Unit 4

Department of Biotechnology, Dayananda Sagar College of Engineering
UNIT-4
TRANSGENIC SCIENCE AND GENETIC IMPROVEMENT
Topics:
4.1 Genetically Modified Organisms: Introduction
Plants as Bioreactors
4.2 i. Polymers
ii. Edible vaccines
Transgenic Plants
i. Stress tolerant plants
4.3
ii. BT Cotton
iii. Golden Rice
4.4 Transgenic Animals
Biosafety Guidelines
4.5
4.6. BT cotton in India– a case study
Dr N Rajeswari, DSCE, B’lore. Genetic Engineering & Applications (19BT5DCGEA)

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UNIT 4
TRANSGENIC SCIENCE AND GENETIC IMPROVEMENT
I. Transgenic Science in Plant Improvement
 During the last decades, a tremendous progress has been made in the development of transgenic
plants using the various techniques of genetic engineering.
 The plants, in which a functional foreign gene has been incorporated by any biotechnological
methods that generally are not present in the plant, are called transgenic plants.
 Transgenic plants have many beneficial traits like insect resistance, herbicide tolerance, delayed fruit
ripening, improved oil quality, weed control etc.
 Some of the commercially grown transgenic plants in developed countries are: “Roundup Ready”
soybean, ‘Freedom II squash’, ‘High- lauric’ rapeseed (canola), ‘Flavr Savr’ and ‘Endless Summer’
tomatoes. During 1995, full registration was granted to genetically engineered Bt gene containing
insect resistant ‘New Leaf’ (potato), ‘Maximizer’ (corn), ‘BollGard’ (cotton) in USA.
1. BioPharming-Plants as Bioreactors:
Plants are easy to grow and can generate considerable biomass. With these features in mind,
research has been carried out to determine whether transgenic plants can be used for the production
of commercial proteins and chemicals. Unlike recombinant bacteria, which are grown in large
bioreactors, a process that requires highly trained personnel and expensive equipment, crops can be
produced relatively inexpensively by less-skilled workers. In addition, when proteins that are
intended for human use are produced in transgenic plants, there is a significantly reduced risk of
mammalian virus contamination in comparison to proteins that are produced in animal cells grown
in culture. Ultimately, the biggest hurdle to overcome in the production of foreign proteins in plants
is the purification of the product of a transgene from the mass of plant tissue. On a laboratory scale,
plants have been used to produce monoclonal antibodies and antibody fragments; the polymer
polyhydroxybutyrate, which can be used to make a biodegradable plastic-like material; and a
number of potential therapeutic agents and vaccine antigens
a) Polymers:
It is costly to produce the polymer poly (3-hydroxybutyric acid), which is used in the synthesis of
biodegradable plastics, by bacterial fermentation. Consequently, research has been conducted to
determine if the polymer could be produced at a lower cost in plants.
 In bacteria, such as Alcaligenes eutrophus, poly (3-hydroxybutyric acid) is synthesized from acetyl
coenzyme A in three steps catalyzed by three enzymes (see Fig).
 The genes that encode these enzymes are organized on a single operon. Since plants are unable to
process the transcript of an operon with more than one gene, each of the three genes was isolated and
cloned separately into a plasmid.
 The genes were targeted to the chloroplast of the plant A. thaliana because previous experiments had
demonstrated that cytoplasmic synthesis produced only low levels of the polymer, and the transgenic
plants were highly stunted.
 Moreover, chloroplasts can accumulate high levels of starch, another biological polymer, so it was
thought that they would similarly be able to accumulate large amounts of poly (3-hydroxybutyric
acid). Unlike highly valued proteins that are used as therapeutics or specialty chemicals, biological
polymers need to be produced at high levels in plants for production to be economically feasible.
 Each of the three poly (3-hydroxybutyric acid) biosynthesis genes was fused to a DNA fragment that
encodes the chloroplast transit peptide of the small subunit of pea ribulose bisphosphate carboxylase
and was placed under the transcriptional control of the cauliflower mosaic virus 35S promoter.
 Separate plants were transformed with each construct with Ti plasmid binary vectors. Two transgenic
plants, each with a different foreign gene in its genomic DNA, were crossed to form a transgenic
plant with two foreign genes.

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 Then the double-gene-transgenic plant was crossed with a transgenic plant that carried the third
foreign gene, and a transgenic plant carrying all three of the bacterial poly(3-hydroxybutyric acid)
biosynthesis
genes was selected.
 Mature leaves of some of the triple-gene-transgenic plants produced more than 1 mg of poly (3-
hydroxybutyric acid) per gram (fresh weight) of leaf.
 Unfortunately, A. thaliana plants that produced very high levels of poly (3-hydroxybutyric acid)
were severely stunted.
 Nevertheless, this work is a first step in the development of plants that produce large amounts of poly
(3-hydroxybutyric acid). However, to realize the commercial potential of this system, these polymers
will have to be produced in plants other than A. thaliana so that there will be a much greater amount
of plant biomass produced.
b) Edible Vaccines:
Although considerable progress has been made in recent years in the development of new vaccines,
in many countries, either the vaccine itself is too expensive to be used on a large scale or there is a
lack of physical infrastructure (e.g., roads and refrigeration) that makes it impossible to disseminate
the vaccine. Commercial vaccines are expensive to produce and package and require trained
personnel to administer injections. Clearly, it would be advantageous if vaccines could be delivered
inexpensively on a broad scale in an edible form, e.g., as part of a fruit or vegetable. When vaccines
are taken orally, they can directly stimulate the immune system (Fig. 20.22). An edible vaccine, in
contrast to traditional vaccines, would not require elaborate production facilities, purification,
sterilization, packaging, or specialized delivery systems. Moreover, unlike many currently utilized
recombinant protein expression systems, plants glycosylate proteins, a factor that may contribute to
the immunogenicity and stability of a target protein.

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Much of the work on edible vaccines that has been reported so far utilizes potatoes as the delivery
vehicle. Potatoes were originally chosen for this work because they were easy to manipulate.
However, potatoes were never intended to be the vaccine delivery plant; they require cooking to
make them palatable, and cooking destroys (inactivates) most protein antigens. Plants that are being
considered for the delivery of edible vaccines include bananas (although banana trees require
several years to mature), tomatoes (although tomatoes spoil readily), lettuce, carrots, peanuts, and
corn (mainly for “vaccinating” animals).
Fig: Schematic representation of how an

edible vaccine generates an immune response
against an antigen from an infectious agent.
The ingested antigen, which is expressed as
part of a plant, binds to and is taken up by M
cells present in the lining of the intestine and
is then passed to other cells in the immune
system, including macrophages and B cells.
The macrophages display portions of the
antigen to the helper T cells, which in turn
respond by secreting small molecules that
activate B cells to synthesize and release
antibodies that can neutralize the antigen.
 Cholera is an infectious diarrheal disease caused by the enterotoxin produced by the gram-negative
bacterium Vibrio cholerae. Globally, there are more than 5 million cases and 200,000 deaths from
cholera each year.
 To develop an edible vaccine against V. cholerae, potato plants were transformed, using A.
tumefaciens, with the cholera toxin subunit B gene.
 Cholera toxin subunit B binds to an intestinal receptor; subunit A contains the toxin activity.
 One gram of transgenic potato produced approximately 30 μg of subunit B.
 After the transgenic potatoes were cooked in boiling water until they were soft enough to be edible
by humans, approximately 50% of the subunit B protein remained undenatured.
 The cooked potatoes were fed to mice once a week for 4 weeks before the mice were tested for
antibodies against the subunit B protein and for resistance to V. cholerae-caused diarrhea.
 These tests indicated that the mice had acquired a significant level of protection against V. cholerae.
 Moreover, although mucosal antibody titers declined gradually after the last immunization, they were
rapidly restored after an oral boost (an additional feeding) of transgenic potato.
 In an interesting variation of the strategy outlined above, the cholera toxin subunit B and A2 genes
were each fused to different antigen genes and then used to generate transgenic potato plants.
 To create these two fusion proteins, a 22-aminoacid epitope from murine (mouse) rotavirus
enterotoxin NSP4 was fused to the C-terminal end of the cholera toxin subunit B protein, and the
enterotoxigenic
E. coli fimbrial colonization factor CFA/I was fused to the N-terminal end of the cholera toxin
subunit A2 protein (Fig).

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Fig: Schematic representation of the association of

two components of an engineered vaccine. The
cholera toxin B subunit is synthesized as a fusion
protein to the rotavirus peptide, the cholera toxin A2
subunit is fused to the enterotoxigenic E. coli
fimbrial colonization factor, and the cholera toxin B
and A2 peptides are non-covalently attached to one
another. Both fusion proteins are made in transgenic
potatoes and combine to form a cholera toxin-like
protein, as shown.
 Normally, the A2 peptide links the A1 peptide, which has the toxic activity, with the subunit B
peptide, which has the binding activity.
 Transgenic potatoes that expressed the two fusion proteins were fed to mice, which generated
antibodies against cholera toxin subunit B protein, murine rotavirus enterotoxin NSP4, and E. coli
fimbrial colonization factor CFA/I and were protected against rotavirus-caused diarrhea.
 This approach holds great promise for the development of inexpensive and readily available vaccines
for a wide range of diseases.
 It has been estimated that Shiga toxin-producing strains of E. coli cause approximately 100,000 cases
of hemorrhagic colitis a year. About 6% of those infections produce severe complications, including
kidney failure.
 Similar to cholera toxin, the Shiga toxin contains one A subunit, which encodes the catalytic, or
toxin, activity, per five B subunits, which act (together) to bind to cellular surface receptors.
 To develop an oral vaccine against type 2 Shiga toxin (type 2 is responsible for the most severe
disease
in humans), the genes for a genetically inactivated version of the Shiga toxin A and B peptides were
both cloned and expressed in tobacco cells. To test the ability of transformed tobacco plants that
synthesized the modified Shiga toxin to protect mice against the toxin, scientists infected mice with
Shiga toxin-producing strains of E. coli.
 Before the introduction of the Shiga toxin-producing bacteria, some of the mice were fed leaves from
transgenic tobacco plants expressing the inactivated Shiga toxin once a week for 4 weeks, while
other mice were untreated.
 One week after the introduction of the toxic E. coli strain, all of the mice that were not fed the
antigen-producing tobacco had died. By contrast, 2 weeks after treatment with the toxic E. coli
strain, all of the orally vaccinated mice were still alive. This experiment serves as a proof of the
concept that oral administration of the inactivated Shiga toxin is a highly effective means of
protecting animals against Shiga toxin-producing E. coli. Of course, for a human oral vaccine, a
more suitable host plant, such as tomato or banana, would be desirable.
2. Resistance to Abiotic and Biotic stresses:

 Plants have evolved genetically based physiological strategies to react to changing environmental
conditions, be they abiotic or biotic.
 Most plants complete their life cycle in a single location and are therefore plagued by challenges such
as nutrient acquisition, pathogen attack, and environmental stresses.
 In response to nutrient limitation, some plants undergo physiological and developmental adaptations to
actively scavenge limited nutrients from their environment. These changes include adjustments in root
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architecture, induction of genes encoding high-affinity transporters (e.g. phosphate, nitrate and
sulphate transporters), rhizosphere acidification, and exudation of organic acids.
 However, typically plants simply tolerate stressful environments, which often improve over time.
They are not equipped to survive their life cycle under constant stress.
 Environmental factors which limit plant exploitation of the environment include Light, Oxidative
Stress, Cold, Heat, Nutrition, Water, Salinity, Toxic concentrations of Metals and Pathogens.
WHY IS THERE A NEED FOR STPs?
 Agricultural plant science has had two main goals for decades: to increase yield and quality of
agricultural products and to improve the protection of crops from diseases or limitations caused by
pathogens, insects, nutrients and stress. These goals have significant economic implications, which
are affected by environmental conditions
 One way of doing this is to develop crops that are more tolerant to such stresses as drought, flooding,
heat, radiation, salinity, chilling and freezing, so that new land can be brought under cultivation.
With the use of stress tolerant plants, there will be the possibility of using a lower quality of
irrigation water with a higher salt content. It may be possible to use less water over the course of the
season which would be an important factor as farmers face increasing competition for water
resources from municipal users.
 The goals of agricultural plant science are to increase crop productivity and the quality of
agricultural products and to protect the environment by maintaining a system of sustainable
agriculture that preserves the ecological basis of plant production. The inherent diversity among
plant species demonstrates clearly that plants are able to adapt to environmental stresses using
genetically based programs. Crop improvement, i.e. the optimization of plant features and
performance according to agricultural needs, has been undertaken for hundreds of years:
agronomists, breeders, and gardeners have used classical plant breeding methods based on selection
of natural variants to improve genetic sources.
 Methods and techniques developed in molecular biology in recent years, especially reliable
transformation systems for essentially all crops and growing numbers of complete genome sequences
of higher plants are providing tools to support plant breeding strategies and allowing scientists to
tackle as yet unsolved problems or to speed up breeding programs. Such tools will extend plant
breeding by introducing new, unanticipated traits, in order to develop plants in which both crop
productivity and stress tolerance are enhanced
Previous Methods
 Classical breeding programs develop new traits by combining different germ plasmas in order to
exploit natural or artificially induced diversity and, subsequently, to select for desired properties.
 Classical genetic methods based on crosses and selection schemes have made enormous
contributions towards stress-related crop improvement, tolerance to stress is generally considered a
quantitative trait, and it is difficult to isolate specific genes involved in stress tolerance
 The problem with traditional plant breeding is that it is time consuming and laborious; it is difficult
to modify single traits; and it relies on existing genetic variability. However, genetic engineering can
now be used as a relatively fast and precise means of achieving improved stress tolerance. Many
organisms have evolved traits that enable them to survive in extreme environments, and thus the
gene(s) that confer these properties can potentially be introduced into higher plants.
i) Development of Abiotic Stress and Senescence Tolerant Plants:
Abiotic stress is defined as the negative impact of non-living factors on the living organisms in a
specific environment. The non-living variable must influence the environment beyond its normal
range of variation to adversely affect the population performance or individual physiology of the
organism in a significant way.
Approaches for Resistance against Abiotic Stress:
a) Improving protection from stress. Eg. Oxidative stress is protected By SOD enzyme.
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b) Reducing sensitivity to stress. Eg. Drought tolerance, salt tolerance and chilling tolerance.
Genes Induced By Abiotic Stresses:
The product of genes whose expression is induced by abiotic stresses are classified in two groups.
Proteins that protect cell from dehydration.
 Enzyme involved in production of osmoprotectants
 Late embryogenesis abundant proteins
 Antifreeze proteins
 Chaperones
 Detoxifying enzymes
Proteins involved in inducing transcription of stress responsive genes.
 TFs
 Protein kinases
 Enzymes involved in phosphoinositide metabolism
a. Oxidative Stress Tolerant plants:

 Unlike many animals, plants cannot physically avoid adverse environmental conditions, such as high
levels of light, ultraviolet (UV) irradiation, heat, high salt concentrations, or drought, so
physiological strategies have evolved to cope with these stresses.
 At the molecular level, one of the undesirable consequences of physiological stress is the production
of oxygen radicals. Thus, investigators reasoned that if they could create plants that were able to
tolerate increased levels of oxygen radicals, these plants should also be able to withstand various
forms of environmental stress.
 A variety of biotic stresses, including salt, freezing, and drought, as well as exposure to pollutants,
stimulate the formation of reactive oxygen species in plant cells. These toxic molecules damage
membranes, membrane-bound structures, and macromolecules, including proteins and nucleic acids,
especially in the mitochondria and chloroplast, resulting in oxidative stress.
 A common type of potentially damaging oxygen radical is the superoxide anion. Within a cell under
oxidative stress, the enzyme superoxide dismutase detoxifies superoxide anion by converting it to
hydrogen peroxide, which in turn is broken down to water by various cellular peroxidases or
catalases.
Fig: Conversion of superoxide anion to

hydrogen peroxide and then to water
and oxygen.
• Plants have several different isoforms of the enzyme superoxide dismutase.

• Cu/Zn SOD found in cytoplasm and chloroplast
• Mn -SOD found in mitochondria
• Fe- SOD found in chloroplast
• Ni-SOD found in prokaryote
 In one study, tobacco plants that were transformed with a superoxide dismutase gene that was under
the control of the 35S promoter from cauliflower mosaic virus had reduced oxygen radical damage
under stress conditions compared with control plants.
 Transgenic tobacco plants that carried the cDNA for a chloroplast-localized Cu/Zn superoxide
dismutase under the control of the 35S promoter from cauliflower mosaic virus were much more
resistant to high-light damage than non-transformed plants.
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 When they were tested, the transgenic plants retained 94% of their photosynthetic activity under
conditions in which nontransformed plants lost all of their activity.
 In another experiment, transgenic plants with cloned Mn superoxide dismutase targeted to their
chloroplasts were three- to fourfold less sensitive to oxidative damage caused by ozone than
nontransformed plants.
 Oxidative stress may also be reduced if the level of oxidized glutathione within a plant is increased.
Glutathione peroxidase catalyzes the conversion of glutathione to oxidized glutathione by reacting
with organic peroxides and reducing them to organic alcohols.
Fig: Oxidation of glutathione to oxidized

glutathione and simultaneous reduction of an
organic peroxide to an organic alcohol,
catalyzed by glutathione peroxidase. -Glu,
glutamic acid residue linked to the next amino
acid through the gamma carboxyl group rather
than through the alpha amino group, as in a
usual peptide linkage; Cys, cysteine; Gly,
glycine.
 To test this idea, a tobacco cDNA encoding an enzyme with both glutathione S-transferase and
glutathione peroxidase activities was isolated. Transgenic tobacco plants that expressed glutathione
peroxidase were created using the isolated cDNA under the control of the 35S promoter, and the
construct was introduced into plants with a binary Ti plasmid system.
 The transformed plants had approximately twice the level of enzyme activity found in
nontransformed plants. Seedlings of these transgenic plants grew significantly faster than control
seedlings when exposed to either chilling or salt stress. The efficacy of this system remains to be
demonstrated in the field.
b. Salt Stress:
 Production of osmoprotective compounds: Osmoprotectants helps plants in two ways by -
a. Acting as a cytoplasmic osmolytes.
b. Protecting and stabilizing macromolecule from damage induced by abiotic stresses.
Genes for Glycinebetaine Biosynthesis-
- Effective osmolyte accumulated during water stress by Bacteria, Cyanobacteria and members
of Chenopodiacae.
- Several crop like potato, tomato, rice, tobacco do not accumulate it but can be made to
accumulate by transgenesis.
- It is obtained in two step-
Choline Monoxygenase Betaine aldehyde dehydrogenase

Choline Betaine Aldehyde Betaine
(CMO) (BADH)
Two enzyme are involved in glycinebetaine biosynthetic pathway.

 Choline Dehydrogenase (CDH) in E. coli and Choline monoxygenase in Spinach.
 Betainealdehyde dehydrogenase (BADH).

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- Bacterial CDH is most useful enzyme it not only catalyze the oxidation of choline into
betainealdehyde but also convert BA into glycinebetaine.
- E. coli betA gene encoding CDH has been cloned and used in transgenesis.
 Expression of catalase in salt tolerant transgenic plants: Catalases (CAT) are haem-containing
tetrameric enzymes involved in the removal of H 2O2. Plant catalases are involved in photorespiratory
functions and scavenging of H2O2 during β-oxidation of fatty acids in germinating seeds and also
during salt stress and other abiotic stress conditions. Catalase enzymes that can remove the Reactive
Oxygen Species (ROS) produced by the salt stress play an important role in tolerance. Thus
quenching of H2O2 was an important factor for salt tolerance as observed in cyanobacteria were able
to produce salt tolerant japonica rice (at 100 mM salt concentration) by over expressing the catalase
gene katE. katE, a catalase gene of Escherichia coli was introduced into the indica rice cultivar
Kasalath. Transgenic rice plants at a very young stage (three-four days) were able to grow up to 15
days in 100 mM NaCl solution and seven days in 250 mM NaCl solution whereas, control plants
died within five days in 100 mM and seven days in 50 mM NaCl. The katE gene was integrated into
BR5 rice plants using an Agrobacterium tumefaciens-mediated method. The introduced katE gene
was actively expressed in the transgenic BR5 rice plants and catalase activity in transgenic rice was
approximately 150% higher than in non-transgenic plants. Under NaCl stress conditions, the
transgenic rice plants exhibited high tolerance compared with non-transgenic rice plants.
Expression of glutathione s-transferase: Glutathione S-transferases (GSTs) are a family of
multifunctional enzymes that play important roles in oxidative stress resistance. The Key Laboratory
of Plant Stress, P. R. China, obtained a GST from Suaeda salsa cDNA library (accession number
BE859255) and showed that it played an important role in salt stress resistance proved that the
expression of Suaeda salsa GST gene in transgenic rice (Oryza sativa L. cv) could confer resistance
to salt stress. Overexpression of a tobacco glutathione S-transferase with glutathione peroxidase
activity (GST/GPX) in transgenic tobacco (Nicotiana tabacum L.) enhanced seedling growth under
salt stress conditions. The Suaeda salsa glutathione S-transferase gene (GST) introduced into
Arabidopsis under the control of the cauliflower mosaic virus 35S promoter. The expression of the
GST gene promoted a higher level of salt tolerance in vivo in transgenic Arabidopsis plants. Southern
and northern blot analyses confirmed that GST was transferred into the arabidopsis genome and the
GST and GPX activities in transgenic plants (GT) were much higher than in wild-type plants (WT)
Strategies to improve salt stress tolerance: Recent advances in plant genome mapping and
molecular biology techniques offer a new opportunity for understanding the genetics of salt stress-
resistance genes and their contribution to plant performance under salt stress. These biotechnological
advances will provide new tools for breeding in salt stress environment. Molecular genetic maps
have been developed for major crop plants, including rice, wheat, maize, barley, sorghum and potato,
which make it possible for scientists to tag desirable traits using known DNA landmarks. Molecular
genetic markers allow breeders to track genetic loci controlling stress resistance without having to
measure the phenotype, thus reducing the need for extensive field-testing over time and space.
Moreover, gene pyramiding or introgression can be done more precisely using molecular tags.
Together, molecular genetic markers offer a new strategy known as marker assisted selection.
Another molecular strategy which depends on gene cloning and plant transformation technology is
genetic engineering of selected genes into elite breeding lines. What makes a particular goal
attainable or unattainable in genetic engineering experiments is the availability of the following three
inputs: (1) the gene of interest, (2) an effective technique for transferring the desired gene from one
species to another and (3) promoter sequences for regulated expression of that gene. Amongst these,
the first is considered a rate-limiting factor. Arrays of salt stress-induced genes have been isolated.
Salt stress-responsive genes can be analyzed following targeted or non-targeted strategy. The
targeted approach relies upon the availability of relevant biochemical information (i.e., in terms of
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defined enzyme, protein, a biochemical reaction or a physiological phenomenon). The non-targeted

strategy to obtain a desired gene is indirect. This strategy, for instance, includes differential
hybridization and shotgun cloning. The list of genes, whose transcription is up regulated in response
to stress, is rapidly increasing. Understanding of the mechanisms which regulate gene expression and
the ability to transfer genes from other organisms into plants will expand the ways in which plants
can be utilized. To exploit the full potential of these approaches, it is essential that the knowledge is
applied to agriculturally and ecologically important plant species.
ii) Biotic Stress: Biotic stress is stress that occurs as a result of damage done to an organism by other
living organisms, such as bacteria, viruses, fungi, parasites, beneficial and harmful insects, weeds,
and cultivated or native plants. The types of biotic stresses imposed on an organism depend the
climate where it lives as well as the species' ability to resist particular stresses. Biotic stress remains a
broadly defined term and those who study it face many challenges, such as the greater difficulty in
controlling biotic stresses in an experimental context compared to abiotic stress.
a) Insect Resistant Plants:
Insect resistant transgenic plants are intrinsically resistant to insect predators.
Increasing Expression of the B. thuringiensis Protoxin
Strategies used to confer resistance against insect predators:
 A gene for insecticidal protoxin produced by one of the several species of Bacillus
thuringienesis.
 Genes for plant proteins such as α amylase inhibitors, protease inhibitors and lectins are
effective.
 Increasing the Expression of Bacillus thuringienesis protoxin:
 Bacillus thuringienesis protoxin does not persist in the environment, nor is it hazardous to mammals.
Thus, it is safe means of protecting plants.
 A number of economically significant pests of crop plants feed on internal plant tissues and are thus
unlikely to be inhibited by B thuringienesis preparations that have been sprayed onto the surface of
the plant.
 To avoid this problem, genes for B thuringienesis (BT) can be expressed in plants. This limits the
environmental distribution of the toxin and avoids the problems associated with its limited
environmental stability and the timing of toxin application.
 Utilizing the BT potoxin to create a transgenic plant that expresses and synthesizes functional form
of this insecticide at sufficient levels to prevent damage by insect predators.
 Initial experiments by B thuringienesis subspecies kurstaki insecticidal protein genes cry1Aa,
cry1Ab, and cryAc were not particularly well expressed in plants.
 To raise the level scientist truncated the gene so that only the N-terminal portion of the insecticidal
protein was produced---this part of the protoxin contains the toxin—and inserted a strong promoter
to direct gene expression. Under these conditions, there was a significant increase the level of ease in
the level of insecticidal toxin produced, affording transgenic plants some protection against damage
from insect predation.
 Minimum sequence that encoded toxin was determined. To this end, the amino acid sequences of
protoxin from various strains of B thuringienesis were compared to determine the common
insecticidal domain. The N-terminal portion of the protoxin molecule which is highly conserved
(~98%) and the C-terminal region is more variable (~45% conserved).
 The insecticidal toxin activity resides within the 1 st 646 aminoacids from the N-terminus of the 1,156
amino acid protoxin.
 This segment of protoxin gene that encodes the highly conserved amino acid was cloned and
expressed in bacteria, the shortened protein was as active as the native (protoxin) from in protecting
plants against lepidopteran insects in laboratory trails.
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 Eg., Transgenic tomato plants, alfalfa, apple, peanut, canola, cotton, potato, rice, eggplant, grape etc.
Fig:
1. Insect eats Bt crystals and spores.
2. The toxin binds to specific receptors in the
gut and the insects stops eating.
3. The crystals cause the gut wall to break
down, allowing spores and normal gut
bacteria to enter the body.
4. The insect dies as spores and gut bacteria
proliferate in the body.
Cry gene designation Toxic to these insect orders
CryIA(a), CryIA(b), CryIA(c) Lepidoptera

Cry1B, Cry1C, Cry1D Lepidoptera
CryII Lepidoptera, Diptera
CryIII Coleoptera
CryIV Diptera
CryV Lepidoptera, Coleoptera
• Bt crystals/insecticidal crystal proteins (ICP), are protein crystals formed during sporulation in some
Bt strains. Bt produces proteins that aggregate to form a crystal.
• These crystal proteins are toxic to very specific species of insects yet harmless to humans and the
natural enemies of many crop pests (beneficial insects). There are more than 150 insects that are
known to be susceptible in some way to Bt.
• The crystal proteins bind specifically to certain receptors in the insect's intestine. Not all insects carry
the same receptors allowing for high species specificity.
• Humans and other vertebrates do not have these receptors in their bodies, so the toxin is unable to
affect us.

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Increasing expression of the B. thuringiensis protoxin

• By site directed mutagenesis: Increasing expression of the B. thuringiensis protoxin by site
directed mutagenesis. Isolated insecticidal toxin gene was modified by site-directed mutagenesis to
change any DNA sequences that could inhibit efficient transcription or translation in n plant host.
• By codon bias changes: Increasing expression of the B. thuringiensis protoxin by codon bias
changes. A “fully” modified version of the insecticidal toxin gene was designed and chemically
synthesized. This gene was also modified to eliminate any potential messenger RNA (mRNA)
secondary structure or chance plant polyadenylation sequences, which might decrease gene
expression
• Under rbcs promoter: Increasing expression of the B. thuringiensis protoxin under ribulose
bisphospate carboxylase (rbcs) promoter. Fully modified protoxin gene was placed under the
control of the promoter for the gene that encodes for the small subunit of the enzyme rbcs and
downstream from the chloroplast transit peptide sequence of this enzyme, so that the overproduced
protoxin became localized within the chloroplast.
b) Other Strategies for Protecting Plants against Insects:

Plants have evolved general insect defense mechanisms that are sufficient for plant survival but
not always effective enough to keep the damage to a level that would be acceptable for crop
plants.
i) Protease inhibitors: Introduction of the potato proteinase inhibitor II gene provides rice plants with
protection against the pink stem borer (Sesamia inferens), a major insect pest of rice. Infestation of
rice plants by pink stem borers causes severe damage to the plants, often resulting in a hollow stem
and dead panicles with no seeds. A plasmid carrying the potato proteinase inhibitor II gene under the
control of its own promoter and transcription termination region was constructed. The plasmid also
contained the first intron from the rice actin gene inserted between the promoter and the potato
proteinase inhibitor II coding region (Fig. 19.4). This construct was introduced into rice suspension
cells by microprojectile bombardment, and transgenic plants were generated. When pink stem borer
larvae were artificially applied, 70 to 100% of the wild-type plants were severely damaged by insect
predation, while only 15 to 20% of the transgenic plants were damaged. Since plant proteinase
inhibitors are common components of both human and animal food and are readily inactivated by
cooking, their introduction into new crops can be regarded as safe.
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ii) α- amylase inhibitor gene: A gene for α -amylase inhibitor from the common bean isolated, placed
under transcriptional control of the strong seed-specific promoter for the bean phytohemagglutinin
gene, and used to transform pea plants (Pisum sativum). Peas are usually quite susceptible to damage
by both cowpea weevils and azuki bean weevils. However; transgenic pea plants that expressed the α
-amylase inhibitor were resistant to both of these insects.
d) Herbicide Resistant Plants: A significant fraction of global crop production is lost through weed
infestation every year, despite the expenditure of $10 billion on more than 100 different chemical
herbicides. In addition, many herbicides do not discriminate weeds from crop plants; others must be
applied early, before the weeds take hold; and some persist in the environment. The creation of
herbicide resistant crop plants is one way to overcome some of these drawbacks.
A number of different biological manipulations that would cause a crop plant to be herbicide
resistant can be envisioned.
 Inhibit uptake of the herbicide.
 Overproduce the herbicide-sensitive target protein so that enough of it remains available for
cellular functions despite the presence of the herbicide.
 Introduce a bacterial or fungal gene that produces a protein that is not sensitive to the herbicide
but performs the same function as the plant (herbicide-sensitive) protein.
 Reduce the ability of a herbicide-sensitive target protein to bind to a herbicide. Endow plants
with the capability to metabolically inactivate the herbicide.
Modification of the target by developing resistance to herbicides:

 Eg., Resistance against Glyphosphate (N-Phsphono Methyl Glycine).The most widely used herbicide
is glyphosate, which is considered to be safe, cheap, effective, and “environmentally friendly”
because it is readily degraded to nontoxic compounds in the soil. Glyphosate, trademarked as
Roundup by the Monsanto Corporation, inhibits a key enzyme in the shikimate pathway, 5-
enolpyruvylshikimate-3-phosphate synthase (EPSPS) that plays an important role in the synthesis of
aromatic amino acids in both bacteria and plants. Plants resistant to this herbicide have been
developed by putting an EPSPS-encoding gene from a glyphosate-resistant strain of E. coli under the
control of plant promoter and transcription termination–polyadenylation sequences and cloning the
construct into plant cells. Transgenic soybean, corn, canola, tobacco, petunia, tomato, potato, and
cotton plants that produce an amount of the resistant E. coli EPSPS sufficient to replace the inhibited
plant enzyme are resistant to the effects of glyphosate. Thus, in these cases, the crop plant would not
be affected by glyphosate treatment, whereas the weeds would be. Crops that have been engineered
to be resistant to glyphosate by this approach are said to be “Roundup ready.”
Detoxification or degradation of herbicide: Detoxification or degradation of the herbicide is the

basis of selective effect of herbicide, so that the herbicide kills the weeds but not the crop.
A number of detoxifying enzymes have been identified in plants and microbes. Some of these
enzymes are
 Glutathione-S-Transferase (GST) in maize which detoxidies herbicide—Atrazine.
 Nitilase: An enzyme that catalyzes the hydrolysis of nitriles to carboxylic acids and ammonia.
Resistance due to inactivation of bromoxynil (3, 5-dibromo-4-hydroxybenzonitrile), a herbicide that
acts by inhibiting photosynthesis, has been achieved for some plants. In this case, resistant plants
were created by the introduction of a bacterial gene that encodes the enzyme nitrilase, which can
inactivate bromoxynil before the herbicide can act. The gene bxn for nitrilase was isolated from the
soil bacterium Klebsiella ozaenae and placed under the control of the light-regulated promoter from
the small subunit of the enzyme ribulose bisphosphate carboxylase before it was transferred to
tobacco plants. As expected, the transgenic plants expressed nitrilase activity in their shoots and
leaves, but not in their roots, and were resistant to the toxic effects of the herbicide.

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Transgenic tomato, potato, soybeans, cotton, corn plants were developed.
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Examples: (short notes)
BT Cotton:
 BACILLUS thuringiensis or Bt is a naturally occurring soil bacterium used by farmers to control
Lepidopteran insects because of a toxin it produces. Through genetic engineering, scientists have
introduced the gene responsible for making the toxin into a range of crops, including cotton.
 Cotton and other monocultured crops require an intensive use of pesticides as various types of pests
attack these crops causing extensive damage. Over the past 40 years, many pests have developed
resistance to pesticides.
 So far, the only successful approach to engineering crops for insect tolerance has been the addition of
Bt toxin, a family of toxins originally derived from soil bacteria.
 The Bt toxin contained by the Bt crops is no different from other chemical pesticides, but causes
much less damage to the environment. These toxins are effective against a variety of economically
important crop pests but pose no hazard to non-target organisms like mammals and fish.
 Three Bt crops are now commercially available: corn, cotton, and potato. As of now, cotton is the
most popular of the Bt crops: it was planted on about 1.8 million acres (728437 ha) in 1996 and
1997.
 The Bt gene was isolated and transferred from a bacterium bacillus thurigiensis to American cotton.
The American cotton was subsequently crossed with Indian cotton to introduce the gene into native
varieties.
 The Bt cotton variety contains a foreign gene obtained from bacillus thuringiensis. This bacterial
gene, introduced genetically into the cotton seeds, protects the plants from bollworm (A.
lepidoptora), a major pest of cotton. The worm feeding on the leaves of a BT cotton plant becomes
lethargic and sleepy, thereby causing less damage to the plant.
 Field trials have shown that farmers who grew the BT variety obtained 25%–75% more
cotton than those who grew the normal variety.
 Also, BT cotton requires only two sprays of chemical pesticide against eight sprays for normal
variety. According to the director general of the Indian Council of Agricultural Research, India uses
about half of its pesticides on cotton to fight the bollworm menace.
 Use of BT cotton has led to a 3%–27 increase in cotton yield in countries where it is grown.
Golden Rice
 One of the most promising developments in the controversial field of genetic engineering is the
success obtained in breeding a nutritionally enriched rice variety now popularly being referred to as
'golden rice'.
 This golden rice is a genetically modified rice which contains genes that produce high levels of beta
carotene and related compounds.
 Beta carotene is contained in yellow fruits like carrots (from which it gets its name) and mangoes
and in vegetables like spinach. Beta carotene and other related compounds are converted in the
human body to the crucially needed vitamin A.
 Unfortunately, many in the developing world that do not have access to fruits and vegetables, suffer
from chronic vitamin A deficiency which results in night blindness.
 Night blindness plagues millions of undernourished people in Asia, including India, crippling their
lives. According to the WHO, vitamin A deficiency hits the poor in 96 countries of the world,
resulting in over five lakh blind children every year. This blindness is irreversible, these children will
never see.

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 The significance of this red- gold rice containing carotenoid genes obtained from the daffodil flower
is the potential it offers to counter vitamin A deficiency and prevent the severely debilitating
condition of night blindness.
 This promising rice variety contains enough precursors of vitamin A in one average portion of rice as
to prevent night blindness through ordinary dietary intake.
 The creators of golden rice are Ingo Potrykus of the Technical University, Zurich and Peter
Beyer of the University Freiburg. The research effort spanning ten years and costing several million
dollars was financed by the Rockefeller Institute.
Making of Golden Rice:
• Golden rice is a variety of rice (Oryza sativa) produced through genetic engineering to biosynthesize
the precursors of beta-carotene (pro-vitamin A) in the edible parts of rice.
• Golden rice was designed to produce Vitamin A precursor beta-carotene in the part of rice that
people eat, the endosperm.
• The rice plant can naturally produce beta-carotene, which is a carotenoid pigment that occurs in the
leaves and is involved in photosynthesis. However, the plant does not normally produce the pigment
in the endosperm since the endosperm is not a tissue in which photosynthesis takes place.
• Golden rice was created by transforming rice with two beta-carotene biosynthesis genes:
 psy (phytoene synthase) from daffodil (Narcissus pseudonarcissus)
 crt1 from the soil bacterium Erwinia uredovora
• IR-64 was selected as an experimental Rice plant.
• Three precisely well-defined genes that produce β-carotene (a provitamin Molecule) were
incorporated.
• Two genes were from daffodil plant (horticulture flowering plant) that produce β-carotene.
• One gene from bacterium Erwinia uredovera. This bacterial crt1 gene was an important inclusion to
complete the pathway, since it can catalyze multiple steps in the synthesis of carotenoids, while these
steps require more than one enzyme in plants.
• These genes were introduced into plasmids along with an endosperm specific promoter gene, so that
they are only expressed in the endosperm.
• This rDNA was introduced into A tumifacieans bacterium.
• Transformed were cultured to produce clone of these genes
• These cells and the rice cell embryos were brought together. Bacteria infect the embryos.
• These embryos with new genes were cultured to get rice plants which produced golden yellow seeds.
• The end product of the engineered pathway is lycopene, but if the plant accumulated lycopene the
rice would be red.
• Recent analysis has shown that the plant's endogenous enzymes process the lycopene to beta-
carotene in the endosperm, giving the rice the distinctive yellow colour for which it is named.
• The original Golden rice was called SGR1, and under greenhouse conditions it produced 1.6 µg/g of
carotenoids.
• This provitamin in rice in converted into Vitamin A when consumed in the body.
• Simplified overview of the carotenoid biosynthesis pathway in golden rice: The enzymes
expressed in the endosperm of golden rice, catalyze the biosyntheis of beta-carotene from
geranylgeranyl diphosphate. Beta-carotene is assumed to be converted to retinal and subsequently
retinol (vitamin A) in the animal gut.

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Antisense Technology
 Antisense refers to short DNA or RNA sequences, termed oligonucleotides, which are designed to be
complementary to a specific gene sequence. The goal is to alter specific gene expression resulting
from the binding of the antisense oligonucleotide to a unique gene sequence.
 Antisense technology was first effectively used in plants to alter the levels of various degradative
enzymes or plant pigments.
 In principle, antisense technology is supposed to prevent protein production from a targeted gene.
Proposed mechanisms include triplex formation, blocking RNA splicing, preventing transport of the
mRNA antisense complex into the cytoplasm, increasing RNA degradation, or blocking the initiation
of translation.
 Both RNAi (RNA interference) and antisense RNA induce destruction of mRNA in the cytoplasm
and inhibit or block production of protein for a particular function (e.g. inducing fruit ripening,
causing cancer etc.).

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1. RNAi: Double stranded RNA is introduced into a cell and gets chopped up by the enzyme dicer to
form siRNA. siRNA then binds to the RNA-induced silencing complex (RISC) and is unwound. The
anitsense RNA complexed with RISC binds to its corresponding mRNA which is then cleaved by the
enzyme slicer and makes it inactive.
2. Antisense RNA: Antisense RNA is a RNA strand which has a mirror image of nucleotide bases of
an mRNA strand. When an artificial gene (DNA) is introduced into a cell, it produces an antisense
RNA complementary to the cell's own mRNA and forms RNA duplex with the mRNA. The
formation of double stranded RNA inhibits gene expression ( = No production of functional
protein because protein synthesis requires single stranded mRNA molecule as a template).
Applications
i. Cancer gene therapy: RNAi and antisense RNA technologies have been used in reducing
expression of cancer causing genes in human cancer cell lines.
ii. Control of fruit ripening: Antisense RNA technology has been used to suppress expression of fruit
ripening genes to make the fruits stay longer in vine and extend marketing period. e.g. the flav'r
sav'r tomato (the first genetically modified food crop introduced in US market in 1995 by a
company called Calgene).
Low Ripening Tomato:
In Tomato in the later stages of ripening polygalactouronase gene is switched on coding for
polygalactouronase enzyme which breaks down polygalactouronic acid component of cell walls
resulting in softening which results in spoilt tomato.
• Time scale: ~8wks from start to finish with color & flavor changes associated with ripening.
• About this time the # of genes involved in later stages of ripening are switched on.
• Partial inactivation of polygalactouronase gene will slow tomato ripening
• Transgenic tomatoes were prepared containing antisense construct of the gene PG resulting in
reduced expression of polygalactouronase and slow ripening and fruit softening, improving
shelflife.
• 730 Bps Restriction fragment from normal polygalactouronase gene was isolated
• Attach poly A signal in beginning with a Caulifower mosaic virus promoter was ligated
• This construct was inserted into a Ti plasmid vector

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• The recombinant Ti plasmid with the gene was transfected to the tomato plant. Once in
plant--- synthesis of antisense RNA complimentary to polygalactouronase mRNA takes place
• This prevents translation of the target mRNA
Examples of Gene Substraction
• Polygalacturonase: Delay in fruit ripening
• Polyphenol oxidase: prevention of discoloration in fruits and vegetables
• Starch synthase: Reduction of starch content
• Chalcone synthase: Modification of flower color.
Bioethics in Plant genetic Engineering:

There are issues and concerns regarding the use of transgenic crops and their effects on the health
and the environment in general. The major concerns about GM crops and GM foods are:
a) Effect of GM crops on biodiversity and environment- As the GM crops are created artificially, there
is no natural process of evolution in their development. Hence, there is a question of this affecting
the biodiversity and overall effect on the environment.
b) The risk of transfer of transgene from GM crops to pathogenic microbes- Antibiotic marker genes
are used to identify and select the modified cells. If GM food containing antibiotic resistance marker
gene is consumed by animals and humans, there is a risk that the transgene will transfer from GM
food to microflora of human and animals. This may lead to the gut microbes to become resistant to
antibiotics.
c) The transfer of genes from animals into Gm crops for molecular farming may change the
fundamental vegetable nature of plants.
d) The GM crops may bring about changes in evolutionary patterns. The plants adapt to the changing
environment in the natural way by changing their genes and developing better races with superior
traits which ultimately leads to the development of evolved races and varieties. What will be the
evolutionary pattern of the GM crops? There are concerns about the effect of transgene flow from
GM crops to other non-GM plants and the alteration of these non-GM crops.
e) There is a risk of transferring allergens (usually glycoproteins) from GM food to human and animals.
f) There is a risk of “gene pollution” i.e. transfer of transgene of GM crop through pollen grains to
related plant species and development of super weeds.
g) There are also some religious issues related to the consumption of transgenic plants with animal
genes introduced into them, especially, for some strict vegetarian people and some ethnic groups
with certain food preferences and restrictions.
h) There is a need to study thoroughly as to how the genetically engineered plants will affect the
ecological balance, once they are released in the environment
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II. TRANSGENIC ANIMALS
Definition: 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.
Transgenic animals have a number of uses:

 Basic medical research: transgenic animals are used to study the function of specific factors in
biological systems.
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 Livestock improvement: Resistance to Infection, Growth efficiency.

 Toxicology: as sensitive test animals for the detection of toxic substances
 Genetics of evolution and embryology
 Molecular biology: analysis of the regulation of gene expression following a specific genetic
change
 Pharming: Production of biological preparations and testing the effects of medicines. Currently, for
example, we can produce large quantities of human enzymes (e.g. alpha-1-antitrypsin) in the milk
of transgenic sheep. The enzymes can then be used to treat people that have defects in their own
production of these substances.
 Diagnostic human immunoglobulins (monoclonal antibodies),
 Nutraceuticals.
 Biotechnology: production of specific proteins
 Xenotransplantation: Develop animals as organ donors
What is a transgene?
A transgene is an artificial gene for a known protein, a relatively short piece of DNA. The desire is to
insert the piece with synthetic, recombinant DNA into the DNA that is already present in the genome
of a living plant or animal.
The transgene must be expressed in the individual that receives it. The transgene usually codes for a
whole protein, but it can also code for a part of a protein that can change the protein's function. The
transgene is often linked to another gene whose only function is to help in the expression of the
transgene in the receiver, a so-called promoter and/or enhancer. The point of the promoter/enhancer
is mostly to ensure that the transgene is expressed in specific organs. We already know that this
promoter/enhancer functions in certain organs, while it does not work in other organs. In this
manner, genes can, for example, be linked to the prolactin-receptor promoter and be expressed
specifically in the mammary gland.
How are transgenic animals made?

Transgenic animals are usually made by one of three methods. Today these techniques are available
in the mouse, but are being developed in other species:
a) microinjection in a fertilized egg,
b) blastocyst injection, or
c) with the help of a retrovirus.
The first two techniques have somewhat different areas of use.

a) Microinjection involves the injection of the transgene into a fertilized egg such that it is in place
before cell differentiation starts. Cell differentiation is a process by which the cells organize
themselves and form organs, such as the liver, kidneys, and so on. All cells in all organs are
transgenic in the new individual following microinjection. The method gives little opportunity to
decide where the transgene is located in the genome of the recipient animal. The transgene can
destroy a vital gene in the cell in which it is injected. Thus, most eggs do not survive or do not
express the transgene. However, between 1% and 30% of eggs produce a live transgenic animal.
b) Blastocyst injection involves placing the transgene initially in an embryonic stem cell (ES cell). ES
cells are cells in culture that are able to develop into all types of tissues. After the cells are injected
with the transgene, and it has been demonstrated that the transgene is present in the ES cells, they are
injected into blastocysts.
The blastocyst stage is an early stage of fetal development at which the fetus is no more than a
cellular sac with a central cavity (both blast and cyst mean bud or sac). This is also long before cell
differentiation begins. The injected ES cells become part of the animal and the animal is a mosaic - a
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mixture - of transgenic and original cells. In the reproductive organs, the mosaic system results in
some sex cells being transgenic, while others are not. Through the inter-mating of mosaic animals
and the demonstration of transgenes in offspring, a homozygous transgenic animal can be
established.
c) A third method is to use a virus to insert the transgene into the mammalian cell. It is usual to use a
retrovirus for this, as such viruses can insert themselves into the host's genome. The virus genome is
then recombinant. Retrovirus-manipulated animals are also mosaic, and further breeding must occur
to establish a homozygous transgenic animal. Such animals can need special housing and
management because of the possibility of spread of infection.
Examples of producing Transgenic Animals:
Normal mice cannot be infected with polio virus. They lack the cell-surface molecule that, in
humans, serves as the receptor for the virus. So normal mice cannot serve as an inexpensive, easily-
manipulated model for studying the disease. However, transgenic mice expressing the human gene
for the polio virus receptor
 can be infected by polio virus and even
 develop paralysis and other pathological changes characteristic of the disease in humans.
Two methods of producing transgenic mice are widely used:
 transforming embryonic stem cells (ES cells) growing in tissue culture with the desired
DNA;
 injecting the desired gene into the pronucleus of a fertilized mouse egg.
The Embryonic Stem Cell Method (Method "1")
Embryonic stem cells (ES cells) are harvested from the inner cell mass (ICM) of mouse blastocysts.
They can be grown in culture and retain their full potential to produce all the cells of the mature
animal, including its gametes.
1. Make your DNA

Using recombinant DNA methods, build molecules of DNA containing
 the structural gene you desire (e.g., the insulin gene)
 vector DNA to enable the molecules to be inserted into host DNA molecules
 promoter and enhancer sequences to enable the gene to be expressed by host cells
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2. Transform ES cells in culture

Expose the cultured cells to the DNA so that some will incorporate it.
3. Select for successfully transformed cells.
4. Inject these cells into the inner cell mass (ICM) of mouse blastocysts.
5. Embryo transfer
 Prepare a pseudopregnant mouse (by mating a female mouse with a vasectomized male).
The stimulus of mating elicits the hormonal changes needed to make her uterus receptive.
 Transfer the embryos into her uterus.
 Hope that they implant successfully and develop into healthy pups (no more than one-third
will).
6. Test her offspring
 Remove a small piece of tissue from the tail and examine its DNA for the desired gene. No
more than 10–20% will have it, and they will be heterozygous for the gene.
7. Establish a transgenic strain
 Mate two heterozygous mice and screen their offspring for the 1:4 that will be homozygous
for the transgene.
 Mating these will found the transgenic strain.
The Pronucleus Method (Method "2")
1. Prepare your DNA as in Method 1
2. Transform fertilized eggs
 Harvest freshly fertilized eggs before the sperm head has become a pronucleus.
 Inject the male pronucleus with your DNA.
 When the pronuclei have fused to form the diploid zygote nucleus, allow the zygote to divide
by mitosis to form a 2-cell embryo.
3. Implant the embryos in a pseudopregnant foster mother and proceed as in Method 1.
An Example
This image (courtesy of R. L. Brinster and R. E. Hammer) shows a transgenic mouse (right) with a
normal littermate (left). The giant mouse developed from a fertilized egg transformed with a
recombinant DNA molecule containing:
 the structural gene for human growth hormone
 a strong mouse gene promoter
The levels of growth hormone in the serum of some of the transgenic mice were several hundred
times higher than in control mice.
Random vs. Targeted Gene Insertion

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The early vectors used for gene insertion could, and did, place the gene (from one to 200 copies of it)
anywhere in the genome. However, if you know some of the DNA sequence flanking a particular
gene, it is possible to design vectors that replace that gene. The replacement gene can be one that
 restores function in a mutant animal or
 knocks out the function of a particular locus.
In either case, targeted gene insertion requires
 the desired gene
 neor, a gene that encodes an enzyme that inactivates the antibiotic neomycin and its relatives,
like the drug G418, which is lethal to mammalian cells;
 tk, a gene that encodes thymidine kinase, an enzyme that phosphorylates the nucleoside
analog gancyclovir. DNA polymerase fails to discriminate against the resulting nucleotide
and inserts this nonfunctional nucleotide into freshly-replicating DNA. So ganciclovir kills
cells that contain the tk gene.
Step 1
Treat culture of ES cells with preparation of vector DNA.
Results:
 Most cells fail to take up the vector; these cells will be killed if exposed to G418.
 In a few cells: the vector is inserted randomly in the genome. In random insertion, the entire
vector, including the tk gene, is inserted into host DNA. These cells are resistant to G418 but
killed by gancyclovir.
 In still fewer cells: homologous recombination occurs. Stretches of DNA sequence in the
vector find the homologous sequences in the host genome and the region between these
homologous sequences replaces the equivalent region in the host DNA.
Step 2
Culture the mixture of cells in medium containing both G418 and ganciclovir.
 The cells (the majority) that failed to take up the vector are killed by G418.
 The cells in which the vector was inserted randomly are killed by gancyclovir (because they
contain the tk gene).
 This leaves a population of cells transformed by homologous recombination (enriched several
thousand fold).
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Step 3
Inject these into the inner cell mass of mouse blastocysts.
Transgenic Sheep and Goats
Until recently, the transgenes introduced into sheep inserted randomly in the genome and often
worked poorly. However, in July 2000, success at inserting a transgene into a specific gene locus
was reported. The gene was the human gene for alpha1-antitrypsin, and two of the animals
expressed large quantities of the human protein in their milk.
This is how it was done.
Sheep fibroblasts (connective tissue cells) growing in tissue culture were treated with a vector that
contained these segments of DNA:
1. 2 regions homologous to the sheep COL1A1 gene. This gene encodes Type 1 collagen. (Its absence
in humans causes the inherited disease osteogenesis imperfecta.)
This locus was chosen because fibroblasts secrete large amounts of collagen and thus one would
expect the gene to be easily accessible in the chromatin.
2. A neomycin-resistance gene to aid in isolating those cells that successfully incorporated the vector.
3. The human gene encoding alpha1-antitrypsin.
Some people inherit two non- or poorly-functioning genes for this protein. Its resulting low level or
absence produces the disease Alpha1-Antitrypsin Deficiency (A1AD or Alpha1). The main
symptoms are damage to the lungs (and sometimes to the liver).
4. Promoter sites from the beta-lactoglobulin gene. These promote hormone-driven gene expression in
milk-producing cells.
5. Binding sites for ribosomes for efficient translation of the mRNAs.
Successfully-transformed cells were then
 fused with enucleated sheep eggs and
 implanted in the uterus of a ewe (female sheep).
 Several embryos survived until their birth, and two young lambs have now lived over a year.
 When treated with hormones, these two lambs secreted milk containing large amounts of alpha1-
antitrypsin (650 µg/ml; 50 times higher than previous results using random insertion of the
transgene).
On June 18, 2003, the company doing this work abandoned it because of the great expense of
building a facility for purifying the protein from sheep's milk. Purification is important because even
when 99.9% pure, human patients can develop antibodies against the tiny amounts of sheep proteins
that remain.
However, another company, GTC Biotherapeutics, has persevered and in June of 2006 won
preliminary approval to market a human protein, antithrombin, in Europe. Their protein — the first
made in a transgenic animal to receive regulatory approval for human therapy — was secreted in the
milk of transgenic goats.
Transgenic Chickens
Chickens
 grow faster than sheep and goats and large numbers can be grown in close quarters;
 synthesize several grams of protein in the "white" of their eggs.
Two methods have succeeded in producing chickens carrying and expressing foreign genes.
 Infecting embryos with a viral vector carrying
o the human gene for a therapeutic protein
o promoter sequences that will respond to the signals for making proteins (e.g.
lysozyme) in egg white.
 Transforming rooster sperm with a human gene and the appropriate promoters and checking
for any transgenic offspring.

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Preliminary results from both methods indicate that it may be possible for chickens to produce as
much as 0.1 g of human protein in each egg that they lay.
Not only should this cost less than producing therapeutic proteins in culture vessels, but chickens
will probably add the correct sugars to glycosylated proteins — something that E. coli cannot do.
Transgenic Pigs
Transgenic pigs have also been produced by fertilizing normal eggs with sperm cells that have
incorporated foreign DNA. This procedure, called sperm-mediated gene transfer (SMGT) may
someday be able to produce transgenic pigs that can serve as a source of transplanted organs for
humans.
Transgenic Primates
In the 28 May 2009 issue of Nature, Japanese scientists report success in creating transgenic
marmosets. Marmosets are primates and thus our closest relatives (so far) to be genetically
engineered. In some cases, the transgene (for green fluorescent protein) was incorporated into the
germline and passed on to the animal's offspring. The hope is that these transgenic animals will
provide the best model yet for studying human disease and possible therapies.
Marker assisted selection for Livestock improvement:

 The idea behind marker assisted selection is that there may be genes with significant effects that may
be targeted specifically in selection.
 Some traits are controlled by single genes (e.g. hair colour) but most traits of economic importance
are quantitative traits that most likely are controlled by a fairly large number of genes.
 However, some of these genes might have a larger effect. Such genes can be called major genes
located
at QTL (quantitative trait locus). Although the term QTL strictly applies to genes of any effect, in
practice it refers only to major genes, as only these will be large enough to be detected and mapped
on the genome. Following the pattern of inheritance at such QTL might assist in selection.
 Marker assisted selection (MAS) is a combined product of traditional genetics and molecular
biology.
 MAS allows for the selection of genes that control traits of interest.
 Combined with traditional selection techniques, MAS has become a valuable tool in selecting
organisms for traits of interest, such as color, meat quality, or disease resistance.
 Marker assisted selection is the process of using the results of DNA testing in the selection of
individuals
to become parents for the next generations.
 The information from the DNA testing, combined with the observed performance records for
individuals, is intended to improve the accuracy of selection and increase the possibility of
identifying organisms carrying desirable and undesirable traits at an earlier stage of development.
 Complex traits, including many of economic importance, are controlled by many genes and are
influenced by the environment.
 When an animal has a favorable performance record for a certain trait, it means that based on
pedigree and phenotype, the animal has inherited a greater than average number of good alleles of
each gene affecting that specific trait. Marker used for are:
Molecular Markers
Until recently, researchers relied on information about how animals, plants, and their relatives
perform to make observations about the genes they possess. Today, researchers can use molecular
markers to find genes of interest that control how plants and animals perform. Some molecular
markers are pieces of DNA that have no known function or impact on animal and plant performance.
Other markers may involve the gene of interest itself.
Linked Markers
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One type of molecular marker is called a linked marker. Using well-designed experiments, scientists
can find molecular markers that are located very close to major genes of interest. The molecular
marker is said to be linked to that gene. Linked markers are only near the gene of interest on the
chromosome and are not part of the DNA of the gene of interest.
Direct Markers
A second kind of molecular marker is one that is part of the gene of interest. Direct markers are
easier to work with after they are found, but they often are more difficult to find than linked markers.
Marker-Assisted Selection
Three common technologies used as molecular markers are: restriction fragment length
polymorphisms, simple sequence repeats, and single nucleotide polymorphisms.
Examples:
a) The Story of Bovine Leukocyte Adhesion Deficiency (BLAD):
 By the year 1988, a genetic disease specific to Holstein cattle was claiming an ever-increasing
number of animals. Because Holsteins are a major breed in milk production throughout the world,
the disease was causing serious economic loss to the milk industry.
 The disease, called bovine leukocyte adhesion deficiency or BLAD, is characterized in young calves
by their inability to fight off common bacterial infections like pneumonia.
 Death usually occurs at an early age. BLAD is caused by a hereditary genetic mutation that disrupts
the function of a protein on white blood cells called leukocytes.
 Bulls are selected for breeding by evaluating the milk production of their female offspring. When a
bull has female offspring with superior milk production, its sperm are collected for use in artificial
insemination (AI). The benefit of AI is that one bull of superior genetics can improve the
performance of herds on many farms.
 One of the risks of AI is that if a sire is a heterozygous carrier of an undesirable recessive allele, that
allele can be spread undetected to many progeny.
 Breeders needed a reliable test to identify cattle that were heterozygous carriers. The test that was
developed was marker assisted selection.
 Breeders needed a reliable test to identify cattle that were heterozygous carriers. The test that was
developed was marker assisted selection.
 Scientists found that the deleterious recessive allele for BLAD had two mutations in the CD18 gene.
One of the mutations did not affect the amino acid sequence, but the second mutation caused an
incorrect amino acid to be produced. In that second mutation, the nucleotide guanine (G) replaced
adenine (A) so the amino acid glycine is produced instead of aspartic acid.
 The DNA strands from each allele contain the site of the BLAD mutation during the PCR process.
When these PCR products are treated with the restriction (cutting) enzyme TaqI, the enzyme
recognizes the TCGA sequence and cuts between the T and C nucleotides. Each strand will generate
DNA fragments consistent with the presence or absence of the restriction site TCGA on the strand. A
normal DNA sequence will contain a TaqI restriction site and generate two fragments, one of 26 base
pairs (bp) and the other 32 bp. In the case of the BLAD mutation, the restriction site for TaqI is lost.
Since there is no restriction site for TaqI on the mutation, a single fragment of 58 bp (the size of the
PCR product) remains. The presence of the 26, 32 and 58 bp fragments indicate the carrier.
 The protein with the amino acid change prevents leukocytes from reaching and destroying the
invading antigen by interfering with their ability to adhere to the blood vessel walls at the area of
infection. This is why calves with BLAD cannot fight infections and die early in life.
 Using molecular marker technology, it has been possible to identify the heterozygous carriers of
BLAD and remove those individuals from the breeding stock.

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 As a result, the disease has been virtually eliminated from the Holstein cattle breed. The defective
allele causing BLAD has not been found in breeds other than Holsteins. However, a similar form of
the genetic disorder has been described in humans.
b) Masitits:
 Although mastitis in cattle is an important factor for dairy economy and animal welfare and although
udder health parameters have a substantial genetic variability, in many countries there is little or no
improvement of udder health in the conventional commercial breeding programs.
 Infection of Udder caused by several different pathogens, which is a common disease in dairy cattle.
 The consequence is decreased milk yield, increased veterinary treatment, and in many cases death of
cattle.
 Linkage mapping citation studies for the data collected from half-sibling cows from the same sire
have identified as an association between putative QTL on chromosome 3, 4, 14, 27 associated with
incidence of mastitis.
 More commonly, SCSs (somatic cell scores)—indicator of incidence of mastitis
 The SCSs is a measure of infalmmatory response occurring in the udder, possibly following the
invasion of a pathogen and, thus does not necessarily indicate clinical or sub-clinical infection.
 Direct link between QTL for SCSs, and resistance and susceptibility to mastitis was not
demonstrated.
 For poorly defined traits, such as mastitis resistance, it is difficult to select candidate genes, as
different pathogens and physiological mechanisms contribute to observed variation.
 One possible candidate locus is the major histocompatibility locus (MHC).
 The MHC region includes genes that code for cell surface proteins involved in binding peptides (eg.,
peptides from pathogens) and presenting them correctly to cells of immune system so that an
appropriate immune response can be initiated.
 Thus MHC is a good candidate locus when considering variation in the immune response or
resistance to disease challenge.

c) Gene mapping in Fur-bearing animals:
 The genetic map of mink consists of 74 genes marking all chromosomes except the Y and the fox
map contains 35 genes marking 15 of 16 autosomes and the X chromosome.
 However, until recently there has been little information about the structure of fur color coding genes
and their chromosomal localizations in mink and fox.
 Comparisons of the mink gene map with those of man and other mammals have revealed the
presence of giant conserved gene region, presumably derived from a common ancestral genome.
 The fox genome contains fewer such regions. The existence of conserved regions of syntenic genes
in phylogenetically remote species allows comparative mapping to be used as a powerful tool for a
targeted search for the location of important genes in for-bearing animals.
Short Notes:
Nutraceuticals
What are Nutraceuticals?
 A nutraceutical is a food with a medical-health benefit, including the prevention and treatment of
disease. The term was coined in the late 1980s by Stephen DeFelice, M.D., founder and chairman of
the Foundation for Innovation in Medicine.

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 Such foods also commonly are referred to as functional foods, signifying they and/or their
components may provide a health benefit beyond basic nutrition. Examples include fruits and
vegetables as well as fortified or enhanced foods. While all foods are functional in that they provide
nutrients, nutraceuticals contain health-promoting ingredients or natural components that have a
potential health benefit for the body. “Functional” attributes of many traditional foods are being
discovered, while new food products are being developed with beneficial.
 The concept of nutraceuticals is not entirely new, although it has evolved considerably over the
years. In the early 1900s, food manufacturers in the United States began adding iodine to salt in an
effort to prevent goiter (an enlargement of the thyroid gland), representing one of the first attempts at
creating a functional component through fortification. Today, researchers have identified hundreds of
compounds with functional qualities, and they continue to make new discoveries surrounding the
complex benefits of phytochemicals (non-nutritive plant chemicals that have protective or disease
preventive properties) in foods.
 Nutraceuticals are hugely popular among consumers in the U.S. and other parts of the world.
American sales for 2003 were an estimated $31 billion, and that figure is expected to grow
substantially over the following several years. Nutraceuticals are one of the fastest-growing segments
of the food industry, especially among affluent baby boomers.
 In Japan, England and other countries, nutraceuticals already have become part of the dietary
landscape. Consumer interest in the relationship between diet and health has increased the demand
for information on nutraceuticals. Rapid advances in science and technology, increasing health care
costs, changes in food laws affecting label and product claims, an aging population and rising
interest in attaining wellness through diet are among the factors fueling U.S. interest in
nutraceuticals. Credible scientific research indicates many potential health benefits from food
components. These benefits could expand the health claims now permitted to be identified by the
Food and Drug Administration (FDA).
Traditional vs. Nontraditional
 Nutraceuticals on the market today consist of both traditional foods and non-traditional foods.
Traditional nutraceuticals are simply natural, whole foods with new information about their potential
health qualities. There has been no change to the actual foods, other than the way the consumer
perceives them. Many — if not most — fruits, vegetables, grains, fish, dairy and meat products
contain several natural components that deliver benefits beyond basic nutrition, such as lycopene in
tomatoes, omega-3 fatty acids in salmon or saponins in soy. Even tea and chocolate have been noted
in some studies to contain health-benefiting attributes.
 Nontraditional nutraceuticals, on the other hand, are foods resulting from agricultural breeding or
added nutrients and/or ingredients. Agricultural scientists are able to boost the nutritional content of
certain crops through the same breeding techniques that are used to bring out other beneficial traits in
plants and animals — everything from beta-carotene-enriched rice to vitamin-enhanced broccoli and
soybeans. Research currently is being conducted to improve the nutritional quality of many other
crops.
 Foods specially formulated with nutrients or other ingredients include products such as orange juice
fortified with calcium, cereals with added vitamins or minerals and flour with added folic acid. In
fact, more and more foods are being fortified with nutrients and other physiologically active
components (such as plant stanols and sterols) as researchers uncover more evidence about their role
in health and disease-risk reduction.
 Tomatoes and salmon are two types of food that researchers have found to contain benefits beyond
basic nutrition — in this case, lycopene and omega-3 fatty acids, respectively.
APPLICATIONS OF NUTRACEUTICALS
 Current Applications

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 Numerous nutraceuticals currently are on the market. The following chart represents a sample of
available nutraceuticals, their components and their potential human health benefits.
CLASS/COMPONENTS SOURCE POTENTIAL BENEFIT

Carotenoids
1. Beta-carotene Carrots, various fruitsNeutralizes free radicals, which may damage
cells; bolsters cellular antioxidant defenses
2. Lycopene Tomatoes and processed May contribute to maintenance of prostate
tomato products health
Dietary Fiber
Insoluble fiber Wheat bran May contribute to maintenance of a healthy
digestive tract
Fatty Acids
Monosaturated fatty acids Tree nuts May reduce risk of coronary heart disease
Flavonoids
Flavonols Onions, apples, tea, Neutralize free radicals, which may damage
broccoli cells; bolster cellular antioxidant defenses
Isothiocyanates
Sulforaphane Cauliflower, broccoli, May enhance detoxification of undesirable
cabbage, kale, horseradish compounds and bolster cellular antioxidant
defenses
Phenols
Caffeic acid, ferulic acid Apples, pears, citrus fruits, May bolster cellular antioxidant defenses; may
some vegetables contribute to maintenance of vision and heart
health
Plant Stanols/Sterols
Stanol/sterol esters Fortified table spreads, May reduce risk of coronary heart disease
stanol ester dietary
supplements
Polyols
Sugar alcohols (xylitol, Some chewing gums and May reduce risk of dental caries (cavities)
sorbitol, mannitol, lactitol) other food applications
Prebiotics/Probiotics
Lactobacilli, Yogurt, other dairy and May improve gastrointestinal health and
bifidobacteria nondairy applications systematic immunity
Phytoestrogens
Is flavones (daidzein, Soybeans and soy-based May contribute to maintenance of bone health,
genistein) foods healthy brain and immune functions; for
women, maintenance of menopausal health
Soy Protein
Soy protein Soybeans and soy-based May reduce risk of coronary heart disease
foods
III. PRODUCTION OF ANTIBIOTICS BY MICROORGANISMS

 ‘Antibiotics’ are biochemicals secreted by microorganisms which, in low concentration, inhibit the
growth or kill other microorganisms, i.e., the antibiotics are ‘antimicrobial agents of microbial
origin’.

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 Antibiotics are produced by a number of microorganisms and inhibit the growth of other
microorganisms even at very low concentrations. As such, the antibiotics have found wide
application in chemotherapy, plant pathology, food preservation, veterinary medicine and as research
tools in biochemistry and molecular biology. At present about 7000 antibiotics are known and about
100 of these are produced commercially by microbial fermentation process.
 The first antibiotic to be isolated was penicillin which was discovered by Alexander Fleming in 1929
and was produced on large scale using cultures of Penicillium notatum during 1940's. During World
War II, the demand for chemotherapeutic agents to treat wound infections led to the development of
a production process for penicillin and the beginning of an era of antibiotic research. This continues
to be the most fascinating area of microbial biotechnology even today.
 Fungi, bacteria and actinomycetes are important antibiotic producing organisms. Most species of
Streptomyces are quite active in the production of a variety of antibiotics. The status of the
production of different antibiotics in India.
Some Microorganisms and Antibiotics Produced by Them -
Organism Antibiotic
Pencillium Penicillin
Bacillus Licheniformis Bacitracin
Cephalosporium acremonium Cephalosporin
Nocordia uniformis Norcardins
S. caespitosus Actinomycins
Streptomyces antibiotieus Mitomycin
S. erythreus Erythromycin
S. griseus Streptomycin, cycloheximide
S. virginae Virginiamycin.
The most important aspect of microbial antibiotic production is the selection of high yielding strain
and a suitable fermentation medium. Since antibiotics are the secondary metabolites of an organism,
it is necessary to limit growth of the microorganism and to divert the organism from primary to
secondary metabolism. This is usually achieved by designing the medium in such a way that a key
nutrient is exhausted at appropriate time, producing the desired metabolic switch or diversion.
Steps for commercial production of antibiotics:
i) Raw Materials
The compounds that make the fermentation broth are the primary raw materials required for
antibiotic production. This broth is an aqueous solution made up of all of the ingredients necessary
for the proliferation of the microorganisms. Typically, it contains a carbon source like molasses, or
soy meal, both of which are made up of lactose and glucose sugars. These materials are needed as a
food source for the organisms. Nitrogen is another necessary compound in the metabolic cycles of
the organisms. For this reason, an ammonia salt is typically used. Additionally, trace elements
needed for the proper growth of the antibiotic-producing organisms are included. These are
components such as phosphorus, sulfur, magnesium, zinc, iron, and copper introduced through water
soluble salts. To prevent foaming during fermentation, anti-foaming agents such as lard oil,
octadecanol, and silicones are used.
ii) The Manufacturing Process
Although most antibiotics occur in nature, they are not normally available in the quantities necessary
for large-scale production. For this reason, a fermentation process was developed. It involves
isolating a desired microorganism, fueling growth of the culture and refining and isolating the final
antibiotic product. It is important that sterile conditions be maintained throughout the manufacturing
process, because contamination by foreign microbes will ruin the fermentation.

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Starting the culture

 Before fermentation can begin, the desired antibiotic-producing organism must be isolated
and its numbers must be increased by many times. To do this, a starter culture from a sample
of previously isolated, cold-stored organisms is created in the lab. In order to grow the initial
culture, a sample of the organism is transferred to an agar-containing plate. The initial culture
is then put into shake flasks along with food and other nutrients necessary for growth. This
creates a suspension, which can be transferred to seed tanks for further growth.
 The seed tanks are steel tanks designed to provide an ideal environment for growing
microorganisms. They are filled with the all the things the specific microorganism would
need to survive and thrive, including warm water and carbohydrate foods like lactose or
glucose sugars. Additionally, they contain other necessary carbon sources, such as acetic
acid, alcohols, or hydrocarbons, and nitrogen sources like ammonia salts. Growth factors like
vitamins, amino acids, and minor nutrients round out the composition of the seed tank
contents. The seed tanks are equipped with mixers, which keep the growth medium moving,
and a pump to deliver sterilized, filtered air. After about 24-28 hours, the material in the seed
tanks is transferred to the primary fermentation tanks.
Fermentation
 The fermentation tank is essentially a larger version of the steel, seed tank, which is able to
hold about 30,000 gallons. It is filled with the same growth media found in the seed tank and
also provides an environment inducive to growth. Here the microorganisms are allowed to
grow and multiply. During this process, they excrete large quantities of the desired antibiotic.
The tanks are cooled to keep the temperature between 73-81° F (23-27.2 ° C). It is constantly
agitated, and a continuous stream of sterilized air is pumped into it. For this reason, anti-
foaming agents are periodically added. Since pH control is vital for optimal growth, acids or
bases are added to the tank as necessary.
Isolation and purification
 After three to five days, the maximum amount of antibiotic will have been produced and the
isolation process can begin. Depending on the specific antibiotic produced, the fermentation
broth is processed by various purification methods. For example, for antibiotic compounds
that are water soluble, an ion-exchange method may be used for purification. In this method,
the compound is first separated from the waste organic materials in the broth and then sent
through equipment, which separates the other water-soluble compounds from the desired one.
To isolate an oil-soluble antibiotic such as penicillin, a solvent extraction method is used. In
this method, the broth is treated with organic solvents such as butyl acetate or methyl isobutyl
ketone, which can specifically dissolve the antibiotic. The dissolved antibiotic is then
recovered using various organic chemical means. At the end of this step, the manufacturer is
typically left with a purified powdered form of the antibiotic, which can be further refined
into different product types.
Refining
 Antibiotic products can take on many different forms. They can be sold in solutions for
intravenous bags or syringes, in pill or gel capsule form, or they may be sold as powders,
which are incorporated into topical ointments. Depending on the final form of the antibiotic,
various refining steps may be taken after the initial isolation. For intravenous bags, the
crystalline antibiotic can be dissolved in a solution, put in the bag, which is then hermetically
sealed. For gel capsules, the powdered antibiotic is physically filled into the bottom half of a
capsule then the top half is mechanically put in place. When used in topical ointments, the
antibiotic is mixed into the ointment.
 From this point, the antibiotic product is transported to the final packaging stations. Here, the
products are stacked and put in boxes. They are loaded up on trucks and transported to

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various distributors, hospitals, and pharmacies. The entire process of fermentation, recovery,
and processing can take anywhere from five to eight days.
Quality Control
Quality control is of utmost importance in the production of antibiotics. Since it involves a
fermentation process, steps must be taken to ensure that absolutely no contamination is introduced at
any point during production. To this end, the medium and all of the processing equipment are
thoroughly steam sterilized. During manufacturing, the quality of all the compounds is checked on a
regular basis. Of particular importance are frequent checks of the condition of the microorganism
culture during fermentation. These are accomplished using various chromatography techniques.
Also, various physical and chemical properties of the finished product are checked such as pH,
melting point, and moisture content.
In the United States, antibiotic production is highly regulated by the Food and Drug Administration
(FDA). Depending on the application and type of antibiotic, more or less testing must be completed.
For example, the FDA requires that for certain antibiotics each batch must be checked by them for
effectiveness and purity. Only after they have certified the batch can it be sold for general
consumption.
The Future
Since the development of a new drug is a costly proposition, pharmaceutical companies have done
very little research in the last decade. However, an alarming development has spurred a revived
interest in the development of new antibiotics. It turns out that some of the disease-causing bacteria
have mutated and developed a resistance to many of the standard antibiotics. This could have grave
consequences on the world's public health unless new antibiotics are discovered or improvements are
made on the ones that are available. This challenging problem will be the focus of research for many
years to come.
Properties of useful antibiotic:

To be truly useful, an antibiotic must possess following qualities in it:
(i) Antibiotic should be of “broad spectrum”, i.e., it should have the ability to inhibit/kill a
number of different types of pathogenic microorganisms.
(ii) Is should check the development of resistant forms of pathogenic microorganisms
It should not result in undesirable side effects in the host.
(iii) It should not disturb the “normal” microbial flora of the host. Such a disturbance may
upset the balance of nature.
Examples:
a) Antibiotics of Fungal Origin Penicillin:
Alexander Flemming discovered penicillin secretion by the mould Penicillium notatum in 1929. He
reported that a contaminating colony of the fungus lysed adjacent colonies of staphylococci; but the
lytic agent seemed too unstable to be useful. However, when Chain (1939) purified the active
material, called penicillin, it proved remarkably effective in certain infections.Penicillin is not a
single chemical compound but a group of substances of related structure and activity. There are six
penicillins : Penicillin G (benzyl penicillin V (phenoxymethyl penicillin), Penicillin F (A2 – pentenyl
penicillin), penicillin X (p-hydroxybenzyl penicillin), penicillin K(n-heptyl penicillin) and
penicillion O (allyl-mercaptomethyl penicillin).
Penicillin is selective for Gram-positive bacteria, some spirochaetes and the Gram-negative
diplococci (Neisseria).
b) Cephalosporins
These are a group of antibiotics obtained from Cephalosporium acremonium, a marine fungus. They
are effective against gram-negative bacteria. Cephalosporidine, cephaloglycin and cephalexin are
some derivative compounds prepared from cephalosporins. These antibiotics, like penicillin, block
the cell-wall synthesis.
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c) Antibiotics of Bacterial Origin Antibiotics Obtained from Streptomyces, an actinomycete

Streptomycin
The antibiotics is obtained from Streptomyces griseus. Its antibiotic activity was first reported by
Waksman in 1944. Streptomycin is a “broad spectrum” antibiotic; inhibits many gram-negative
bacteria, several species of Mycobacterium including. M. tuberculosis. It also inactivates
bacteriophages. Streptomycin in commercially produced by the strains of the bacterium is a
fermentation medium consisting of soybean meal, glucose and mineral salts. The pH of the medium
is maintained at 7.5. The fermentation takes place under submerged conditions at 25 to 30°C for 5-7
days. When the fermentation is complete, the masses of bacterium are separated and the antibiotics is
obtained from the fermentation broth using organic solvents.
IV. Monoclonal Antibodies:

Introduction: Antibodies are proteins produced by the B lymphocytes of the immune system in
response to foreign proteins, called antigens. Antibodies function as markers, binding to the antigen
so that the antigen molecules can be recognized and destroyed by phagocytes. The part of the antigen
that the antibody binds to is called the epitope. The epitope is thus a short amino acid sequence that
the antibody is able to recognize. Two features of the antibody-epitope relationship are key to the use
of monoclonal antibodies as a molecular tool.
 specificity -- the antibody binds only to its particular epitope
 sufficiency -- the epitope can bind to the antibody on its own, i.e. the presence of the whole
antigen molecule is not necessary
 Each B cell in an organism synthesizes only one kind of antibody. In an organism, there is an
entire population of different types of B cells and their respective antibodies that were
produced in response to the various antigens that the organism had been exposed to. However
to be useful as a tool, molecular biologists need substantial amounts of a single antibody (and
that antibody alone). Therefore we need a method to culture a population of B cells derived
from a single ancestral B cell, so that this population of B cells would allow us to harvest a
single kind of antibody. This population of cells would be correctly described as monoclonal,
and the antibodies produced by this population of B cells are called monoclonal antibodies.

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In contrast, antibodies obtained from the blood of an immunized animal are called polyclonal
antibodies.
Production of monoclonal antibodies
 The production of monoclonal antibodies was pioneered by Georges Kohler and Cesar Milstein in
1975. Let us see how their method, now tried and tested for over 20 years, would be applied in a
particular case.
 In order for us to isolate a B lymphocyte producing a certain antibody, we first have to induce the
production of such a B cell in an organism. For example, if we need an antibody for avian SERCA2
protein, we would inject the protein into a mouse. This is typically done in two doses, an initial
"priming" dose and a second "booster" dose 10 days later (Campbell MA, pers. comm.). Since the
protein is of foreign origin, the mouse immune system recognizes it as such and soon some of the B
cells in the mouse would begin production of the antibody to avian SERCA2.
 A sample of B cells is extracted from the spleen of the mouse and added to a culture of myeloma
cells (cancer cells). The intended result is the formation of hybridomas, cells formed by the fusion
of a B cell and a myeloma cell. The fusion is done by using polyethylene glycol, a virus or by
electroporation (Campbell MA, pers. comm.)
 The next step is to select for the hybridomas. The myeloma cells are HGPRT- and the B cells are
HGPRT+. HGPRT is hypoxanthine-guanine phosphoribosyl transferase, an enzyme involved in the
synthesis of nucleotides from hypoxanthine, an amino acid (Biotech Resources, 1995-7). The culture
is grown in HAT (hypoxanthine-aminopterin-thymine) medium, which can sustain only HGPRT+
cells (Biotech Resources, 1995-7). The myeloma cells that fuse with another myeloma cell or do not
fuse at all die in the HAT medium since they are HGPRT-. The B cells that fuse with another B cell
or do not fuse at all die because they do not have the capacity to divide indefinitely. Only
hybridomas between B cells and myeloma cells survive, being both HGPRT+ and cancerous.
 The initial collection of B cells used is heterogenous, i.e. they do not all produce the same antibody.
Therefore the hybridoma population too does not produce a single antibody. There is also another
complication. A hybridoma cell is initially tetraploid, having been formed by the fusion of two
diploid cells. However the extra chromosomes are somehow lost in subsequent divisions in a random
manner (Campbell MA, pers. comm.). This means that we cannot be certain that the hybridomas will
all produce the desired antibody or even any antibody at all. Screening is required to decide which
hybridoma cells are actually producing the desired antibody.
 Each hybridoma is cultured and screened after doing SDS-PAGE (sodium dodecyl sulfate -
polyacrylamide gel electrophoresis) and Western blots. The probe used is the epitope of the antibody
that is desired, which may be labeled by radioactivity or immunofluorescence. Once we are sure that
a certain hybridoma is producing the right antibody, we can culture that hybridoma indefinitely and
harvest monoclonal antibodies from it.
Uses of monoclonal antibodies

Monoclonal bodies have a variety of academic, medical and commercial uses.
Antibodies are used in several diagnostic tests to detect small amounts of drugs, toxins or hormones,
e.g. monoclonal antibodies to human chorionic gonadotropin (HCG) are used in pregnancy test kits
(Biotech, 1989). Another diagnostic uses of antibodies is the diagnosis of AIDS by the ELISA test.
 Antibodies are used in the radioimmunodetection and radioimmunotherapy of cancer, and some
new methods can even target only the cell membranes of cancerous cells (Chaudhari et al, 1994).
A new cancer drug based on monoclonal antibody technology is Ritoxin, approved by the FDA in
November 1997.
 Monoclonal antibodies can be used to treat viral diseases, traditionally considered "untreatable".
In fact, there is some evidence to suggest that antibodies may lead to a cure for AIDS.

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 Monoclonal antibodies can be used to classify strains of a single pathogen, e.g. Neisseria
gonorrhoeae can be typed using monoclonal antibodie.
 Researchers use monoclonal antibodies to identify and to trace specific cells or molecules in an
organism, e.g. developmental biologists at Tyhe University of Oregon use monoclonal antibodies
to find out which proteins are responsible for cell differentiation in the respiratory system.
 OKT3, an antibody to the T3 antigen of T cells, is used to alleviate the problem of organ rejection
in patients who have had organ transplants.
Monoclonal vs Polyclonal Antibodies

A continuing question facing the developer of an immunoassay is whether to use monoclonal (MAb)
or polyclonal (PAb) antibodies in the assay. Polyclonal antibodies are the natural mixture of
antibodies resulting from the immune response to an antigen. A family of antibodies results, each
binding specifically to a different antigenic determinant (or part of a determinant) on the same
antigen. Subsets of polyclonal antibodies can also exist in which all the antibodies are specific for
one antigenic site (epitope), but having varying avidities for that site.
Whereas such diversity in the immune response may have evolved as a protective means to
the host animal, PAbs present problems to the immunoassay developer looking for high antibody
specificity and low total protein. For example, in most crude antisera,>90% of the antibodies present
have no or very low avidity for the antigen. This means that extensive purification must be used to
isolate the specific antibodies. Routine purification measures cannot, however, separate the resulting
specific antibodies further according to the specific determinant they bind to on the antigen. As a
result, it is difficult to use the antibodies from the same preparation in an assay requiring two
antibodies owing to differences in avidities between the antibodies and cross-reactivity problems.
3. Clearing Oil spills:

 Oil spills: An oil spill is a release of a liquid petroleum hydrocarbon into the environment due to
human activity, and is a form of pollution. The term often refers to marine oil spills, where oil is
released into the ocean or coastal waters.

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 Oil spills include releases of crude oil from tankers, offshore platforms, drilling rigs and wells, as
well as spills of refined petroleum products (such as gasoline, diesel) and their by-products, and
heavier fuels used by large ships such as bunker fuel, or the spill of any oily refuse or waste oil.
Spills may take months or even years to clean up.
 Cleanup and recovery from an oil spill is difficult and depends upon many factors, including the type
of oil spilled, the temperature of the water (affecting evaporation and biodegradation), and the types
of shorelines and beaches involved.
Methods for cleaning up include:
 Bioremediation: use of microorganisms or biological agents to break down or remove oil.
 Bioremediation Accelerator: Oleophilic, hydrophobic chemical, containing no bacteria, which
chemically and physically bonds to both soluble and insoluble hydrocarbons. The bioremedation
accelerator acts as a herding agent in water and on the surface, floating molecules to the surface of
the water, including solubles such as phenols and BTEX, forming gel-like agglomerations.
Undetectable levels of hydrocarbons can be obtained in produced water and manageable water
columns. By overspraying sheen with bioremediation accelerator, sheen is eliminated within
minutes. Whether applied on land or on water, the nutrient-rich emulsion creates a bloom of local,
indigenous, pre-existing, hydrocarbon-consuming bacteria. Those specific bacteria break down
the hydrocarbons into water and carbon dioxide, with EPA tests showing 98% of alkanes
biodegraded in 28 days; and aromatics being biodegraded 200 times faster than in nature they also
sometimes use the hydrofireboom to clean the oil up by taking it away from most of the oil and
burning it.
 Cleanup and recovery from an oil spill is difficult and depends upon many factors, including the
type of oil spilled, the temperature of the water (affecting evaporation and biodegradation), and
the types of shorelines and beaches involved.
 Pseudomonas putida is a gram-negative rod-shaped saprotrophic soil bacterium. Based on 16S
rRNA analysis, P. putida has been placed in the P. putida group, to which it lends its name.
 It is the first patented organism in the world. Because it is a living organism the patent was
disputed and brought before the United States Supreme Court in the historic court case Diamond
v. Chakrabarty which the inventor, Ananda M. Chakrabarty, won. It demonstrates a very diverse
metabolism, including the ability to degrade organic solvents such as toluene. This ability has
been put to use in bioremediation, or the use of microorganisms to biodegrade oil.
Superbug: solution to the oil pollution

Over 10 million metric tons of petro-pollutants enter the sea ecosystem each year and killing birds,
shell fish, fishes, invertibrates and other sea animals and planktons. This poses more adverse effect
on marine life and food chain.
To solve the problem of sea petro-pollution, the Indian-born American scientist, Dr. Anand Mohan
Chakravarty succeded in producing a microbacterial SUPERBUG by genetically engineered strain
of Pseudomonas putida. It is capable of utilising complex chemical compounds like hydrocarbons. It
is also called oil eating bug.
Petro-products, including oils, genrally contains octane, xylene, camphor and naphthalene. No single
strain of P. putida is able to digest all these four petro-chemical; for this needed four strains of P.
putida with four different plasmid DNA's. Dr. Anand introduced four plasmid DNA's from four
different strains into one single cell of Pseudomonas putida and formed a SUPERBUG.
Pseudomonas is an aerobic and gram-negative bacterium belongs to Pseudomonadaceae family of

bacteria. P. aeruginosa and P. fluorescens are fluorescent pigment forming bacteria.

35
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This superbug utilise a number of toxic chemicals like chlorobenzenes, 2.4-D, 2.4.5-T, and petro-
chemicals like octane, camphor, etc. This discovery of superbug really open the door for checking
oil-pollution in seas.

36

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Department of Biotechnology, Dayananda Sagar College of Engineering

4.6. BT cotton in India– a case study

Dr N Rajeswari, DSCE, B’lore. Genetic Engineering & Applications (19BT5DCGEA)

Dr N Rajeswari, DSCE, B’lore. Genetic Engineering & Applications (19BT5DCGEA)

Dr N Rajeswari, DSCE, B’lore. Genetic Engineering & Applications (19BT5DCGEA)

Fig: Schematic representation of how an

Dr N Rajeswari, DSCE, B’lore. Genetic Engineering & Applications (19BT5DCGEA)

Fig: Schematic representation of the association of

2. Resistance to Abiotic and Biotic stresses:

a. Oxidative Stress Tolerant plants:

Fig: Conversion of superoxide anion to

• Plants have several different isoforms of the enzyme superoxide dismutase.

Fig: Oxidation of glutathione to oxidized

- It is obtained in two step-

Choline Monoxygenase Betaine aldehyde dehydrogenase

Two enzyme are involved in glycinebetaine biosynthetic pathway.

Dr N Rajeswari, DSCE, B’lore. Genetic Engineering & Applications (19BT5DCGEA)

defined enzyme, protein, a biochemical reaction or a physiological phenomenon). The non-targeted

Cry gene designation Toxic to these insect orders

CryIA(a), CryIA(b), CryIA(c) Lepidoptera

Dr N Rajeswari, DSCE, B’lore. Genetic Engineering & Applications (19BT5DCGEA)

Increasing expression of the B. thuringiensis protoxin

b) Other Strategies for Protecting Plants against Insects:

Modification of the target by developing resistance to herbicides:

Detoxification or degradation of herbicide: Detoxification or degradation of the herbicide is the

Dr N Rajeswari, DSCE, B’lore. Genetic Engineering & Applications (19BT5DCGEA)

Transgenic tomato, potato, soybeans, cotton, corn plants were developed.

Dr N Rajeswari, DSCE, B’lore. Genetic Engineering & Applications (19BT5DCGEA)

Dr N Rajeswari, DSCE, B’lore. Genetic Engineering & Applications (19BT5DCGEA)

Dr N Rajeswari, DSCE, B’lore. Genetic Engineering & Applications (19BT5DCGEA)

Dr N Rajeswari, DSCE, B’lore. Genetic Engineering & Applications (19BT5DCGEA)

Bioethics in Plant genetic Engineering:

Transgenic animals have a number of uses:

 Livestock improvement: Resistance to Infection, Growth efficiency.

How are transgenic animals made?

The first two techniques have somewhat different areas of use.

1. Make your DNA

2. Transform ES cells in culture

Random vs. Targeted Gene Insertion

Dr N Rajeswari, DSCE, B’lore. Genetic Engineering & Applications (19BT5DCGEA)

Dr N Rajeswari, DSCE, B’lore. Genetic Engineering & Applications (19BT5DCGEA)

Marker assisted selection for Livestock improvement:

Dr N Rajeswari, DSCE, B’lore. Genetic Engineering & Applications (19BT5DCGEA)

Dr N Rajeswari, DSCE, B’lore. Genetic Engineering & Applications (19BT5DCGEA)

Dr N Rajeswari, DSCE, B’lore. Genetic Engineering & Applications (19BT5DCGEA)

CLASS/COMPONENTS SOURCE POTENTIAL BENEFIT

III. PRODUCTION OF ANTIBIOTICS BY MICROORGANISMS

Dr N Rajeswari, DSCE, B’lore. Genetic Engineering & Applications (19BT5DCGEA)

Dr N Rajeswari, DSCE, B’lore. Genetic Engineering & Applications (19BT5DCGEA)

Starting the culture

Dr N Rajeswari, DSCE, B’lore. Genetic Engineering & Applications (19BT5DCGEA)

Properties of useful antibiotic:

c) Antibiotics of Bacterial Origin Antibiotics Obtained from Streptomyces, an actinomycete

IV. Monoclonal Antibodies:

Dr N Rajeswari, DSCE, B’lore. Genetic Engineering & Applications (19BT5DCGEA)

Uses of monoclonal antibodies

Dr N Rajeswari, DSCE, B’lore. Genetic Engineering & Applications (19BT5DCGEA)

Monoclonal vs Polyclonal Antibodies

3. Clearing Oil spills:

Dr N Rajeswari, DSCE, B’lore. Genetic Engineering & Applications (19BT5DCGEA)

Superbug: solution to the oil pollution

Pseudomonas is an aerobic and gram-negative bacterium belongs to Pseudomonadaceae family of