene of interest DHFR

Transfect
Dfhr - cells

Grow in Culture a Grow in Culture a

Nucleoside Colony of 0.05 uM Mtx Colony of
Free medium cells cells
Methotrexate (MTX) Selection

Grow in Culture a Grow in Culture a

0.25 uM Mtx Colony of 5.0 uM Mtx Colony of
cells cells

Foreign gene
expressed in
high level in
CHO cells
 Mammalian Systems
Disadvantages Advantages
• Selection takes time • Can express large
(weeks for set-up) proteins (>50 kD)
• Cell culture is only • Correct glycosylation &
sustainable for limited signal peptide removal,
period of time generates authentic
• Set-up is very time proteins
consuming, costly, • Has chaperonins to help
modest yields fold “tough” prtns
 Mammalian Cell Successes
• Factor IX
• Factor VIII
• Gamma interferon
• Interleukin 2
• Human growth hormone
• Tissue plamsinogen activator (TPA)
 Conclusion
• Isolation of gene of interest
• Introduction of gene to expression vector
• Transformation into host cells
• Growth of cells through fermentation
• Isolation & purification of protein
• Formulation of protein product
 What About the Term
Genetic Engineering?

Genetic engineering is the basic tool set of biotechnology

Genetic engineering involves:

 Isolating genes
 Modifying genes so they function better
 Preparing genes to be inserted into a new species
 Developing transgenes
 What is a transgenic?

Concept Based on the Term Transgene

Transgene – the genetically engineered gene added to a species

Ex. – modified epsp synthase gene (encodes a protein

that functions even when plant is treated with Roundup)

Transgenic – an organism containing a transgene

introduced by technological (not breeding) methods

Ex. – Roundup Ready Crops

 Why are transgenics important?

We can develop organisms that express a “novel” trait

not normally found in the species

Extended shelf-life tomato (Flavr-Savr)

Herbicide resistant soybean (Roundup Ready)

 Agriculture Transgenics On the Market

Insect resistant cotton – Bt toxin kills the

cotton boll worm
• transgene = Bt protein
Source: USDA

Insect resistant corn – Bt toxin kills the

European corn borer
• transgene = Bt protein
Normal Transgenic
 Herbicide resistant crops
Now: soybean, corn, canola
Coming: sugarbeet, lettuce, strawberry
alfalfa, potato, wheat
(transgene = modified EPSP synthase or
phosphinothricin-N-acetyltransferase
Source: Monsanto

Virus resistance - papaya resistant to

papaya ringspot virus
• transgene = virus coat protein
 Biotech chymosin; the enzyme used
to curdle milk products
Source: Chr. Hansen
• transgene = genetically engineered enzyme

bST; bovin somatotropin; used to increase

milk production
• transgene = genetically engineered enzyme

Source: Rent Mother Nature

Some Ag Biotech Products Are Discontinued

Poor Quality
• FlavrSavr tomatoes (Calgene)

Negative Consumer Response

• Tomato paste (Zeneca)

Negative Corporate Response

• NewLeaf (Monsanto)

Universal Negative Publicity

• StarLink corn (Aventis)
 Next Generation of Ag Biotech Products

Golden Rice – increased Vitamin A content

(but not without controversy)
transgene = three pathway enzymes

Sunflower – white mold resistance

transgene = oxalate oxidase from wheat
Source: Minnesota
Microscopy Society
 Turfgrass – herbicide resistance;
slower growing (= reduced mowing)

Bio Steel – spider silk expressed in goats; used to

make soft-body bullet proof vests (Nexia)
Biotechnology is Not Just on the Farm

Disease Treatment

Diagnostics
 
Environmental Cleanup
 
Human Applications
 Human Applications
• Pharmaceutical products
New solutions to old problems
 
• Disease diagnosis
Determine what disease you have or may get 

• Gene therapy
Correcting disease by introducing a corrective
gene
 Biotechnology and Health

Product Use
Insulin Diabetes
Interferon Cancer
Interleukin Cancer
Human growth hormone Dwarfism
Neuroactive proteins Pain

The genes for these proteins are:

• Cloned
• Inserted into bacteria
• Product isolated using biofermentation
Environmental Applications

Bioremediation - cleanup contaminated

sites; uses microbes designed to degrade
the pollution

Indicator bacteria – contamination can be

detected in the environment
Future Health-related Biotech Products

Vaccines – herpes, hepatitis C, AIDS, malaria

Tooth decay – engineered Streptococcus mutans,

the bacteria that destroys enamel
 Edible Vaccines
Transgenic Plants Serving Human Health Needs
• Works like any vaccine
• A transgenic plant with a pathogen protein gene is developed
• Potato, banana, and tomato are targets
• Humans eat the plant
• The body produces antibodies against pathogen protein
• Humans are “immunized” against the pathogen
• Examples:
Diarrhea
Hepatitis B
Measles
 A Popular Term We Need To Know

GMOs - Genetically modified organisms

• GMO - an organism that expresses traits that result

from the introduction of foreign DNA

• Originally a term equivalent to transgenic organism

 Important Plant Improvement Methods

• Breeding
Crossing two individuals from the same species;
produces a new, improved variety;
Source: USDA not a biotechnology procedure

• Transformation
Adding a gene from another species; the
essential biotechnology procedure to produce
transgenics
Source: USDA
 Interspecific Cross
Wheat Rye

Triticale
New species, but
NOT biotechnology
products
 Mutagenesis

A useful procedure to produce a new trait

But the normal gene is modified

A transgene is not involved

 The product of mutagenesis is not a GMO

Mutagenesis Changes the DNA Sequence

Mutagenesis
Treatment
Susceptible
Normal
Gene
ATTCGA

Resistant
Mutant
Gene
ATTGGA
 The Roundup Ready Story

• Glyphosate is a broad-spectrum herbicide

• Active ingredient in Roundup herbicide
• Kills all plants it come in contact with
• Inhibits a key enzyme (EPSP synthase) in an amino acid pathway

• Plants die because they lack the key amino acids

• A resistant EPSP synthase gene allows crops

to survive spraying
 Roundup Sensitive Plants
Shikimic acid + Phosphoenol pyruvate
+ Glyphosate

X
Plant
EPSP synthase

Without amino acids,

X
3-Enolpyruvyl shikimic acid-5-phosphate
(EPSP)
plant dies

X
X
Aromatic
amino acids
 Roundup Resistant Plants
Shikimic acid + Phosphoenol pyruvate
+ Glyphosate
RoundUp has no effect;
Bacterial enzyme is resistant to herbicide
EPSP synthase

3-enolpyruvyl shikimic acid-5-phosphate

(EPSP)
With amino acids,
plant lives

Aromatic
amino acids
 The Golden Rice Story

• Vitamin A deficiency is a major health problem

• Causes blindness
• Influences severity of diarrhea, measles

• >100 million children suffer from the problem

• For many countries, the infrastructure doesn’t exist

to deliver vitamin pills

• Improved vitamin A content in widely consumed crops

an attractive alternative
 -Carotene Pathway Problem in Plants
IPP (Isopentynyl diphosphate)

Geranylgeranyl diphosphate

Phytoene synthase

Phytoene
Problem: Phytoene desaturase
Rice lacks
these enzymes ξ-carotene desaturase

Lycopene
Lycopene-beta-cyclase
Normal
Vitamin A  -carotene
“Deficient” (vitamin A precursor)
Rice
 The Golden Rice Solution
-Carotene Pathway Genes Added

IPP

Geranylgeranyl diphosphate

Daffodil gene Phytoene synthase

Phytoene
Vitamin A
Phytoene desaturase
Pathway Single bacterial gene;
is complete performs both functions
and functional ξ-carotene desaturase

Lycopene
Daffodil gene Lycopene-beta-cyclase

Golden  -carotene
Rice (vitamin A precursor)
 Introducing the Gene
or
Developing Transgenics

Steps

1. Create transformation cassette

2. Introduce and select for transformants

 Transformation Cassettes

Contains

1. Gene of interest
• The coding region and its controlling elements

2. Selectable marker
• Distinguishes transformed/untransformed plants

3. Insertion sequences
• Aids Agrobacterium insertion
 Transformation Steps

Prepare tissue for transformation

• Tissue must be capable of developing into normal plants
• Leaf, germinating seed, immature embryos

Introduce DNA
• Agrobacterium or gene gun

Culture plant tissue

• Develop shoots
• Root the shoots

Field test the plants

• Multiple sites, multiple years
 Delivering the Gene
to the Plant
• Transformation cassettes are developed in the lab

• They are then introduced into a plant

• Two major delivery methods

• Agrobacterium

Tissue culture
• Gene Gun required to generate
transgenic plants
The Lab Steps
The Next Test Is The Field
Herbicide Resistance

Non- transgenics

Transgenics
Final Test of the Transgenic
Consumer Acceptance

RoundUp Ready Corn

Before After
• In nature, plant cells often live in close association with
certain bacteria, which may provide a convenient vehicle
for introducing cloned DNA into plants. 
• Agrobacterium tumefaciens, for example, attaches to the
cells of dicotledonous plants and causes the formation of
plant tumours known as galls. 
• This bacterium introduces a circular DNA molecule called
the Ti (tumour -inducing) plasmid into the plant cell in a
manner similar to bacterial conjugation. 
• The plasmid DNA then recombines with the plant DNA. 
Since the Ti plasmid has been isolated, new genes can be
inserted into it using recombinant DNA techniques and
the Ti genes causing tumours can be disrupted.   
• The resulting recombinant plasmid can then transfer desired genes into
plant cells.
An especially useful characteristic of plants for transgenic studies is the
ability of cultured plant cells to give rise to mature plants. 
• Meristemativ (growing) cells from dissected plant tissue or cells within
excised parts of a plant will grow in culture to form callus tissue, an
undifferntiated lump of cells. 
• Under the influence of plant growth hormones, different plant parts (roots,
stems, and leaves) develop from the callus and eventually grow into whole
fertile plants. 
• When an agrobacterium containing a recombinant Ti plasmid infects a
cultured plant cell, the newly incorporated foreign gene is carried into the
plant genome. 
• A. tumefaciens readily infects dicots (petunia, tobacco, carrot) but not
monocots; reliable techniques for introducing genes into monocots are still
being developed.
• Direct introduction of DNA by electroporation has been successful in rice
plants, (which are monocots), and the future looks bright for the
manipulation of other commercially important monocotyledonous crop
plants. 
• Also available for gene transfer experiments are cells of a tiny, rapidly
growing member of the mustard family called Arabidopsis thaliana. 
• This plant appears to be well suited to genetic analysis of a variety of
developmental and physiological processes. 
• It takes up little space, is easy to grow, and has a small genome, and genes
defined by mutations can be cloned by positional cloning strategies. 
• A transgenic animal is one that carries a foreign gene that has
been deliberately inserted into its genome. The foreign gene is
constructed using recombinant DNA methodology. In addition
to a structural gene, the DNA usually includes other
sequences to enable it
• to be incorporated into the DNA of the host and
• to be expressed correctly by the cells of the host.
• Transgenic sheep and goats have been produced that express
foreign proteins in their milk.
• Transgenic chickens are now able to synthesize human
proteins in the "white" of the eggs.
• These animals should eventually prove to be valuable sources
of proteins for human therapy.
• To date, there are three basic methods of producing
transgenic animals:
• DNA microinjection
• Retrovirus-mediated gene transfer
• Embryonic stem cell-mediated gene transfer
• 1. DNA Microinjection
• The mouse was the first animal to undergo successful gene
transfer using DNA microinjection. This method involves:
• transfer of a desired gene construct (of a single gene or a
combination of genes that are recombined and then cloned)
from another member of the same species or from a different
species into the pronucleus of a reproductive cell
• the manipulated cell, which first must be cultured in vitro (in a
lab, not in a live animal) to develop to a specific embryonic
phase, is then transferred to the recipient female
• 2. Retrovirus-Mediated Gene Transfer
• The second method produces chimeras, altered animals with mixed
DNA.
• A retrovirus is a virus that carries its genetic material in the form of
RNA rather than DNA. This method involves
• retroviruses used as vectors to transfer genetic material into the
host cell, resulting in a chimera, an organism consisting of tissues or
parts of diverse genetic constitution
• chimeras are inbred for as many as 20 generations until
homozygous (carrying the desired transgene in every cell)
transgenic offspring are born
• The method was successfully used in 1974 when a simian virus was
inserted into mice embryos, resulting in mice carrying this DNA.
• 3. Embryonic Stem Cell-Mediated Gene Transfer
• The presence of transgenes can be tested at the embryonic
state in this third method.
• This method involves:
• isolation of totipotent stem cells (stem cells that can develop
into any type of specialized cell) from embryos
• the desired gene is inserted into these cells
• cells containing the desired DNA are incorporated into the
host’s embryo, resulting in a chimeric animal
• Unlike the other two methods, which require live transgenic
offspring to test for the presence of the desired transgene, this
method allows testing for transgenes at the cell stage.
 Medical Applications
• Transplant organs may soon come from transgenic animals.
• a) xenotransplantation
Patients die every year for lack of a replacement heart, liver,
or kidney. For example, about 5,000 organs are needed each
year in the United Kingdom alone.
• Transgenic pigs may provide the transplant organs needed to
alleviate the shortfall.
• Currently, xenotransplantation is hampered by a pig protein
that can cause donor rejection but research is underway to
remove the pig protein and replace it with a human protein.
• Milk-producing transgenic animals are especially useful for
medicines.
• b) nutritional supplements and pharmaceuticals
Products such as insulin, growth hormone, and blood anti-clotting factors
may soon be or have already been obtained from the milk of transgenic
cows, sheep, or goats.
• Research is also underway to manufacture milk through transgenesis for
treatment of debilitating diseases such as phenylketonuria (PKU),
hereditary emphysema, and cystic fibrosis.
• In 1997, the first transgenic cow, Rosie, produced human protein-enriched
milk at 2.4 grams per litre. This transgenic milk is a more nutritionally
balanced product than natural bovine milk and could be given to babies or
the elderly with special nutritional or digestive needs. Rosie’s milk
contains the human gene alpha-lactalbumin.
• A transgenic cow exists that produces a substance to help human red cells
grow.
• c) human gene therapy
Human gene therapy involves adding a normal copy of a gene
(transgene) to the genome of a person carrying defective
copies of the gene. The potential for treatments for the 5,000
named genetic diseases is huge and transgenic animals could
play a role. For example, the A. I. Virtanen Institute in Finland
produced a calf with a gene that makes the substance that
promotes the growth of red cells in humans.
What are the ethical concerns surrounding transgenesis?
• These ethical issues, better served in their own article, include questions
such as:
• Ethical concerns must be addressed as the technology grows, including the
issue of lab animal welfare.
• Should there be universal protocols for transgenesis?
• Should such protocols demand that only the most promising research be
permitted?
• Is human welfare the only consideration? What about the welfare of other
life forms?
• Should scientists focus on in vitro (cultured in a lab) transgenic methods
rather than, or before, using live animals to alleviate animal suffering?
• Will transgenic animals radically change the direction of evolution, which
may result in drastic consequences for nature and humans alike?
• Should patents be allowed on transgenic animals, which may hamper the
free exchange of scientific research?