Concept and Scope of Biotechnology

Introduction

Biotechnology is defined as any technique that uses living organisms or parts of

organisms to make or modify products, to improve plants and animals or to develop
microorganisms for specific uses. Biotechnology is advancing by leaps and bounds and
it has more applications in our day today life from pharmaceutical development to food
production and the treatment of polluting waste.

Life
Sciences
Technology Biotechnology

The evolution of Biotechnology

1919 Hungarian Agronomist Karl Ereky coined the term Biotechnology

1928-Scottish bacteriologist Alexander Fleming discovers the antibiotic use of Penicilin
1943- Canadian scientist Oswald Theordore Avery discovers that DNA is the carier of
genes
1953-8iologist James Watson and Francis Crick describes the double helix of DNA
1969-An enzyme is synthesized in vitro for time in history
1972- Paul Berg, a biochemist, utilized a restriction enzyme to cut DNA into fragments.
He employed a ligase enzyme to join two DNA strands concurently to forma hybrid
circular molecule. This was the first recombinant DNA (rDNA) molecule synthesized.
1978- Recombinant insulin-Herbert w Boyer at UcSF synthesized synthetic human
insulin by introducing the insulin gene into the bacterium Escherichia coli.
1980-The U.S. patent for gene cloning is awarded to Cohen and Boyer.
1983-First genetically engineered transgenic plant is presented and The Polymerase
Chain Reaction (PCR) technique is conceived.
1997-Scientists introduced the world to Doly, the sheep. the first cloned mammal
1998-Draft of the human genome map
2000- The US Supreme Court granted that genetically modified living organisms could
be patented. Kary Mulis and other researchers at UC Berkeley, California, established a
tool for multiplying DNA sequences in vitro using the polymerase chain reaction (PCR).
2010-A group of researchers fromJ. Craig Ventere Institute created the first synthetic
cell
2012- Thirty-one-year-old Zac Vawter successfully uses a nervous system-controlled
bionic leg to climb the Chicago Willis Tower.
2013- the first bionic eye is produced in US giving hope to blind people world wide
2019- Scientists for the first time reported the use of the CRISPR technology to edit
human genes to treat cancer patients with whom standard treatments were not
successful.
2020-Scientists develop a CRISPR-Cas12a-based gene editing system that can probe
and control several genes at once and can implement logic gating to e.g. detect cancer
cells and execute therapeutic immunomodulatory responses.
2020,14 March-Biotechnology innovations lead the fight against the SARS-CoV-2
2020, 7 October- The Nobel Prize in Chemistry is awarded to Emmanuelle Charpentier
and Jennifer A. Doudna for their work on genome.
2021- CRISPR-dCas9 epigenome editing method for a potential treatment of chronic
pain, an analgesia that represses Nav1.7 and showed therapeutic potential in three

mouse models of pain.

2021-Development of a vaccine of mRNAs for the body build 19 proteins in tick saliva
which, by enabling quick development of erythema (itchy redness) at the bite site,
protects guinea pigs against Lyme disease from ticks.
2021- A team of scientists reports a new form of biological reproduction in the, <1 mm
sized, xenobots that are made up of and are emersed in frog cells.
The development of biotechnology can be divided into broad stages like:

Stage 1-Ancient biotechnology (8000-4000 BC). Early history as related to food, shelter
and domestication of animals.
Stage 2- Classical biotechnology (2000 BC; 1800-1900 AD) which built on ancient
biotechnology starts with fermentation which promotes food production and medicine.
Stage3- Moden biotechnology (1977) Manipulates genetic information in organisms,
genetic engineering and various technologies enable us to improve crop yield and food
quality in agricuture and to produce a broader array of products in industries.

Types of Biotechnology

Like the colours in rainbow, the different biotechnology applications grouped into seven
colours

1. Red Biotechnology-Medical (the development of insulin, growth hormone,

vaccines, artificial organs, regenerative therapy)
2. Green Biotechnology (Disease resistant and weather resistant plants
3. White Biotechnology (industrial Biotechnology (Sel-healing concrete,. plants
that change colour when detects an explosive, clothing and footwear made with
synthetic spider web etc.
4. Yellow Biotechnology (Food production, GM foods)
5. Blue Biotechnology (Exploit marine resources)
6. Groy Biotechnology (Bioremediation, ecological recovery)
7. Gold Biotochnology (Bioinformatics-storing, analysing and separating biological
information.
Today biotechnology help us fight hunger, disease, produce more safely products,
reduce our ecological footprint and save energy.

Animal Blotechnology
Animal biotechnology is a branch of biotechnology in which Iliving organisms were
engineered for pharmaceutical, agricultural or industrial applications. All animals like
livestock, poultry, fish, insects, companion animals and laboratory animals were
included under animal biotechnology. It provides new tools for increasing livestock
productivity. improving human health, animal health and welfare. Biotechnology
improves the food we eat, improves an animal's impact on the environment, enhances
ability to detect, treat and prevent diseases. Animal biotechnology contributes to animal
production by improving the environmental component of the production systems as
well as by improving the genetic make-up of livestock.

Biotechnology for the improvement of Environmental component

Improve forage species to increase their productivity or nutritional value
microorganisms are engineered to produce food aditives
Improve the gut micro flora
production of therapeutic compounds like vaccines from genetically engineered

microorganisms
Biotechnology for the genetic improvement
Focuses the areas like:

Reproductiva blotechnology
Methods developed to increase the reproductive potential of livestock include:
1. Artificial insemination (Al):
2. Multiple ovulation and embryo transfer (MOET)
3. Oocyte harvesting (OPU), in vitro oocyte maturation (1VM), in vitro fertilisation

(IVF)
4. Nuclear transfer or embryo cloning
5. Embryo transfer (EM)
6. Gamete and embryo cryopreservation
7. Marker assisted selection (MAS)

Transgenesis
Transgenesis is the process of introducing a gene from one organism into the genome
of another organism. A transgenic animal is one which has been genetically altered to
have specific characteristics it otherwise would not have. The first advanced eukaryotes
to prove amenable to such genetic modifications were mice subsequently, a variety of
species including fish and fruit flies have been subject to transgenic manipulation.
GloFish (2008) (Fig.1) are a type of transgenic zebrafish (Danio rerio) that have been
modified through the insertion of a green fluorescent protein (gfp) gene. Not all GloFish
are green, however. Rather, there are several gfp gene constructs, each encoding a
different colored phenotype, from fluorescent yellow to fluorescent red.

Fig.2.Multi-coloured Glow fish (Courtesy of www.glofish.com. All rights reserved.)

Transgenic technologies are used for improving milk production and the meat in fam
animals as well as for creating models of human diseases. Transgenic animals are used
for the production of proteins for human medical use
Reproductive Cloning
Reproductive cloning is a method used to make a clone or an identical copy of an entire
multicellular organism. clones can be produced by splitting an earty embryo similar to
identical twins and cloning of adult animals is usually done by a process called somatic
cell nuclear transfer (SCNT). Reproductive cloning involves the implantation of a cloned
embryo into a real or an artficial uterus. The embryo develops into a fetus that is then
caried to tem. Reproductive cloning techniques underwent significant change in the
1990s, by the birth of Dolly. generated through the process of SCNT.
Somatic Cell Nuclear Transfer (SCNT)

a a Finm Dorsat ene

asut coih Carac****

onor

das

Fig.3. Dolly: The cloning of a sheep,1996. (2015 Encyclopedia Britanica,Inc)

Reproductive cloning techniques underwent significant change in the 1990s, following

the birth of Dolly, who was generated through the process of SCNT. This process
entails the removal of the entire nucleus from a somatic cell of an organism inserting the
nucleus into an egg cell that has had its own nucleus removed. Once the somatic
nucleus is inside the egg, the egg is stimulated with a mild electrical current and begins
dividing. Thus, a cloned embryo, essentially an embryo of an identical twin of the
original organism, is created. For reproductive cloning, the embryo is implanted into a
uterus of a surrogate female, where it can develop into a live being.

Therapeutic Cloning
Therapeutic cloning enables the cultivation of stem cells that are genetically identical to
a patient. The differentiated cells then could be transplanted into the patient to replace
diseased or damaged cells without the risk of rejection by the immune system.
Embryonic stem cells, have the unique ability to generate all types of cells in an
organism, which can be used to replace injured or diseased tissues. It helps to lean
more about the molecular causes of disease by studying embryonic stem cell lines from
cloned embryos derived from the cells of animals or humans with different diseases.
And also differentiated tissues derived from ES cells are excellent tools to test new
therapeutic drugs. Stem cells used for the treatment of Alzheimer disease, Parkinson
disease, diabetes mellitus, stroke, and spinal cord injury etc. In addition, stem cells
could be used for in vitro studies of normal and abnormal embryo development or for
testing drugs to see if they are toxic or cause birth defects. Therapeutic cloning can also
use SCNT to reprogram somatic cells into undifferentiated cells or embryonic stem cells
for different therapeutic purposes, such as the treatment of degenerative diseases or
traumatic injuries, or to corect genetically predisposed conditions. somatic cells can be
reprogrammed into a pluripotent stage by means of expressing some transcription
factors to get induced pluripotent stem (IPS cells) an ideal target for therapeutic coning.
A brief history of cloning
1938- First idea of cloning by Hans Spemann in a Tantastic experiment" to replace the
nucleus of an egg cell with the nucleus of another cell and development of embryo from
that cell.
1952-An attempt to clone Rana pipiens frog by Robert Briggs and Thomas King.
1970-Xenopus laevis frog by John B. Gurdon.
1981 Karl lmenese and Peter Hope cloned a mouse using nucleus from a mouse
embryo.
1994- Neal Firsttried to clone a sheep.He took the nucleus from an embryonic cell and
obtained a sheep embryo.

1995 Two sheep are cloned (Moran and Megan),first animals cloned from
differentiated cells by means of nuclei transfer.
1996-The first mammal cloned from a cell taken from an adult animal, Dolly the sheep
by lan Wilmut and Keith Campbell.
1997-Gene, the first cloned domestic cattle in the world was bom at the American
Breeders Service facilties in Deforest, Wisconsin, United States.
1998-The first cloned mouse, Cumulina.
1999- Daisy. Holstein heifer was cloned by Dr. Xiangzhong (Jery) Yang using ear skin
cells from a high-merit cow named Aspen at the University of Connecticut followed by
three additional clones, Amy, Betty, and Cathy. Brahman bull, was loned from Chance,
a beloved celebrity bull was borm in August, 1999 at Texas A&M University.
1999 The first cloned rhesus monkey. Tetra (female) by embryo splitting (artificial
twinning).
2000 The first cloned pig (5 Scottish piglets (Jose, Josúe, Juan, Amber and Josefor
organ donation.

2000-Texas A&M University cloned a Black Aberdeen Angus bul.

2001-Downen TX 63 684 (Megan) was cloned from Boer goat. European mouflon lamb
was the first cloned endangered species to live past infancy.
2001-Gaur, a species of wild cattle, was the first endangered species to be cloned.
2001 cdoned Holsteins, Hereford, Jersey cows.
2001-A cat cloned, named Copy Cat.
2002 Konrad Hochedlinger and Rudolf Jaenisch clone mice from T lymphocytes.
2003-A Javan banteng calf was cloned from frozen cells using a cow as a surogate
mother and raised at the San Diego Wild Animal Parks Infant Isolation Unit.
2003-Worlds first cloned rat, Ralph (male).
2003-A rabbit is cloned in France and Southern Korea
2003 Idaho Gem, a mule is cloned
2003-A deer (Dewey)., a horse (Prometea)anda rat (Ralph) cloned, Pyrenean ibex.
2004-Little Nicky, first commercially cloned cat.
2005-Snuppy, an Afghan hound puppy, was the first dog to be cloned, in South Korea.
2005- Snuwolf and Snuwolfy, arctic wotf was cloned by South Korean scientists.
2006- Scamper, an extremely successful barrel racing horse, a gelding. was cloned.
The resulting stallion, Clayton, became the first cloned horse to stand at stud.
2008- A Labrador dog cloned
2009 Injaz, a female camel cloned from ovarian cells of a female killed for meat in
2005 at the Camel Reproduction Center in Dubai, United Arab Emirates.
2009 Samrupa, the world's first Murrah buffalo calf cloned using a simple "Hand

guided cloning technique".

2009- Garima-I and Garima-ll, buffalo calf cloned using an "Advanced Hand guided
Cloning Technique"
2009- The world's first pashmina goat clone, Noori, was produced at Centre of Animal
Biotechnology at Sher-e-Kashmir University of Agricultural Sciences and Technology of
Kashmir (SKUAST), Indian Administered Kashmir.
2010- A cloned male bufalo calf Shresth was bom at the NRDI.
2010-Got, the first Spanish Fighting Bull was cloned by Spanish scientists.
2011-coyotes, using domestic dogs as suTogate mothers by Sooam Biotech, Korea.
2013 Mahima, buffalo was inseminated with frozen-thawed semen of a progeny
tested bull and gave birth to a female calf, in January, 2013.
2017- A gene-edited dog, named Longlong byn Sinogene, a Beijing. China-based
biotechnology company.
2017- Zhong Zhong and Hua Hua (female crab-eating macaques)
2019- Garlic, the first Chinese commercially cloned cat .
2019-The first batch of monotocous cloned police dogs were borm
2020-Elizabeth Ann, a black-footed ferret female, was born on December 10, 2020, at
the Fish and Wildlife Service's Black-footed Feret Conservation Center in Colorado.
She is a clone of a female named willa, who died in the mid-1980s and left no living
descendants.
2020- Kurt, the world's first successfuly cloned Przewalski's horse was born.

Goal of Animal Blotechnology

Goal 1: Providing Nutritious Food for a Growing Human Population

Encompassing global food security. improving nural economies and development,
increasing productivity of agricultural enterprise and exports of agricutural products,
and reducing trade deficits.
Goal 2: Improving Sustainablity of Animal Agriculture
Improve environmental sustainability, economic sustainability and preserve germplasm
and genetic diversity.
Goal 3: Increasing Animal Fitness and Improving Animal Welfare
Improve animal fitness through adaptation to local and regional conditions, biotic and
abiotic stresses such as climate change, diseases and pests, and optimizing the
microbiome.
Goal 4: Meeting Consumer Needs and Choices
Enable consumer choices such as cultural or traditional foods, heathy choices (lean
and tender meat products), nutritional enhancements, and food raised through desired
farming practices (organic, no antibiotics ever).

Advancing Biotechnology to Improve the Sustainability and Efficlency of Animal

Production

Biotechnologies such as genomic selection are often combined with advanced

reproductive techniques such as cloning, embryo transfer, and artificial insemination to
accelerate the rate of genetic gain in animal breeding programs. Genetic engineering
has resulted in only a single approved application for an animal to be used for human
consumption, the fast-growing AquAdvantage Atdantic salmon (FDA, 2017), even
though the methodology was developed almost 30 years ago.

Fig.4. The genetically modified fish, 'AquAdvantage' salmon

Samon is first transgenic animal to win US approval for food in 04 August 2017.They gave it
a growth-homone gene from Chinook salmon (Oncorhynchus tshawytscha), along with
genetic regulatory elements from a third species, the ocean pout (Zoarces americanus).
The genetic modifications enable the salmon to produce a continuous low level of
growth hormone. The genetically modified fish, called 'AquAdvantage' salmon, were
engineered by Aquaßounty Technologies of Maynard, Massachusetts, Canada to
express higher levels of a growth hormone than wild salmon.

This situation was changed with the development of new gene editing technologies.
Gene editing techniques have the capacity to reduce threats from current and emerging
zoonotic pathogens; reduce required levels of production inputs; alleviate animal stress
and distress from disease, heat stress, the production environment, and other factors;
and improve growth efficiency, animal well-being, nutrition, and product quality. The
current methodologies of genome/gene editing using site-directed nucleases and base
editors (e.g. CRISPRICas9) facilitate the production of small edits such as single base
changes and small insertions/deletions.

Technology involved Animal Blotechnology

Gene Knockout
A gene knockout (KO) is a genetic technique in which one of an organism's gene is
made inoperative or "knocked out". knockouts are used to study gene function, usually
by investigating the effect of gene loss. Knockouts are accomplished through
techniques like Homologous recombination and site-specific nucleases. site specific
nucleases have been successfully used for gene editing, including zinc finger nuclease
(ZFN), transcription activator-like effector nuclease (TALEN), and the clustered regulartly
interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas) protein 9
system.
Conditional self-knockout

Deletions can be induced in a specific organ at a specific time in development, rather

than being deleted from birth. Knockout studies yield a great deal of information about
gene function. The gene may be activated during embryonic development, but the
deleterious effects can be observed in the adult organism. The CreHoxP recombination
system is the most commonly used system for conditional gene knockout.

Conditional salf-knockout in retinal degeneration

In humans, Otx2 has been found to play a role in retinal disease and ocular
malformations. In a conditional knockout study of Otx2 in mice, a progressive alteration
of retinal pigment epithelium (RPE) and photoreceptor cells was noticed and resulted
complete loss of photoreceptors. It was interpreted that Obx2 plays a direct role in
maintaining those cells, and specifically the process of melanogenesis. The findings had
a significant impact on the study of human disease, as it was proven that many
degenerative diseases of the retina are not developmental pathologies, but rather occur
in adults due to other processes.

Mario R. Capecchi Sir Martin J. Evans Oliver Smithies

Prize share: 1/3 Prize share: 1/3 Prize share: 1/3

Fig.5.The Nobel Prize in Physiology or Medicine 2007 was awarded jointty to Mario R.
Capecchi, Sir Martin J. Evans and Oliver Smithies tor their discoveries of principles for
introducing specific gene modifications in mice by the use of embryonic stem cells-
Knockout mice."
In vitro breeding schemes based on an embryo-stem cell technology

TheFuture: Invitrobreeding(IVB)
Lenome seiection of superior bulls and cows

-J years

wweks
MATING mbryos
tstablish

SELECTION
VB:3-4 months
erem Genomic selection ef supenor
trom ESCE male and temaie ESG
23 months CLONING

MEIOSIS

Fig.6. Efficient isolation of pluripotent embryonic stem cells (ESCs) from cattle embryos
allows the development ot in vitro breeding schemes besed on an embryo-stem cell-
gemete cycle, including an intermediate genomic selection to provide directiona!
selection of genetic progress. f such scheme could be accomplished, would
significantly decrease the generation interval and allow for increased selection intensity
leading to accelerated genetic progress. IVF, in vitro fertilization; ET, Embryo transfer.
Image from Van Eenennaam (2018). Reproduced with permission from the author's
entry in the Encyclopaedia of Food Securty and Sustainability

ESCs may have dramatic effects on the rate of genetic gain in terms of reducing the
cattle generation interval from 2-3 years to 3-4 months, offering a way to dramatically
reduce the generation interval and conceptually enable a tenfold increase in the rate of
genetic gain.
CRISPR-Cas9
CRISPRICas9 is one of gene-editing tools that allows modification of DNA in a genome,
the complete set of genetic instructions in an organism. One of the reasons for its
popularity is that it makes it possible to camy out genetic engineering on an
unprecedented scale at a very low cost. How it differs from previous genetic engineering
techniques is that it alows for the introduction or removal of more than one gene at a
time. This makes it possible to manipulate many different genes in a cell line, plant or
animal very quickly, reducing the process from taking a number of years to a matter of
weeks.
CRISPR-based genome editing9

Cells or zygotes Diversity

Diversity ohased technologies
of genome editing mediated by CRISPRR
Potentialapplications

Cas9 Indels and transgeniC

Human disease
DNA models
Ow
dCass BE DNA single-base editing
Xenotransplantation
31 Transcriptional activation
Sheep dCaseA
Transcription Bioreactors
dCas9 R Transcriptional repression
*****
NHPS
Cas13 Knockdownn Agricultural
breeding
RNA
Dog
Cas1 BE RNA single-base editing

Fig.7. Major strategies to recruit DNA- and RNA-targeting and modifying enzymes via
the CRISPRICas systems, and their potential applications in large animals to life
science fields. Left panel: large animals including pig, cow, sheep, monkey and dog are
discussed in this review. Middle panel: CRISPR-based technologies have been
developed to edit DNA and RNA, and regulate transcription. Right panel: potential
applications of genome-edited large animals in modelling human diseases, offering
xenotransplant organs, livestock breeding and more.
LERZE

Flg.8.The Nobel Prize in Chemistry 2020 was awarded jointly to Emmanuele

Charpentier and Jennifer A. Doudna Tor the development of a method for genome
editing-CRISPRICas9 genetic scissors "by Royal Swedish Academy of Sciences, on 7
October 2020.
First monkeys with customized mutations bom, Milestone for targeted gene-editing
technology promises better models for human diseases.

Flg.9. Twin cynomolgus monkeys born in China are the first with mutations in specific
target genes. 30 January 2014
Xingxu Huang, a geneticist at the Model Animal Research Center of Nanjing University
in China, and his coleagues successfully engineered twin cynomolgus monkeys
(Macaca fascicularis) with two targeted mutations using the CRISPRICas9 system.
Gene-edited cattle produce no homs
The scientists used the transcription activator like effector nucleases(CRISPR) DNA
editing technique to introduce a natural allele linked to horn lessness into dairy cow
embryos in 2016.

Fig.10. 2-month-old Spotigy homless dairy cow

 APPLICATIONS
1.Animal Blotechnoiogy to Advance Human Health
Animal-made phamaceuticals (AMPs) transfom biotech animals into "factories" to
produce therapeutic proteins in their milk, eggs, and blood, which can be used in the

development of biophamaceuticals.
2.Biotechnology to Improve Animal Health
Artificial insemination, embryo transfer, in vitro fetilization, genetic mapping and cloning
helped famers enhance breeding, resulting in healthier herds. New vaccines,
diagnostic tests and practices can help farmers treat animal diseases, while reducing
food borne pathogens at the farm level.
3.Biotechnology to Develop More Nutritlous Food
Food quality improved by introducing desirable traits through new genes into fam
livestock and poultry.
4.Conservation of Environment and Animals
Biotechnology can help produce environmentally friendly animals, as well as conserve
endangered species. Genetic studies of endangered animals can also resut in
increased genetic diversity which can result in healthier populations of species.
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