CHAPTER ONE

1.0 INTRODUCTION

Over the last three decades biotechnology has advanced to a level where it is generally feasible
to make particular changes to the genome, and therefore to the expressed characteristics of
living organisms. The product of such a change is called a transgenic or a genetically modified
organism. A transgenic bacteria is an bacteria whose genome has been altered by the inclusion
of foreign genetic material. The purpose of adding a new gene to an organism’s genome is to
have the organism produce a protein or set of proteins that it did not produce before the gene
was added or manipulated (Aine and Meghan, 2005).

Improvement of bacteria production by transgenesis is still in infancy and despite its intensive
use, bacteria transgenesis is still suffering from technical limitations. The generation of
transgenesis has recently become easier or possible for different species thanks to the use of
transposons or retrovirus, to incubation of sperm which DNA followed by fertilization by
intracellular sperm injection or not and to the use of the cloning technique using somatic cells
in which genes has been added or in activated. All these techniques are expected to offer
experimenters new and more precise models to study gene function even in large bacterias.
Improvement of breeding by transgenesis has become more reasonable including through the
precise allele replacement in farm bacterias (Louis, 2002).

Transgenic bacterias represent unique models that are custom tailored to address specific
biological questions.

Hence, the ability to introduce functional genes into bacterias provides a very powerful tool
for dissecting complex biological processes and systems. This has made it possible to explore
the regulation of gene expression as well as the regulation of cellular and physiological
processes. Significant uses of live transgenic mammals are in the arenas of agricultural,
biological, biotechnological from transgenic bacterias to humans and biomedical sciences
including production of pharmaceuticals and in the field of organ transfer (Wall and Seidel,
1992). Therefore the objectives of this review paper are:

_ To highlight the application of transgenic bacterias in biomedicine

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_ To review available literatures on the methods of producing transgenic bacteria

_ To assess and describe the feature prospect regarding transgenic bacterias.

Biotechnology is not new. Cheese, bread, alcoholic beverages and yoghurt are products of
(traditional) biotechnology and have been known for centuries. But recent developments in
molecular biology have given biotechnology new meaning, new prominence, and new
potential. It is (modern) biotechnology that has captured the attention of the public and can
have a dramatic effect on the world economy and the society. Genetic engineering which is an
example of modern biotechnology is the process of transferring individual genes between
organisms or modifying the genes in an organism to remove or add a desired trait or
characteristic. Through genetic engineering, genetically modified (GM) crops or organisms are
formed. These GM crops or GMOs are used to produce biotech-derived foods. It is this
specific type of modern biotechnology that seems to generate the most attention and concern
by consumers and consumer groups. What is interesting is that modern biotechnology is far
more precise than traditional forms of biotechnology. It allows for the transfer of only one or a
few desirable genes, thereby permitting scientists to develop crops with specific beneficial
traits and reduced undesirable traits. Traditional biotechnology such as cross-pollination in
corn produces numerous, nonselective changes.

Actually, it can more accurately be said to originate with the publication of the Cohen and
Boyer paper in 1973. Since then, the field of agricultural biotechnology (Ag Biotech) has
grown rapidly over the last half-century.

Modern agricultural biotechnology has offered opportunities to produce more nutritious and
better tasting foods, higher crop yields and plants that are naturally protected from diseases
and insects. Genetic modifications have produced fruits that can ripen on the vine for better
taste, yet have longer shelf lives through delayed pectin degradation. Similarly, introducing
genes that increase available iron levels in rice three-fold is a potential remedy for iron
deficiency, a condition that affects more than two billion people and causes anemia in about
half that number. Most of the today’s hard cheese products are made with a biotech enzyme
called chymosin, produced by genetically engineered bacteria which are considered more pure
and plentiful than its naturally occurring counterpart, rennet, which is derived from calf
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stomach tissue (Kass, 2005). More so, the starch content of Russet Burbank potato has been
successfully increased in 1992, by Monsanto Company through gene transfer. Such a gene
from a bacterium increased the starch content of the potato. The advantage is that the higher
starch content reduces oil absorption during frying , thereby lowering the cost of processing
French fries and chips, with a concomitant reduction of the fat content in the finished product.

Sub-Saharan Africa is considered one of the world’s food insecure regions. The available
statistics indicates worsening scenario. Africa’s overall food production capacity is said to be
increasing at the rate of 1.4% while its population is expanding at about 2.4% per year (FAO,
2000). The continuing decline in food production will have to be reversed if massive food
insecurity, poverty, social and political instability are to be averted. Green revolution type
technologies requiring increased land, water and fertilizer use may not be appropriate for sub-
Saharan Africa due to resource limitations and population pressure. Biotechnology application
may be considered to be more appropriate to solving our agricultural and poverty reduction
problems, so as it minimizes inputs while increasing yields (Alhassan, 2002). Reduced
pesticide use can mean more abundant and diverse bacteria and beneficial insect populations,
cleaner water, fewer containers for disposal, and reduced fuel use for pesticide applications.
Improved weed control enhances conditions for no-till farming, which greatly reduces soil
erosion and pre-emergent pesticide use.

Improved crop yields mean that the acreage available for production is used in a more efficient
way (Bridges et al., 2003). Biotechnology is currently changing the way plants and bacterias
are grown, boosting their value to growers, processors, and consumers. The most common
transgenic varieties available today are those that tolerate proprietary brands of herbicides, and
those that contain insecticide genes.

In the developing nations, small and peasant farmers are the primary producers of staple foods.
This sector, which is so important for food production, is itself characterized by poverty and
hunger. Thus rural areas in the developing nations are today characterized by extreme
inequalities in access to land, insecurity of land tenure and in the quality of land farmed.
Furthermore, by keeping wages and living standards low, the elite guarantees that healthy
domestic markets will never emerge. The result is a downward spiral into deeper poverty and

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marginalization. One irony of our world is that food and other farm products flow from areas
of hunger and need to areas where money is concentrated.

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 CHAPTER TWO

2.0 TRANSGENIC BACTERIA DESCRIPTION AND CONSTRUCTION

A transgenic bacteria is an bacteria that carries foreign DNA along with its original DNA. The
foreign gene is inserted into the bacteria’s genome by methods of recombinant DNA
techniques. This allows the bacterial to have an altered DNA that will produce chemicals that
it otherwise would not produce through natural means. This process can yield many benefits
such as creating disease models for mimicking human disorders, to providing organ donors for
transplantations, to serving as food, to enhancing bacteria milk that produces life saving
pharmaceuticals in it. This chapter will discuss the methods for creating transgenic bacterial,
and the assays used for their screening.

2.1 DNA

DNA, or deoxyribonucleic acid, is one of the two types of molecules that encode genetic
information (RNA being the other). DNA is the material that stores and transfers genetic
characteristics in all life forms, and is the primary component of chromosomes. DNA is a long
double-stranded polymer that is held together by hydrogen bonds between base pairs of
nucleotides. DNA is polymerized from monomer units called nucleotides, denoted as rungs in
the ladder in the figure. These nucleotides are found in four different structures: adenine and
guanine are purin bases; thymine and cytosine are pyrimidine bases. The adenine always pairs
with thymine, and guanine always pairs with cytosine. The nucleotides form a paired structure
which creates a double helix. The DNA is compacted with proteins, including histones, to
make chromosomes (DNA, 2005).

Genes are the basic hereditary factor in DNA. It is a segment of DNA that contributes to the
organism’s phenotype or function. Genes have distinct sequences of nucleotides that allow the
DNA segment to produce its necessary proteins. The region of the gene that codes for the
production of a protein are called exons. These exons contain codes that are used in specific
portions for the completion of the protein. Introns are the parts of a gene that are initially
transcribed into the primary RNA transcript along with the exons, but are subsequently

5
removed to make the mature protein are called introns. The introns are removed when the
exons are spliced together.

2.1.1 RECOMBINANT DNA TECHNOLOGY

Recombinant DNA technology was first developed in the early 1970's by Paul Berg, Herbert
Boyer, and Stanley Cohen, when they were investigating ways to recombine the DNAs of
microorganisms. In order to produce a transgenic bacteria, its DNA must be manipulated along
with foreign DNA. This method of genetic engineering, manipulation of DNA, is called
recombinant DNA (rDNA). This transgenic methodology has now been applied to many other
organisms such as fungi, mice, plants, pigs, sheep, fish, and even cows. Recombinant
technology may be used to better understand human illnesses (as with Alzheimer’s mouse) or
even to have bacterial produce essential life saving drugs by inserting genes encoding
pharmaceutical enzymes into an bacteria genome. This technology is the first step in cloning
genes.

Recombinant DNA technology allows for short specific portions of DNA to be inserted into a
vector which aids the DNA amplification. Plasmids are commonly used as vectors since they
are easy to manipulate. Plasmids are molecules of DNA that are found in bacteria but occur
separately from the bacterial chromosome (Figure-2). Plasmids are small, circular, and usually
carry only few genes. The inserted DNA (shown as blue in the figure) replicates in the
plasmid's cytoplasm, so a large number of DNA copies are created. The DNA may then be
purified from the bacteria, and inserted into the host bacteria.

Restriction enzymes are used to excise a piece of human DNA encoding the transgene (upper
right in the figure). The restriction enzymes cut the DNA at specific sequences, and this allows
the DNA to be inserted at the proper position in the vector. A ligase enzyme is then used to
seal the two pieces of DNA together, creating the recombinant DNA molecule. To better
understand this process, think of the restriction enzymes as enzymatic scissors for cutting the
DNA, and the ligase enzyme as glue to hold the mixed DNAs in place.

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2.2 METHODS OF TRANSGENESIS

The establishment of stable transgenic bacterias implies that the foreign DNA is present in
gametes or one cell embryos to allow its transmission to progeny. To reach this goal, the
foreign gene can be transferred using different methods according to bacteria species.
Currently, many techniques are used to create these useful bacterias, and the five most popular
techniques; DNA microinjection into the oocyte pronucleus, DNA transfer using ES cells,
DNA transfer using retro viral vector, the use of transposon and DNA transfer by nuclear
transfer (Louis, 2002).

2.2.1 DNA MICROINJECTION

DNA microinjection has become the most commonly applied method for gene transfer in
bacterias. Using DNA microinjection, mouse was the first bacteria to undergo successful gene
transfer. Microinjection of embryos with DNA has been the traditional approach for
generating transgenic livestock. For processes such as cellular or pronuclear injection the
target cell is positioned under the microscope and two micromanipulators one holding the
pipette and other one, holding a micro capillary needle usually between 0.5 to 5 μm in
diameter (larger if injecting stem cells into an embryo) are used to penetrate the cell membrane
and/or the nuclear envelope (David and Matthew, 2013).

2.2.2 THE USE OF TRANSPOSONS

Transposons are short genomic DNA regions (About 1-3 kb) that autoreplicate and are
randomly integrated as multiple copies within the same genome. Integration is achieved by the
integrase enzyme encoded by the transposon itself. Foreign DNA can be introduced into the
transposon to replace the integrase gene, and the recombinant transposon can be microinjected
into one-cell embryos along with the transposon integrase enzyme.

Using this method, the foreign gene can be integrated into the embryo genome insects, silk
worms, chicken, fish and mammals (Sumiyama et al., 2010).

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2.2.3 DNA TRANSFER INTO GAMETES

One logical approach to generate transgenic bacterias may theoretically consist of introducing
foreign DNA in gametes before fertilization. The incubation of spermatozoa in the presence of
DNA followed by invivo or invitro fertilization led to the generation of transgenic mice, fish,
chicken, rabbits, pigs, sheep and cows (Sato et al., 2002).

However, the results obtained with this method are inconsistent. The yield of transgenic
bacterias is usually low and largely unpredictable. Moreover, the integrated DNA is most of
the times profoundly rearranged and no more functional. This phenomenon can seemingly be
greatly attenuated by selecting the most appropriate ejaculates and by removing DNAse by
repeated washing and addition of DNAse inhibitors (Baccetti and Spadafora, 2000).

This method in its improved version might become attractive in future for some species. The
spermatozoa were incubated with the antibody-DNA complex and further used to fertilize
oocyte by in vitro fertilization in mouse, by artificial insemination in chicken and by injection
into uterine horn in pig. In all cases, up to 30% of born bacterias were transgenic and the trans-
genes were expressed and transmitted to progeny without any rearrangement. Interestingly the
same antibody recognized the same antigen in several mammals including human and in lower
vertebrates. This method might greatly simplify gene addition in bacterias (Qian et al., 2001).

2.2.4 THE USE OF RETRO VIRAL VECTORS

A retrovirus is a virus that carries its genetic material in the form of RNA rather than DNA.
Retroviruses are used as vectors to transfer genetic material into the host cell. The result is a
chimera, an organism consisting of tissues or parts of diverse genetic constitution. A chimera
is an bacteria that consists of two or more tissues that have different genetic compositions
produced by genetic engineering. This means that some of the cells are transgenic and some of
the cells in the organism are not (Margawit and Endag, 2003).

The chimeric mouse was constructed by introducing cells from one strain of mouse into the
embryos of another strain of mouse by microinjection into the blastocyst stage embryo which
is a day-5 embryo. The embryo was then implanted into a surrogate mother, and allowed to

8
grow into a chimeric adult mouse that exhibited characteristics of each strain of mouse (Aine
and Meghan, 2005).

Retroviral vectors are under intensive study to tentatively transfer gene to human somatic cells
and proceed to gene therapy. Similar vectors have been designed to transfer foreign genes into
mammalian embryonic cells. A family of vectors capable of infecting chicken primordial germ
cells and of generating transgenic bacterias has been described. This approach appears much
simpler than microinjection and might become used extensively in future (Lois et al., 2002).

2.2.5 GENE TRANSFER USING EMBRYONIC CELLS

Gene replacement by homologous recombination is performed in routine in bacteria and yeast.

It can be achieved in somatic mammalian cells although with a relatively poor efficiency. This
approach is very attractive since it can lead to specific gene inactivation, to targeted point
mutation in an bacteria genome or to the replacement of a given gene by a non-related one.
Cells in which it occurred must be selected and further used to generate a living embryo.

This proved to be feasible on condition to use embryonic stem cells capable of forming
chimeric embryos after microinjection into blastocysts or morula (Collodi, 2001).

Although laborious this protocol has become popular and genes are frequently inactivated in
mouse. Despite repeated efforts, the extension of this method to species other than mouse is
failed. This is clearly due to the fact that the recombined ESCs have more or less the capacity
to participate to the development of chimeric embryos but that transmission of the mutation to
progeny has been observed so far only in two mouse lines (Smithies, 2001).Two recent studies
indicate that chicken cultured primordial germ cells retransferred to embryos can participate to
their development and transmit their genes to progeny (Petitte et al., 2002).

2.2.6 GENE TRANSFER BY NUCLEAR TRANSFER

This method was extended to sheep about 15 years ago but the success was restricted to
experiments in which cells used as nuclear donors were taken from early embryos and not
previously cultured. These observations gave credence to the idea that bacteria cloning by
nuclear transfer was possible only with nuclei from totipotent or pluripotent cells. A

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systematic study carried out in sheep revealed that fully differentiated cells as well as
embryonic or foetal cells could successfully give their genes to generate normal bacterias The
conditions defined for cloning bacterias starting from differentiated cells was retained soon
after to generate transgenic sheep by gene addition (Schnieke et al., 1997). On the other hand,
it has been repeatedly observed that cells from adults and cells cultured for long periods of
time is a less efficient material for cloning by nuclear transfer. The reasons why these
phenomena occur are unknown. Mutation of essential genes for embryo development may
occur progressively in living bacterias and in cultured cells (Kang et al., 2004).

Gene addition by the cloning technique has been extended to goat, cow (Arat et al., 2001).
This suggests that gene replacement will become a reality in several species other than mouse
in the coming years (Dai et al., 2002).

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 CHAPTER THREE

3.0 MAKING OF TRANSGENIC BACTERIAS

There are three types of laboratory bacteria models mentioned in the literature. They are
spontaneous, induced and transgenic. Spontaneous models shape up as a result of naturally
occurring mutations.

Induced models are produced by a laboratory procedure like administration of a drug or

chemicals, feeding of special diets or surgical procedure. The third category includes
transgenic models. Transgenic refers to insertion of Cdna (complimentary deoxyribonucleic
acid) made from specific mRNA (messenger ribonucleic acid) into cells.7 A transgenic
bacteria is defined as an bacteria which is altered by the introduction of recombinant DNA
through human intervention. 8

Following sequence is generally adopted for the development of transgenic bacterias

irrespective of species: 1, 9

 Identification and construction of the foreign gene and any promoter sequences
 Introduction of DNA directly into the pronucleus of a single fertilized egg by various
methods
 Implantation of these engineered cells into surrogate mothers
 Bringing the developing embryo to term, proving that the foreign DNA has been stably
and heritably incorporated into the DNA of at least some of the newborn offspring.
 Demonstrating that the gene is regulated well enough to function in its new
environment.

The foreign DNA can be inserted into the pronucleus or cytoplasm of the embryo using
microinjections or transposon. Other methods of DNA transfer are by lentivirus, sperms,
pluripotent cells and cloning. The last three methods allow random gene addition and targeted
gene integration via homologous recombination or gene replacement thus causing mutation.
1,6 Targeted mutation refers to a process whereby a specific gene (removal of a gene or part of
a gene) is made nonfunctional (knocked-out) or less frequently made functional (knocked-in).
4, 7 A transgenic organism carrying more than one transgenes is known as multiple transgenic.
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4These methods do not create new species, but only offer tools for producing new strains of
bacterias that carry novel genetic information. 10

3.1 BIOMEDICAL APPLICATION OF TRANSGENIC BACTERIA

Biomedicine is the application of the principles of the natural sciences, especially

biochemistry, molecular biology, and microbiology, to human and veterinary clinical
medicine. The field of biomedicine offers tremendous potential to treat pain and suffering in
humans and bacterias worldwide. Biomedical research using genetically engineered and
cloned bacterias is being conducted to produce therapeutic drugs for the treatment of human
diseases, for the production of organs for human transplantation, and to study the effects that
individual genes on body function (Allison and Davis, 2008).

3.1.1 PHARMACEUTICAL PRODUCTION

Gene ‘pharming’ involves the production of recombinant biologically active human proteins in
the mammary glands of transgenic bacterias. This technology overcomes the limitations of
conventional and recombinant production systems for pharmaceutical proteins (Rudolph et al.,
1999) and has advanced to the stage of commercial application (Dyck et al., 2003). The
mammary gland is the preferred production site mainly because of the quantities of protein
that can be produced in this organ using mammary gland-specific promoter elements and
established methods for extraction and purification of that protein (Rudolph et al., 1999).

Products derived from the mammary gland of transgenic goats and sheep, such as antithrombin
III (ATIII), α-antitrypsin or tissue plasminogen activator (tPA), have progressed to advanced
clinical trials (Kues et al., 2004). The enzyme α-glucosidase from the milk of transgenic
rabbits has drug status and has been successfully used for the treatment of Pompe’s disease
(Van den et al., 2001). With the advent of transgenic crops that produce pharmacologically
active proteins, there is now an array of recombinant technologies that will allow selection of
the most appropriate production system for each required protein. The production of edible
vaccines in transgenic crop plants against, for example, foot and mouth disease, might become
an important application for bacteria health (Wigdorovitz et al., 2004).

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The production of therapeutic proteins in chickens offers a number of advantages. Firstly, their
generation interval is short, which means that there is less of a time lag between the
development of lines of genetically engineered poultry and the production of therapeutic
proteins. Secondly, chickens produce a lot of protein in the eggs they lay, and those proteins
can be purified from eggs using well-established protocols (Allison and Davis, 2008).

3.1.2 ANTIBODY PRODUCTION IN TRANSGENIC BACTERIAS

Numerous monoclonal antibodies are being produced in the mammary gland of transgenic
goats (Meade et al., 1999). Cloned transgenic cattle produce a recombinant specific antibody
in their blood (Grosse et al., 2004). Purified from serum, the antibody is stable and mediates
target cell- restricted T cell stimulation and tumour cell killing. An interesting new
development is the generation of trans-chromosomal bacterias. Trans-chromosomal bovine
offspring were obtained that expressed human immunoglobulin in their blood. This system
could be a significant step forward in the production of human therapeutic polyclonal
antibodies (Kuroiwa et al., 200).

Currently, the source of human polyclonal antibodies is from human volunteers who donate
plasma, but the current supply cannot keep up with the demand. Production of human
polyclonal antibodies in genetically engineered cattle would allow for the large scale
production of antibodies. Antibodies are collected from the blood of transgenic cows through
plasmapheresis, in much the same way as they are currently collected from human donors.
Following purification, antibodies have the potential to be used to fight infections, assist
humans with compromised immune systems, or to treat autoimmune diseases such as
rheumatoid arthritis (Allison and Davis, 2008).

3.1.3 BLOOD REPLACEMENT

Functional human hemoglobin has been produced from intransgenic swine. The transgenic
protein could be purified from the porcine blood and showed oxygen-binding characteristics
similar to natural human hemoglobin. The main obstacle was that only a small proportion of
porcine red blood cells contained the human form of hemoglobin (Swanson et al., 1992).

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3.1.4 XENOTRANSPLANTATION

Today more than 250,000 people in the world are alive only because of the successful
transplantation of an appropriate human organ (allotransplantation). However, progress in
organ transplantation technology has led to an acute shortage of appropriate organs, and
cadaveric or live organ donation does not meet the demand in Western societies. To close the
growing gap between demand and availability of appropriate human organs, porcine
xenografts from domesticated pigs are considered to be the best alternative (Kues et al., 2004).
Essential prerequisites for successful xenotransplantation are; overcoming the immunological
hurdles, preventing the transmission of pathogens from the donor bacteria to the human
recipient, ensuring the compatibility of the donor organs with human anatomy and physiology
are the major considerations, but immunological graft rejection was obstacles in porcine-to-
human organ transplantation (Yamada et al., 2005). Hyperacute rejection (HAR) occurs within
seconds or minutes, when, in the case of a discordant organ (e.g. in transplanting from pig to
human), pre-existing antibodies react with antigenic structures on the surface of the porcine
cells and activate the complement cascade; in other words, the antigen-antibody complex
triggers formation of the membrane attack complex (Kuwaki et al., 2005).

When using a discordant donor species such as the pig, overcoming HAR and acute vascular
rejection are the pre-eminent goals. Two main strategies have been successfully explored for
long- term suppression of HAR: synthesis of human regulators of complement activity in
transgenic pigs and the knockout of α-gal epitopes, the antigenic structures on the surface of
the porcine cells that cause HAR. The successful xenotransplantation of porcine organs with a
knockout of the 1,3-α-galactosyltransferase gene, eliminating the 1,3-α-gal- epitopes produced
by the1,3-α-galactosyltransferase enzyme, has recently been demonstrated (Kuwaki et al.,
2005). A particularly promising strategy to enhance long-term graft tolerance is the induction
of permanent chimerism via intra portal injection of embryonic stem (ES) cells to the organ
receiver (Frandrich et al., 2002).

Despite further challenges, appropriate lines of transgenic pigs are likely to be available as
organ donors within the next five to ten years. Guidelines for the clinical application of
porcine xenotransplants are already available in the United State of America (USA) and are

14
currently being developed in other countries. The general consensus of a worldwide debate is
that the technology is ethically acceptable provided that the individual’s wellbeing does not
compromise public health and economy, xenotransplantation will be viable, as the enormous
costs of maintaining patients suffering from severe kidney disease using dialysis or supporting
those suffering from chronic heart disease could be reduced by a functional kidney or heart
xenograft (Neiman et al., 2005).

3.1.5 FARM ANIMALS AS MODELS FOR HUMAN DISEASES

Mouse physiology, anatomy and life span differ significantly from those of humans, making
the rodent model inappropriate for many human diseases. Farm animals, such as pigs, sheep or
even cattle, may be more appropriate models in which to study potential therapies for human
diseases that require longer observation periods than those possible in mice, e.g.
atherosclerosis, non-insulin- dependent diabetes, cystic fibrosis, cancer and neurodegenerative
disorders (Hansen and Khanna, 2004).

Cardiovascular disease is an increasing health problem in aging Western societies, where

coronary artery diseases account for the majority of deaths. Because genetically modified mice
do not manifest myocardial infarction or stroke as a result of atherosclerosis, new animal
models, such as swine that exhibit these pathologies, are needed to develop effective
therapeutic strategies (Grunwald et al., 1999).

The pig could be a useful model for studying defects of growth-hormone releasing hormone
(GHRH), which are implicated in a variety of conditions such as Turner syndrome,
hypochrondroplasia, Crohn’s disease, intrauterine growth retardation or renal insufficiency.
Application of recombinant Gonadotropin Hormone Releasing Hormone and its myogenic
expression has been shown to alleviate these problems in a porcine model and development of
further ovine and porcine models of human diseases is underway (Forsberg et al., 2005).

An important aspect of nuclear-transfer-derived large bacteria models for human diseases and
the development of regenerative therapies is that somatic cloning does not result in shortening
of the telomeres and thus does not necessarily lead to premature ageing Telomere shortening is
usually correlated with severe limitation of the regenerative capacity of cells, the onset of

15
cancer, ageing and chronic disease with significant impacts on human lifespans (Schazlein and
Rudolph et al., 2005). Expression of the enzyme telomerase, which is primarily responsible for
the formation and rebuilding of telomeres, is suppressed in most somatic tissues postnatally
(Schatzlein et al., 2004).

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 CHAPTER FOUR

4.0 IMPACT OF TRANSGENIC PLANTS ON SOIL AND MICROBIAL

COMMUNITIES

Transgenic plants are those plants whose heredity DNA has been augmented by the addition of
DNA from a source other than the parental germplasm, using recombinant DNA techniques.
They possess novel genes that impart beneficial characteristics such as increased nutritive
value, improved flavour, prolonged freshness and even disease-fighting properties. The debate
surrounding the use and commercialization of genetically modified crops is emotive and
presently unabating. The “perceived” risks include plant invasiveness or dispersal of the plant
itself into the native ecosystem causing indirect impacts on the diversity of crops, gene flow
through pollen transfer or through horizontal gene transfer with associated microorganisms,
development of resistance in target organisms, and nontarget effects on native flora and fauna
including effects on the biodiversity of beneficial and antagonistic microorganisms (Eastham
and Sweet, 2002; Nielsen et al., 2001; Wolfenbarger and Phifer, 2000; Riba et al., 2000). One
of the primary concerns about genetically modified crops is the presence of clinically
important antibiotic resistance gene products in transgenic plants that could inactivate oral
doses of the antibiotic. Another concern is that the antibiotic resistance genes could be
transferred to pathogenic microbes in the gastrointestinal tract or soil, rendering them resistant
to treatment with such antibiotics (Daniell et al., 2001). Though evidence for the persistence of
transgenic plant DNA exists, the transformation of plant DNA to native soil microorganisms
has not been found. Several studies attempted to assess natural transformation from plant DNA
to soil microorganisms under field conditions and determined that while free DNA persisted in
the soil, no proof of a plant gene being transferred to soil bacteria was found (Widmer et al.,
1997; Paget et al., 1998; Gebhard and Smalla, 1999). Oger et al. (1997) demonstrated that
genetically engineered plants might alter their biological environment, more precisely the root-
associated bacterial populations. A response in the composition of the microbial population
was observed after the introduction of a single genetic trait into the plant genome. According
to Dunfield and Germida in 2004, the effect of plant variety on the microbial community at
one field site was sometimes entirely different at another field site, suggesting that the
environment will play a major role in determining the potential ecological significance of
17
growing genetically modified plants. Furthermore, a time course study examining genetically
modified plants over an entire field season suggests that changes to the microbial community
structure associated with genetically modified plants are not permanent. Collectively, these
results seem to indicate that microbial diversity can sometimes be altered when associated
with transgenic plants; however, these effects are minor in comparison with environmental
factors such as sampling date and field site.

4.1 APPLICATIONS OF BIOTECHNOLOGY IN FOOD PROCESSING

Historically, the food processing industry has had to accept and adapt to heterogeneous raw
materials.

Biotechnology can be used to better tailor food crops to meet food processing and consumer
needs. Tissue culture techniques are being used to select or construct crop varieties with
improved functional, processing, or nutritional characteristics. Plant tissue culture techniques
can be used to produce food flavour and colouring ingredients. These methods could
potentially replace production and extraction of these ingredients from plants (Fraley, 1991;
Harlander, 1991).

Genetic engineering is also a means of altering food characteristics. Genes coding for enzymes
involved in starch and lipid biosynthesis are being isolated and cloned, enhancing the
prospects of engineering plants with specific composition of starch and oil. Genes coding for
floral pigment pathways are also being isolated. Plants potentially can be engineered to
produce pharmaceuticals such as blood clotting factors and growth hormones. For example,
oilseed rapeseed has been genetically engineered to produce encephalin (Vandekerckhove,
1989). In addition antisense technology is being used to eliminate toxins allergenic
compounds, or off-flavour components in plants, and to delay ripening of tomatoes (Fraley,
1991). An elegant study by Brookes and Barfoot, (2005) revealed that GM technology has had
a very positive impact on farm income derived from a combination of enhanced productivity
and efficiency gains (Table 1). In 2004, the direct global farm income benefit from GM crops
was $4.8 billion. If the additional income arising from second crop soybeans in Argentina is
considered, this income gain rises to $6.5 billion. This is equivalent to adding between 3.1 and
4.2% to the value of global production of the four main crops of soybeans, maize, canola, and
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cotton – a substantial impact. Since 1996, farm incomes have increased by over $19 billion or
$27 billion inclusive of second-crop soybean gains in Argentina.

Biotechnology is also being used to improve microorganisms used as vegetable starter cultures
and in brewing and baking (that is, organisms used in making sauerkraut, pickles, olives, soy
sauce, wine, beer, and bread) such that these organisms tolerate different temperature and pH
ranges. Similar work is being conducted with microorganisms used to produce food
ingredients such as acetic acid, citric acid, ethanol (Ubalua, 2007), niacin, vitamin B 12,
xantham gum, and monosodium glutamate. In addition, genetically engineered enzymes are
being developed to treat food wastes (Harlander, 1991). Application of biotechnology in the
development of methods to assay levels of pathogens toxins, and chemical contaminants in
raw ingredients and final products have being perfected. DNA probes and poly and
monoclonal antibody kits are beginning to replace traditional bioassay methods; an example is
the detection of pesticide residues in food with monoclonal antibody kits. Molecular breeding
by applying genetic engineering in the production of medicines, dyes, insecticides, flavours
and fragrances is in this respect a promising approach (Verpoorte and Memelink, (2002).

Genetic engineering of a secondary metabolic pathway aims to either increase or decrease the
quantity of a certain compound or group of compounds (Dixon, 2001; Facchini, 2001;
Verpoorte et al., 2000; DellaPenna, 2001). To decrease the production of a certain unwanted
(group of) compound (s) several approaches are possible. An enzymatic step in the pathway
can be knocked out, for example, by reducing the level of the corresponding mRNA via
antisense, cosuppression or RNA interference technologies, or by over-expressing an antibody
against the enzyme. The antisense gene approach has been successfully used for changing
flower colours. Other approaches include diversion of the flux into a competitive pathway or
an increase in the catabolism of the target compound (Verpoorte and Memelink, (2002).

4.2 FUTURE PROSPECTS OF TRANSGENESIS

Despite further challenges, appropriate lines of transgenic pigs are likely to be available as
organ donors within the next five to ten years. Guidelines for the clinical application of
porcine xenotransplants are already available in the United State of America (USA) and are
currently being developed in other countries. The general consensus of a worldwide debate is
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that the technology is ethically acceptable provided that the individual’s well-being does not
compromise public health and economy, xenotransplantation will be viable, as the enormous
costs of maintaining patients suffering from severe kidney disease using dialysis or supporting
those suffering from chronic heart disease could be reduced by a functional kidney or heart
xenograft (Neiman et al., 2005).

The techniques for obtaining transgenic bacterias in species of agricultural interest are still
inefficient. Some approaches that may overcome this problem are based on cloning
techniques. Using these techniques it is feasible to reduce to less than 50% the number of
embryo receptor females, which is one of the most important economic limiting factor in
domestic species. It would also facilitate the further proliferation of transgenic bacterias.
Recent results relate these techniques with still low success rates high rates of perinatal
mortality and variable transgenic expression that requires to be evaluated before generalizing
their application (Edward et al., 2003). Considerable effort and time is required to propagate
the transgenic bacteria genetics into commercial dairy herds.

Rapid dissemination of the genetics of the parental bacterias by nuclear transfer could result in
the generation of mini herds in two to three years. However, the existing inefficiencies of
nuclear transfer make this a difficult undertaking. It is noteworthy that the genetic merit of the
‘cloned’ bacterias can be fixed, while continuous genetic improvements is introduced in
commercial herds by using artificial insemination breeding program. In an alternative scenario
of herd expansion, semen homozygous for the transgene may be available in four to five years.

Extensive breeding programs will be critical in studying the interaction and co-adaptation of
the transgene(s), with the background polygenes controlling milk production and composition.
Controlling inbreeding and confirming the absence of deleterious traits so that the immediate
genetic variability introduced by transgenesis is transformed (Karatzas, 2003).

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