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H2 BIOLOGY
APPLICATION SYLLABUS

Topics covered:
1
...
GENE THERAPY

2
...
PLANT CLONING

3
...
Genetic Engineering & GMOs

!

Chapter 1: ICS DNA (Part I)
INTRODUCTION
Genetic engineering centered the idea of isolation, cloning and sequencing of specific DNA from
different sources
...
This allows a large number of proteins to be synthesized
from the gene of interest
COMPONENTS FOR IN VIVO GENE CLONING
Gene cloning: Isolation of individual genes (target DNA or gene of interest), followed by the
generation of recombinant DNA molecules, and then propagated in a host cell to produce multiple
copies of that recombinant DNA molecules and hence the target DNA / gene of interest

Items required creating recombinant DNA molecule:
1
...
Gene of interest
3
...
DNA ligase
5
...
Enzyme DNA ligase is used to form a stable and permanent recombinant DNA molecule
Part 1: Formation of H-bonds
Part 2: Formation of phosphodiester bond
• The phosphodiester bonds between two DNA fragments are reformed by catalytic action of
DNA ligase

Note: Need to use the same restriction enzyme so that both vector and gene of interest can have
complementary sticky ends to allow for formation of H-bonds

Cloning Vector: DNA molecule that can carry foreign DNA into a host cell and independently
replicate in the host cell (most commonly used: plasmids)
Features of cloning vector
1
...

•
•
•

Variety of unique restriction sites (multiple cloning sites / polylinkers)
Contains a variety of unique restriction sites
Can be recognized by a range of restriction enzymes
Allowing a wide range of foreign genes to be cloned

3
...


Distinguishes between host cells which took up reannealed plasmid and recombinant plasmid
Restriction sites are located within selectable markers
...
Size limitation – size of DNA fragment it can effectively carry
2
...
An organism’s entire genome is cleaved into a large number of restriction fragments
2
...
The bacterial clones than make up a gDNA library
4
...
Isolation of mature mRNA - Mature mRNA is mixed with chemically synthesized molecules
called oligo-dT / poly-dT which binds to the 3’poly-A tail by complementary base pairing
...
Synthesis of 1st cDNA strand - Enzyme reverse transcriptase is used to make a DNA copy
from the mRNA, forming an mRNA-cDNA hybrid/heteroduplex
3
...
The 3’ end has a tendency to loop back on itself, creating a hairpin loop acting as a
primer for 2nd cDNA strand synthesis
4
...
S1 nuclease then cleaves the hairpin loop at the end of the
double stranded cDNA
5
...
This is done through DNA ligase, which
forms phosphodiester bonds between DNA fragments
...
Introduction of vectors with cDNA into bacterial host celss and resulting clones of bacteria
cells form cDNA library
Advantages of using cDNA as DNA source
• Mature mRNA sequences have no introns as they have already undergone splicing
• Restriction digestion of whole genome produces many DNA fragments, cDNA represents only a
portion of the cell’s genome corresponding to protein-coding sequences, which is a smaller
proportion

Difference between gDNA and cDNA:
gDNA library
Entire genome is represented

cDNA library
Only portion of genome expressed as proteins is
represented
Larger number of clones required to store library Comparatively smaller number of clones
Same gDNA obtained from all cells of organism Each cell gives different cDNA library as
different mRNA is produced based on cell type,
stage of development, environmental conditions
and functions
Intergenic noncoding regions are stored
Intergenic noncoding regions are not stored
Sequences stored contain introns
Sequences stored do not contain introns
Most clones contains only part of coding Each clone has a continuous coding sequence
sequence of a gene
Most genes are present in only one copy (hard to cDNA has many copies of mRNAs produced in
isolate and identify gene of interest)
large quantities (enriched in certain mRNA
species)
Host cells:
Through bacterial transformation, recombinant DNA molecules are placed within host cells for in
vivo amplification
...
coli that make it a good host organism
1
...
coli used are Generally Regarded As Safe (GRAS)
2
...
coli is easy to grow, requiring little space and nutrition and can be grown on nutrient agar
plates or solution
3
...
coli has very rapid replication time (1 division per 20 minutes)
4
...
coli is also easy to kill and dispose of
5
...
coli is able to take up and tolerate foreign DNA pieces

STEPS INVOVLED FOR IN VIVO GENE CLONING
Main steps:
1
...
Insertion of DNA into cloning vectors
3
...
Selection of clones with cloning vectors and subsequent identification of clones with
recombinant vectors
5
...
Insertion of DNA into cloning vectors:
Procedure
(1) The cloning vector (plasmid with chosen restriction enzyme) and gene of interest is cleaved by
the same restriction enzyme, making the DNA linear
(2) Sticky-ended fragments are incubated with linearized cloning vector molecules under conditions
favoring base pairing (annealing)
(3) Treatment with DNA ligase to link molecules covalently to produce recombinant DNA plasmid
vector
There are two ways annealing of sticky ends by CBP can occur between:
• 2 overhanging ends of a plasmid – forming reannealed plasmid
• Overhanging ends of plasmid and fragment of foreign DNA – forming recombinant plasmid
o Contains DNA fragments with gene of interest OR other DNA fragments
2
...
Host cells are made competent by pre-treating the cells with ice-cold calcium chloride solution,
allowing formation of calcium-phosphate-DNA complex to prevent repulsion of DNA
2
...
Cells are then subjected to heat shock (42oC for 2 minutes), causing them to take up ligation
products – known as transformation, host cells now known as transformants
4
...
Host cells are made competent by pre-treating the cells with ice-cold sterile H2O
2
...
Electricity is applied in a pulse lasting a few microsecond to a millisecond, disturbing the
phospholipid bilayer of the membrane, causing formation of transient pores so DNA are driven
across the membrane through pores
4
...
Selection of clones:
Host cells are then transferred to surface of nutrient agar plates, then they are spread out and
allowed to replicate
...

In this case, it is known as colony hybridisation:
1
...
Cells are lysed to release their DNA, and binds to nitrocellulose – also results in denaturation of
DNA to give ssDNA
3
...
The probe tags the clone by forming H-bonds with
the ssDNA complement on the filter
4
...
Once the colonies are identified, the cells can be grown in liquid culture in a large tank, known
as scaling up – obtaining large quantities of the gene product

Summary of identification of clones:
Replica plating

Nutritional requirement

APPLICATIONS OF GENE CLONING
Advantage of using proteins synthesized through recombinant DNA technology compared to
extracting them from cellular tissue:
1
...
Ease of extraction and purification – prokaryotic cells tend to produce a smaller range of
proteins than animal cells, allowing for easy purification
3
...
Ethical and religious concerns prohibit their use
Synthesis of Human Insulin:
Insulin is synthesized by the β-cells of the islets of Langerhans in the pancreas
...

The leader sequence is removed and the C chain excised after translation, leaving the A and B chain
linked by two disulfide bonds
Insulin displays two characteristics for production by recombinant DNA techniques:
• Human insulin is not modified after translation by addition of sugar molecules (no need for
post-translational modification)
• Insulin is a relatively small protein, with the A chain (21 aa) and the B chain (30 aa)
Two artificial genes are first constructed, for A chain (63 nucleotides) and B chain (90 nucleotides)
1
...
Both recombinant plasmids with two artificial genes are transformed separately into E
...
Large amounts of A and B polypeptides are produced for amplification of protein production –
each expressed independently as fusion proteins (with first few aa residues of β-galactosidase)
4
...
Purified A and B chains are mixed, reduced and reoxidized to form disulfide bonds in insulin

Synthesis of human growth hormone (hGH):
It is also known as somatotrophin, produced and released by the anterior pituitary gland, located on
chromosome 17
...
There were problems with
extracting it from pituitary glands of dead humans:
• Supplies were limited and only children most severely deficient were treated
• Extracts occasionally contaminated with prion causing Creutzfeldt-Jakob disease
Preparation of hGH cDNA fragment
1
...
A pituitary cDNA library is prepared
3
...
Longer segment with codons 24 to 191, is retained for use to construct recombination plasmid
Expression
5
...
coli
6
...
Recombinant plasmid introduced into E
...
Selection and identification of E
...
Lactose used to induce production of hGH
10
...
DNA template – dsDNA sample contains the nucleotide sequence of interest (gene of interest)
and can be gDNA or cDNA
2
...
They (1) flank the sequence of interest and (2) are complementary to
the 3’ end of both template strands
3
...
Thermostable DNA polymerase – Isolated from thermophilic bacterium (Taq polymerase),
stable at high temperature and not denatured by repeated heat treatments in the PCR cycle, thus
no need to add new polymerase after each cycle (saves time and money)
5
...
The PCR is a chain reaction, as the newly synthesized DNA strands serve as templates for
DNA synthesis in subsequent cycles
Practical applications for PCR:
It amplifies the DNA sequence from a small amount of the original sample in a short time, but PCR
only specifically amplifies the section of DNA between two primers, allowing for purification
...
Sensitivity – PCR is extremely sensitive, can amplify sequences from minute amounts of DNA
2
...
Robustness – PCR permits amplification of specific sequences from material in which DNA is
badly degraded or embedded in a medium from which conventional DNA isolation is difficult
Disadvantages
1
...
Infidelity of DNA replication in vitro – DNA polymerases used in PCR often lack 3’ to 5’
exonuclease activity, having an error rate of 1 in 10000 bases misincorporated
3
...
Need for target DNA sequence information – To construct specific oligonucleotide primers,
prior sequence information is required
5
...
Probe molecules are cloned from cDNA or gDNA
2
...
Analysis can then be done using radiography after the process of gel electrophoresis
Techniques utilizing principle of nucleic acid hybridisation:
It is applied in several practical techniques to identify or locate a specific nucleic acid sequence –
1
...
Southern Blotting (DNA sample)
3
...
In situ hybridisation
5
...
Sequence change of restriction sites hence a creation or destruction of a particular restriction site
2
...
Sequence repetitions due to difference in number of tandem repeats in different individuals
(VNTR), altering length of restriction fragments
Therefore, this causes different banding patterns from different individuals

Possible ways of generating RFLPs:

RFLP analysis:
Used to detect differences in DNA sequences of 2 samples or between individuals

If two alleles differ in that either
• A restriction site created or destroyed by a nucleotide change at an RFLP locus on one
chromosome but not its homologue OR
• Distance between 2 given restriction sites is longer on one chromosome than its homologue due
to a greater number of tandem repeats present or DNA insertion
The two RFLP alleles and thus chromosomes can be distinguished by the pattern of restriction
fragments on a Southern blot, detected by a probe
All diploid organisms have two copies of each chromosomes, thus individuals may be homozygous
for each RFLP allele or heterozygous for the RFLP locus

Applications of RFLP analysis:
Riding on the use of RFLPs as genetic markers, RFLP analysis has practical applications like:
1
...
Detection of disease causing alleles through linkage analysis
3
...
g
...
The
HGP also worked on sequencing genomes of important model organisms like bacterium (E
...
Pharmacogenomics
• Genetic differences makes us unique in our physiology and biochemistry, affecting the way we
react to drugs
• Some are dangerous, some are ineffective
• Knowledge about which genes affect a person’s response to a drug, called pharmacogenomics,
may allow doctors to prescribe the right drugs for the right individuals at the right dosage
2
...
Insights through comparative genomics
• It directly compares genetic information between organisms to identify genomic similarities and
differences that enable inferences to be made about genome evolution
• This is applied in gene discovery, development of animal model of human diseases and
elucidation of evolutionary history and relationship between genes, genomes and species
• Further comparative studies will help determine the yet unknown function of thousands of other
genes
4
...
Privacy and confidentiality of genetic information
• Ownership and control of genetic information
2
...

•
•
•

Fairness in use and interpretation of genetic information
By insurers, employers, courts, schools, adoption agencies and military
Parties to whom such information should be granted, and means of using this information
Likelihood of maintaining privacy of test results

4
...
Clinical issues
• Include professional education of doctors and other health service providers and patients, as
well as public education in genetic capabilities, scientific limitations and social risks
• Also implementation of standards and quality-control measures in testing procedures
• Measures to prepare healthcare professionals for new genetics
• Strategies to prepare public to make informed choices
• Means of balancing current scientific limitations and social risk with long-term benefits
6
...
Conceptual and philosophical implications
• Regarding human responsibility, free will versus genetic determinism and concepts of health
and diseases
• Whether people’s genes make them behave in a certain way, and thus can they control themself
• Definition of acceptable diversity
• Line between medical treatment and enhancement
8
...
An unspecialized cell from the embryo, fetus or adult that has
2
...
Can differentiate and give rise to specialized cells that make up tissues and organs of the body
4
...
It reflects the differential
potential of the stem cell
1
...
Pluripotent stem cells: descend from totipotent stem cells
• Can give rise to types of cells that develop from the three germ layers (mesoderm, endoderm
and ectoderm) but cannot form extra-embryonic membrane
• Taken from the inner cell mass of the blastocyst
3
...
This
usually involves the formation of an intermediate progenitor cell – with limited self-renewal ability

Committed stem cells (includes multipotent cells):
They are those that (1) have a more limited pathway of development compared to pluripotent cells
and (2) are destined to produce a specific group of cells
Examples: Hemangioblast stem cell – gives rise to all blood vessels, bloody cells and lymphocytes
• Can either give rise to more specifically committed cells like hematopoietic stem cell OR
generate progenitor cells (precursor cells)
Commitment of stem cells:
Why specific cell types cannot be induced to produce another cell type
1
...
Acetylation of genes required for specific differentiation into a specific cell type
3
...
Specific repressors present to prevent the expression of these genes

Progenitor cell:
A progenitor cell usually shows some evidence of differentiation, is committed to a limited number
of pathways and is more limited in developmental pathways than a multipotent cell

In this way, mitotic division of stem cells (1) preserves a population of undifferentiated cells while
(2) steadily producing a steam of differentiating cells

SECTION 2: EXAMPLES OF NORMAL FUNCTIONS OF STEM CELLS
EMBRYONIC STEM CELLS
Embryonic stem cells are isolated from the inner cell mass (ICM) of the blastocyst and results in the
death of the fetus
Defining properties of ESCs:
1
...
Exhibit and maintain a stable, full (diploid), normal complement of chromosome
3
...
Capable of developing into all fetal tissues during development
5
...
Unlike adult stem cells, they are easy to obtain pure and can be cultivated in large numbers
Fates of ESCs in a developing embryo:
In a developing embryo, the formation of the inner cell mass, ICM, is considered the first event that
distinguishes cells from each other and the cells will continue developing to form the embryo
The ICM is originally a two-layered disc

ADULT STEM CELLS
During adult life, tissues and organs undergo wear and tear and require a constant supply of
replacement cells
...
It is an undifferentiated cell able to renew itself and can differentiate to form major specialized
cell types
2
...
Stem cell numbers in the body are very low
Roles of adult stem cells:
• Maintain and repair the tissue in which they are found
• Replace cells that die because of injury or disease

Defining properties of ASCs:
1
...
They can make identical copies of themselves for long periods of time – known as long-term
self-renewal
3
...
They are clonogenic
Hematopoiesis:
It is a well-studied example of cell differentiation as blood cells at all stages are relatively
accessible and it can be grown in culture
We consider the differentiation of a multipotent (hematopoietic) stem cell
• Primary role of the HSCs is to replace blood cells and they are self-renewing
• They are the precursors of progenitor cells that are committed to one of the hematopoietic
lineages at a later stage and are derived from the mesoderm in the embryo

Erythrocytes, leukocytes and platelets are derived from 3 main lineages
...
The hematopoietic system can be considered as hierarchical system with the
multipotent HSC at the top
...
In hematopoiesis,
the bone marrow stromal cells provide the stem-cell niche
~ END ~

Chapter 4: Gene Therapy
INTRODUCTION
A genetic disease is caused by abnormalities in an individual’s genome
There are four different of genetic disorders
1
...
Multifactorial – caused by the combination of environmental factors and mutations in multiple
genes
3
...
Mitochondrial – caused by mutations in the mitochondrial DNA
Definitions:
Gene therapy: Introduction of the normal copy of a gene into the specific cells of a person carrying
defective / abnormal copies of the gene
Transgene: Gene that has been introduced into a cell or an organism and is different from
endogenous genes (already present in the organism)
Transgenic: Term used to describe the organism carrying the introduced gene
GENE TRANSFER APPROACHES
The delivery and targeting of transgene to proper tissues is crucial to the success of gene therapy
and there are two primary methods of gene transfer – ex vivo and in vivo
Methods of gene transfer:
Ex vivo gene therapy
In vivo gene therapy
Indirect method of transfer – genes transferred Direct method of transfer – genes are transferred
are located outside the patient
directly into tissues and organs
Following transfer of genetic material in the lab
using
techniques
similar
to
bacterial
transformation, cells are then reintroduced
Advantage / Benefit
No fear of rejection of tissue transplant as cells
are originally from the patient

Scientists have primarily relied on using viruses
as vectors, but some gene have been injected
directly into tissues
Advantage / Benefit
No need for surgical removal of cells

Disadvantage / Challenge
Disadvantage / Challenge
Need for specific delivery of transgene (only to
Need for surgical removal of cells
the intended tissues in the body)
So far ex vivo strategies have been more effective than in vivo

Types of cells used in gene therapy:
Germ-line gene therapy
Transgene is introduced into an early embryo or germ cell, mainly the ovum and sperm
• High risk therapy due to potential of introducing new inheritable mutation where all
descendants would be affected
• More controversial as it can be used to control human development in ways not connected to the
disease and is thus banned
Somatic cell gene therapy
Involves methods where cells from target somatic cells are used (stem cells, bone marrow tissue and
endothelial cells)
• Effects limited to one individual thus safeguards against danger of transmitting negative effects
of therapy to future generations
• Critical consideration is that the target cells should ideally have a long in vivo half-life or
significant replicative potential – so biological effects of transfer can be of useful duration
Therefore, stem cells are ideal candidates for somatic cell gene therapy – allowing expression of
transferred gene in a large population of daughter cells
...

Vectors are carrier molecules or particles for delivering the transgene to the target cells / tissues
Important criteria for vector system:
The vector by which a foreign transgene is introduced into target cells needs to be efficient and safe
1
...
Safety – ascertain that the body does not react adversely to the gene product (producing
antibodies) and the introduction has no deleterious effects
Comparing types of vectors for gene therapy:
Retrovirus
Type
vector

of
ssRNA virus

Adenovirus
Viral vector
dsDNA virus

Integration to
host cell
Advantage

Yes
(Using
reverse
transcriptase)
Transgene
can
be
integrated into human
chromosome
Long term stability

Disadvantage

Only relatively short DNA
sequences
Can only infect dividing
cells
Random integration leads
to insertional mutagenesis

No
Longer DNA can be
introduced
High levels of replication
and expression and stable
Can infect non-diving cell
Expression of transgene is
usually
unstable
&
transient
Might
exhibit
strong
immune responses
Involved in malignant
transformation

Liposome

“Naked” DNA
Non-viral vector
Lipid bilayers surrounding Small
plasmids
aqueous core packed with transgene
DNA
No
No
Much longer DNA can be
introduced
No viral genes that cause
disease
Large scale production
Less efficient
Expression of transgene is
transient

with

Much longer DNA can be
introduced
No viral genes that cause
disease
Large scale production
Inefficient in gene transfer

SEVERE COMBINED IMMUNODEFICIENCY (SCID)
Disease description:
Immunodeficiencies are genetic or acquired deficiencies that lead to loss of immune function
...
There are two kinds of SCID
1
...
Autosomal (ADA-) SCID
X-linked SCID
Mutation in the Interleukin 2 Receptor Gamma (IL2RG) gene located on the X chromosome
• It codes for a protein necessary for growth and maturation of lymphocytes
• Mutations prevents these cells from developing and functioning normally – hence the body is
unable to fight off infections
• Males are affected by the X-linked SCID more frequently than females
Autosomal SCID
Inherited disorder that damages the immune system and is caused by mutations in the adenosine
deaminase (ADA) gene on the long arm of chromosome 20
• It is characterized by a deficiency of the enzyme ADA
• ADA gene codes for ADA which eliminates deoxyadenosine when DNA is broken down
• Mutations in the ADA gene reduce the activity of ADA leading to build up of deoxyadenosine
to levels that are toxic to immature lymphocytes
• It is an autosomal recessive inheritance
Treatment options (in general):
There are currently 4 main treatment options for SCID
1
...
Give the child a transplant of bone marrow from a normal histocompatible donor (giving the
child a continuous source of healthy T and B cells)
• T cells in the transplant might attack cells of the child, producing graft-versus-host disease
• Donor cells may be infected with a virus which could overwhelm their immune system first
3
...
Administer gene therapy by giving the patients functional genes

Retroviral gene therapy for SCID:
For both X-linked and ADA- SCID, ex vivo gene therapy trials using retroviral vectors have been
conducted
Prior to treatment, a recombinant retroviral was constructed with normal gene/allele put under
control of a strong viral promoter
• A selectable market is placed next to the normal gene / allele for identification of successfully
transfected cells
Chimeric gene construct
A chimeric gene construct is made incorporating the normal ADA gene / allele

Gene therapy procedure
• (1) - (2): Blood stem cells (HSCs) from the bone marrow of the patient are isolated and grown
in cell culture outside the body
• (3): Cells are transfected with a retroviral vector containing the normal gene
• (4): Successfully transfected cells are selected
• (5): They are then returned to the patient
Gene Therapy trial
In 1991, several children with ADA- SCID were treated with ex vivo gene therapy with retroviral
vectors and also in late 1990s for 15 French baby boys suffering from X-linked SCID
Outcome – after more than 10 years, many of the children
• Can live normal lives at home instead of a sterile bubble
• Have normal (with some exceptions) number of T cells
• Have responded to several childhood immunization, such as tetanus and polio, producing both T
cells and specific antibodies
• Produce sufficient antibodies so that they do not need period infusions of immunoglobulin
Setback – gene therapy for X-linked SCID faced a major setback when five of the boys developed
leukemia due to insertion of retroviral vector into a gene on chromosome 11 implicating in some
case of acute lymphoblastic leukemia

CYSTIC FIBROSIS (CF)
Disease description:
In CF, the basic problem is an abnormality in the glands that produces sweat and mucus
...
Loss of excessive amount of salt in sweat thus resulting in the upset of balance of minerals in
the blood, might cause abnormal heart rhythms and also shock
2
...
Other medical problems like inflation of nasal sinuses, nasal polyps, clubbing of fingers and
toes, pneumothorax, coughing of blood, heart enlargement, abdominal pain and discomfort etc
...

Early attempts of somatic cell gene therapy involved using adenoviral vector, but due to rapid
immune responses it proved ineffective
• Attention then turned to the use of liposomes instead in CF gene therapy
Liposomal gene therapy for CF:
CFTR transgenes are delivered directly into somatic cells by coating them with lipids, which
mediate fusion with cell membrane – known as lipoplexes
Gene therapy procedure
1
...
Normal CFTR gene is inserted into the plasmid using DNA ligase
3
...
Liposomes are taken as nasal spray / aerosol / inhaler and they enter cells of lung tissue and fuse
with cell membranes of the epithelial cells
5
...
Short-lived nature of gene therapy
• Transgene introduced into target cells must be turned on to make the protein it encodes and
must remain functional – cells containing transgene must be long lived and stable
• There are problems like (1) rapidly dividing nature of some target cells and thus transgenes are
lost each time the cell divides, (2) certain viral vectors carrying only small genetic payload and
(3) difficulty of inserting the transgene into billions of target cells
• One way to solve the problem is to integrate the gene into the host genome (technically difficult
and results in problems if inserted into inappropriate positions)
• Sufficient amounts of corrective DNA must be delivered to enough cells to be therapeutically
beneficial
• Yet limitation lies in how the activity of the transgene can be controlled to that the transfected
cells can make appropriate amounts of the gene product at the right time and place

2
...
Immune response
• Anytime a foreign body is introduced into human tissues, the immune system is design to attack
the invader and it might reduce the gene therapy effectiveness or leads to rejection
• The human body tends to suppress gene expression, essentially turning new genes off or
suppressing introduced genes and low levels of gene expression affect gene therapy efforts
• Gene delivery vectors need to be able to avoid the body’s natural surveillance system
• The immune system’s enhanced response to invaders it has encountered also make it difficult to
repeated in patients
4
...
Multigene disorders
• Conditions or disorders that arise from mutations in a single gene are the best candidates for
gene therapy
• However many diseases arise from the combined effects of mutations in many genes – and are
difficult to treat effectively using gene therapy
CONSIDERATIONS FOR USE OF GENE THERAPY
Social considerations:
1
...
Safety concerns
• Existing risks of using gene therapeutic techniques

3
...
Manipulation of human genes
• Primary issue lies with whether human genetic modification is ethically acceptable
• Some critics suggest that tampering with human genes in anyway will inevitably lead to
practices of eugenics – deliberate effort to control genetic makeup of human populations
• Techniques involved can also insert genes into normal human, with the intention to modify or
enhance particular traits
• Others see no fundamental difference between the transplantation of genes into somatic cells
and the transplantation of organs into individuals
2
...
Somatic cell versus germ-line gene therapy
• Controversy whether it is more or less ethical than germ-line gene therapy, and if it should be
used to treat human germ cells in the hope of correcting defects in future generations
• Almost universal agreement that germ-line gene therapy is morally and ethically unacceptable
• Issue to consider the type of circumstances under which the genomes of human germ lines or
embryos should be altered - could be seen as interference with the natural process of evolution
• From biological perspective, elimination of unwanted alleles from gene pool could backfire as it
removes genetic variation, which is important for survival of species if environment changes
4
...
Plantlets
2
...
Callus
4
...
Carbon and energy source
• Sucrose is the most preferred carbon and energy source
• Useful respiratory substrate as plant cells and tissues lack autotrophic ability (enhances
proliferation of cells and generation of green shoots)
2
...
Inorganic nutrients
• Nitrogen – component of all proteins, enzymes and metabolic processes involved in synthesis
and transfer of energy
• Part of chlorophyll, green pigment of plant responsible for photosynthesis
• Magnesium – Part of chlorophyll, essential for photosynthesis
• Helps activate many plant enzymes needed for growth
4
...
Used to start new cultures
2
...
g
...
Explants from almost any plant structure or part-seeds, stems, roots, leaves, storage organs, or
fruits are excised
2
...
Continued subculture at three- to four-week intervals of small cell clusters taken from these
callus masses can maintain the callus culture for long periods
Uses of callus culture
1
...
Preparation of single cell suspension and protoplast
3
...
Viruses can be more easily incorporated into protoplasts
2
...
Genetic engineering – protoplasts can absorb DNA, proteins, and other large macromolecules
allowing new genetic material to be incorporated
Procedure for protoplast culture
Establishment
1
...
Soak pieces in solution containing cellulose, sugar & salts in a petri dish, digesting the cell wall
3
...
Culture the protoplasts on a petri dish with feeder cells embedded in agar
Multiplication
5
...
Transfer microshoots after 2 – 4 weeks into agar medium to induce root development
Acclimatization
7
...
A number of
insect pests prey on corn but the most damaging is the European corn borer
What is Bt?
It is a naturally occurring soil-borne bacterium that produces crystalized proteins that are toxic and
selectively kill specific groups of insects and their larvae
...
Genetic engineers first isolate from the Bt bacterium the foreign gene coding for one of the Bt
proteins toxic to the corn borer and insert into the corn plant genome via the use of Ti plasmids
of Agrobacterium tumefaciens
2
...
The transgene is then inserted into the corn genome after multiplication using vectors and
bacteria
Benefits of Bt corn
• Plants protect themselves against pets – cuts down reliance on chemical insecticides thus
reducing cost, labour and environmental damage
• Bt toxin also breaks down rapidly in the environment as it is naturally occurring
• Harvest yields are much higher and hence cope with rising demand worldwide
• Mode of action of Bt toxin is highly specific and very effective and have no effect on non-target
insects – safe for humans and animals
• Economic losses due to pest infestations are reduced
• Efficient agricultural production contributes to reducing costs in the food production sector
Concerns associated with Bt corn
• Low levels of Bt protein could potentially provide ideal conditions for nature to select for
resistant individuals
• Certain insects may evolve genetic resistance to the Bt protein in transgenic plants
• Non-pest species could be harmed
• Bt protein does not normally occur in the food chain and may cause allergies
• Transgenes in Bt corn plant could potentially be transferred, via pollen or some other route, to
any closely-related weeds

Nutrition: Golden Rice
Refers to a genetically engineered, yellow-orange rice grain that contains beta-carotene which is
converted to Vitamin A by the human body
In developing countries, vitamin A deficiency is a leading cause in blindness in children
• It also makes children more susceptible to infectious diseases
Golden Rice
It is an interesting case of genetic engineering
• Introduces a new biochemical pathway in tissue of the transgenic plant
• Could not be done by conventional breeding as no rice cultivar known produces enzymes in the
endosperm – original constructs used one gene from daffodil and one from bacterium
1
...
Plant does not usually produce the pigment in the endosperm as it is only produced in the leaves
and involved in photosynthesis
3
...
A recombinant DNA plasmid construct with an AFP gene promoter from ocean pout attached to
cDNA for salmon growth hormone is used to produce rapidly growing fish

2
...
AFP promoter can be used to stimulate transcription of “downstream” genes under cold water
conditions
4
...
Genetic markers
• Vectors in genetic engineering contain antibiotic resistance marker genes and enter the
transformed crop
• Concerns that antibiotic resistance gene, if not properly broken down by digestive system when
consumed, may be passed to potentially harmful bacteria and make them resistant
• Flavr Savr tomatoes: The first GM food with kanamycin resistance with longer shelf life
2
...
Possibility of creation of hazardous new pathogens
• If cancer cell genes were transferred into bacteria or viruses, “rogue microbes” can be created
4
...
GM crops might establish themselves as weeds
• Seeds from GM crops modified to withstand unfavorable environmental conditions (herbicide
resistant) may be carried to other places
• May establish themselves as weeds
• GM oilseed rape: contains genes for resistance to herbicide Basta
6
...
Reduced effectiveness of pesticides
• Concern that insects may become resistant to GM crops that can produce their own pesticides
• Bt corn: produces their own toxin Bt toxin
8
...
Issues on acceptability of genetic manipulation of animals and plants
• Whether humans have the right to “play God” and tamper with nature by mixing genes among
species
• Unnatural violation of natural organisms’ intrinsic values
• Whether living organisms have rights in the case of “human rights”
2
...
Religious concerns or dietary restrictions
• Plants that have been modified to carry animal genes face objections from vegetarian groups
• Objections to genes from some species for religious groups (pig genes for Muslim)
4
...
Impact of GE on crop production in developed countries vs 3rd world countries
• Whether richer countries are benefitting at the expense of poorer countries
• World food production may be dominated by a small number of large companies with the
technical know-how
• Terminator technology: genetically switch off a plant’s ability to germinate a second time, thus
forcing farmers to buy a fresh supply of seeds each time
6
...
Issues with consumer rights
• Consumers have a legitimate interest in and right to information with regard to GMO
• Rules to transparent sharing of relevant information and communication of associated risks
ensure protection of consumer rights
• Labeling measures might not be adequate as it is not mandatory, unjust to those who object to
GMOs
• Regulations against mixing of GM foods with non-GM products are also not strictly enforced
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