Faculty of Life and Allied Health Sciences

Ramaiah University of Applied Sciences

Department Biotechnology Programme M.Sc. Biotechnology
Semester III Batch 2020
Course Code 19BTG513A Course Title Animal Biotechnology
Course Leader(s) Dr. Soma Chaki/Dr. Swati Sinha

Assignment
Reg. No. 20LABT091005 Name of Student DEEPANRAJ S P
Section

Max Examiner
Marking System
Marks Marks

A 1.1 Write an essay on role of biotechnology in world food safety 10

Part A

Part-A Max Marks 10

B 1.1 Elucidate the ethical concerns 05

B.1 Max Marks 05

Part B.2

B 2.1 Define mRNA therapeutics 02

Discuss its drawbacks and recent advances to overcome the
B2.2 08
same
B.2 Max Marks 10
Total Marks 25

Please note:
1. Documental evidence for all the components/parts of the assessment such as the

reports, photographs, laboratory exam / tool tests are required to be attached to the
assignment report in a proper order.
2. The marks for all the questions of the assignment have to be written only in the

Component –B: Assignment table.

Course Marks Tabulation
Component-1 (B)
Assignment Examiner Remarks
A
B.1
B.2
Marks (Max 25 )
Signature of Examiner

Assignment
Instructions to students:

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 1. Maximum marks is 25.
2. The assignment has to be neatly word processed as per the prescribed format.
3. The maximum number of pages should be restricted to 10.
4. Restrict your report for Part-A to 4 pages only.
5. Restrict your report for Part-B to a maximum of 6 pages.
6. The printed assignment must be submitted to the course leader.
7. Submission Date: 30.11.2021
8. Submission after the due date is not permitted.
9. IMPORTANT: It is essential that all the sources used in preparation of the assignment
must be suitably referenced in the text.
10. Marks will be awarded only to the sections and subsections clearly indicated as per
the problem statement/exercise/question

Part -A (10 Marks)

Preamble
“Biotechnology in animal farming has the potentiality to produce super-productive animals, which are
as to provide the necessary amount of nutritious milk, animal protein, and fat-less meat. It helps us to
produce crops in the highly saline and nearly drought soils. This is why, it has been postulated that
biotechnology can help meet the basic rights of global population by contributing to food security.”
A.1. Based on the statement, prepare a debate cum essay addressing primarily whether
Biotechnology is the only way to meet the thrust of world food security?

Along with the intensive development of methods of livestock breeding, breeders’ expectations are growing
concerning feed additives that would guarantee such results as accelerating growth rate, protection of health from
pathogenic infections and improvement of other production parameters such as: absorption of feed and quality of
meat, milk, eggs. It is estimated that by 2050 the number of people in the world will reach 9 billion. Constant
growth of the human population is inseparably associated with a growing demand for food of plant and animal
origin. For that reason, scientists are looking for solutions allowing intensification of food production, with
simultaneous reduction of production costs, and in compliance with high standards of quality and safety (for both
people and the environment). Types of used feed additives affect animal health and increased production of high
quality meat, eggs, milk and fish. Animal production is inseparable from nutrition and health of the consumer, and
animal intestinal pathogens, such as Campylobacter, Salmonella, Listeria and Yersinia, are a direct source of food
contamination and a cause of zoonoses. Therefore, new methods of animal breeding are introduced, aimed at
increased quality and safety of meat, while taking animal welfare and respect for the natural environment into
account.
Despite numerous difficulties associated with the registration of feed additives, particularly in the category of
zootechnical feed additives, modern global economy and strong market competition result in the need to introduce
new technologies to animal nutrition. Numerous scientific reports confirm a beneficial effect of probiotics on
animal health, particularly in terms of protection against pathogens, stimulation of immunological response and
increased production capacity. Prebiotics may be used alternatively or support the effect of probiotics.
Interestingly, the use of combination of those components demonstrating a synergistic effect may be even more
efficient in the stimulation of intestinal microbiota and protection of animal health. The greatest problem

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encountered by the scientists who attempt to create synbiotic formulas is selection of appropriate probiotic and
prebiotic (high selectivity of action). Feeds containing probiotic organisms are a great hope for that field of the food
industry. The hope is even greater considering the fact that consumers do not accept animal products originating
from animals in which antibacterial substances had been used. Meeting all expectations requires much work in the
field of scientific research, development of innovative technologies and convincing breeders that the spending on
prebiotic-containing feed will translate to better production effects and higher quality of animal products, and thus
it will guarantee an expected economic profit. It should be underlined that the use of feed additives, such as
probiotics, prebiotics and synbiotics is safe, does not have a negative impact on the natural environment, and
reduces the demand for antibiotic-based growth stimulators. However, the mechanisms of action of probiotic
organisms, prebiotics, as well as their combinations in synbiotics, require further studies.
Increasing the Global Cropland Area:
One of the simplest ways to improve agricultural production with the current technology and crop cultivars
would be to significantly extend the global cropland area. However, new agricultural lands are scarcely available or
can only be obtained at a high environmental cost. Also, land cultivated with irrigation is much more productive
than rainfed cropland. Irrigation systems are currently used to grow crops in about 280 × 10 6 ha. of arable land this
represents just under 20% of the total cultivated land, but produces more than 40% of world food
supplies.Therefore, a significant increase in the area of irrigated arable land would lead to a parallel increase in
food production. Unfortunately, this will not be possible in a world where fresh water for irrigation is becoming an
increasingly scarce resource. Another possibility would be the use of natural habitats of great ecological value, such
as rainforests, but it would be against the necessary sustainability of natural resources and conservation of
biodiversity. The use of our actual high-input agriculture in marginal, low-fertility land will be also unsustainable, as
it will require large inputs of agrochemicals and could not be maintained for a long time. In addition, at the
moment the area available for agriculture is actually being reduced, mostly by the change of land use due to urban
development and industrialization in many emerging economies and developing countries.
Searching for ‘Stress Tolerance’ Genes:
A lot of effort has been invested in the last 20–30 years in the isolation and characterization of genes involved
in stress response pathways, with the expectation that their overexpression in transgenic plants would confer
improved tolerance to abiotic stress. They include, for example, genes encoding ion transporters or enzymes of
osmolyte biosynthesis. Moreover, most stressful environmental factors induce an increase in cellular ‘reactive
oxygen species’ (ROS) levels thus causing, as a secondary effect, oxidative stress in plants. Consequently, another
basic and general response to abiotic stress involves the activation of enzymatic and non-enzymatic antioxidant
systems to mitigate oxidative damage of DNA, membranes and proteins. All these responses are mediated by major
stress-induced changes in gene expression patterns, which also include the synthesis of specific ‘protective’
proteins, such as heat-shock proteins (HSPs), late embryogenesis-abundant (LEA) proteins, osmotin, etc.

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 Part B
B1 Preamble (05 marks)
Elucidate the ethical concerns associated with the application of Biotechnology in using animal as live
Bioreactors for production of therapeutic molecules to serve health care?

Animal biotechnology is one of the main areas of biotechnology which concerns the application of animals,
including transgenic animals, in various fields. Even though these transgenic animals can be useful for improving
the welfare of humans and animals, the well-being of animals being used in the studies may be negatively affected.
Ethical issues relating to experimentation on animals and the production of transgenic animals are discussed in this
review, and included here is a consideration of the phenomena of "conditional ethical blindness". Finding
alternative protocols including the 4 Rs (reduction, refinement, replacement, and responsibility) to minimize the
employment of animals in scientific procedures is one way to solve these problematic issues. These strategies can
be successively utilized for certain animal biotechnological protocols and can be used to intelligently avoid
unethical manipulation of animals. The use of animals in experimental research has increased due to the
advancement of research and development in medical technology. Each year, millions of animals are being
experimentally used worldwide and 90% of these animals are mice and rats, including cats, dogs, rabbits and
primates . The pain, distress and even death of animals during the experimentation have been a dispute
issue for years .
Monkeys are one of the most controversial animal models used in experiments becausethey have a close
genetic relationship to humans. However, less than 1% of animals used in European countries (EU)
aremonkeys. Monkeys are used only if it is not possible to use a non-animal model, or other animal species for the
research being undertaken. However, monkeys are frequently used for testing Covid-19 treatments due to their
similarity to humans and they are valuable as they provide the most reliable source of information.
Animal biotechnology has become an important branch of biotechnology and it is being used in an increasing
number of applications in basic science, medical science and agriculture. The core of animal biotechnology is
addressed on transgenic animals produced through cloning and genetic engineering techniques which are
nowadays becoming extremely advanced and common. There seems to be little doubt that the use of animal
biotechnology benefits human welfare. However, whilst using animals as a model, scientific risk assessment and
use of the precautionary principle, as well as ethical guidelines have to be highly concerned. Some of the ethical
issues are still questionable and responses to them may well depend on individual’s opinion, culture and religion.
‘Conditional ethical blindness’ is also a factor that can determine how individuals, organizations and society see and
respond to the main ethical issues. Therefore, one way to solve these problems is the use of alternative protocols
such as reduction, refinement, replacement and responsibility to minimize the use of animals as well as to avoid
animal ethical controversies. When experimentation with animals cannot be avoided, they must be treated with as
much kindness and compassion as possible. Effective and responsible communication through scientific
community, industry and government stakeholders are also required in order to reach a consensus of society on
the acceptance of genetic engineering and animal cloning.

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 B2 Preamble (10marks)
Messenger RNA (mRNA)-based therapeutics hold the potential to cause a major revolution in the
pharmaceutical industry because they can be used for precise and individualized therapy, and enable
patients to produce therapeutic proteins in their own bodies without struggling with the
comprehensive manufacturing issues associated with recombinant proteins.
B2.1 Define mRNA based therapeutics?

Messenger RNA (mRNA)-based therapeutics hold the potential to cause a major revolution in the
pharmaceutical industry because they can be used for precise and individualized therapy, and enable patients to
produce therapeutic proteins in their own bodies without struggling with the comprehensive manufacturing issues
associated with recombinant proteins. Compared with the current therapeutics, the production of mRNA is much
cost-effective, faster and more flexible because it can be easily produced by in vitro transcription, and the process
is independent of mRNA sequence.

B2.2 The stability, immunogenicity, translation efficiency, and delivery are all pivotal issues need to
be addressed in mRNA based therapy. Discuss?

Improving the stability and translation of mRNA: One of the major challenges of naked mRNA- based therapy is its
short half- life, which is caused by the rapid degradation by abundant extracellular RNases. The half-life of in vitro
transcribed mRNA (IVT mRNA) and its protein products is a crucial factor affecting the pharmacokinetic (PK) and
pharmacodynamics (PD) properties of mRNA-based therapeutics. To optimize the efficiency of mRNA, a variety of
chemical modifications to mRNA structures were explored, including modifications to the 5’-cap, poly (A) tail, 5’-
and 3’-UTRs, and coding region. The poly (A) tail decorates the 3’ end of mature mRNA in eukaryotes. It is produced
by transcribing its DNA template or by using a recombinant poly (A) polymerase post transcriptionally. The latter is
limited because the length of poly (A) tail can vary with each other in the production of mRNA batches, which
makes the reproducible batches with a defined poly (A) length very difficult. The 5’- and 3’-UTRs in mRNA contain
specific regulatory sequence elements that modulate the translation and stability of mRNA. For the protein coding
region of mRNA, codon optimization leads to controllable translation of the sequence to desired protein. Single
synonymous codon substitution may have a significant impact on protein expression, protein folding, and cell
function. Because the same amino acid can be translated from a distinct set of codons, there are multiple choices
to rewrite an mRNA code to produce exactly the same protein. Recently, researchers of Moderna, Inc. observed
that mRNA secondary structure could regulate protein expression by changing the half- life of mRNA translation,
and modified nucleotides that stabilize mRNA spatial structure enabled high protein expression level. Machine
learning is also applied to design the sequence of mRNA to produce more or less desired proteins. Until now, this
technology has been successfully employed in mRNA- based therapeutics, such as the expression of non-viral
proteins and development of infectious disease vaccines. In general, the structural mRNA elements of 5’ -cap, 3’-
poly (A) tail, 5’- and 3’-UTRs and coding region are all modification targets. In order to obtain the best mRNA
therapeutic efficiency, it is necessary to optimize the combination for specific applications.
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Avoiding immunogenicity of mRNA: A great issue along with IVT mRNA is its immunogenicity, because exogenous
RNA will be recognized as a signal of viral infection. Non-immune cells recognize RNA through the retinoic acid-
inducible gene I (RIG-I) receptor and then trigger an innate immune response. In addition to modifying nucleotides
and adding poly (A) tails, optimizing the codons to render the mRNA GC-rich, minimizing U content is another
effective way to eliminate RNA immunogenicity. Therapeutics developed a sequence- engineering without any
chemical modification of mRNA. After the in vitro transcription, a series of purification processes including
concentration, precipitation, extraction, and chromatography are needed to produce mRNA. Sophisticated
techniques such as anion exchange chromatography, size exclusion columns, high performance liquid
chromatography (HPLC) and affinity chromatography are applied to remove dsRNA and truncated transcripts.
mRNA delivery: Efficient and safe delivery of mRNA is one of the biggest challenges in the development of mRNA-
based therapeutics, which is more challenging than delivery of small oligonucleotides.The size of mRNA (300–5,000
kDa, 1–15 kb) is significantly larger than siRNA and miRNA mimic (13-15 kDa), antisense oligonucleotide (4–10 kDa)
and antimiR (4–10 kDa). The N-Acetylgalactosamine (GalNAc)-oligo conjugate exhibited excellent efficiency and
safety of hepatocyte-targeted delivery in vivo, but was non-effective for mRNA delivery. Because of their size,
charge, and degradability, naked mRNA cannot readily pass through the cell membrane and efficiently leak into the
cytoplasm. Researches proved that naked mRNA was taken up by cells via the scavenger-receptor mediated
endocytosis pathway and accumulated in the endosome.

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