SBL100-Lecture

Introduction to genetic engineering

 Genetic Engineering
• Genetic engineering- the direct human manipulation of
an organism's genome using modern DNA technology.
• Produce therapeutic recombinant proteins.
• It involves the introduction of foreign or synthetic genes
into the organism of interest.
• Silencing of the bad genes.
• Transgenic animals & plants.

Tools:
• Using knowledge of DNA and various techniques.
• DNA Extraction – simple chemical procedure to separate DNA.
• DNA Cutting – restriction enzymes cut particular DNA sequences.
• Separating DNA – gel electrophoresis.
• Copy – using polymerase chain reaction “PCR”
 Approaches of genetic engineering
During 1980s, genetic engineers at the Monsanto Corporation
began to produce recombinant bovine growth hormone (rBGH)

by manipulating the DNA sequence (gene) that carries the

instructions for, or encodes, the growth hormone protein.

Growth hormone acts on many different organs to increase the

overall size of the body.

Before the advent of genetic technologies, growth hormone was

procured from the pituitary glands of slaughtered cows and then
injected into live cows.

However, harvesting the growth hormone from the pituitary

glands of cows and humans is laborious, and many cadavers are
necessary to obtain small amounts of the protein.

Genetic engineers at Monsanto realized that they could produce

large quantities of bovine growth hormone in the laboratory, inject
dairy cows, and increase milk production.
A case study: How
engineers produced rBGH
• The gene is sliced from cow
chromosome on which it resides by
exposing the cow DNA.

• Once the gene is removed from the cow

genome it is inserted into a bacterial
structure called a plasmid .

• A plasmid is a circular piece of DNA that

can replicate itself.

• When the cut plasmid and the cut gene

are placed together in a test tube they
reform into a circular plasmid with the
extra gene incorporated.

• The bacterial plasmid has now

genetically engineered to carry a cow
gene.

• rBGH is called a recombinant gene

because removed from its original
location cow genome and recombined
with bacterial plasmid DNA.
restriction enzymes are highly specific molecular scissors. cut DNA at specific sequences:

The first step in the production of the r BGH protein is to transfer the BGH gene from the
nucleus of a cow cell into a bacterial cell.

Bacteria with the BGH gene will then serve as factories to produce millions of copies of this
gene and its protein product—making many copies of a gene is called cloning the gene.
Once inside the cell, plasmids replicate, themselves, as does the bacterial cell, making
thousands of copies of r BGH gene. Using this procedure, scientists grow large amounts of
bacteria

The cloned BGH gene into bacterial cells, now produce the protein encoded by the gene.

The process of protein synthesis is also referred to as gene expression , since proteins are
synthesized when the genes that encode them are turned on.

Proteins are only synthesized when a particular cell needs them.

The scientists were then able to break open the bacterial cells, isolate
the BGH protein, and inject it into cows.
Close to one-third of all dairy cows in the USA now undergo daily
injections with rBGH, produced 20-30% more milk.
Monsanto Corporation has got the FDA approval.
Binding of Teicoplanin drug and its efficacy compared to some other drugs
 FDA Approved Drug Teicoplanin Found Effective Against Main Protease of COVID -19
virus: Study https://home.iitd.ac.in/news-teicoplanin.php

Prof. Ashok Patel, KSBS, IIT Delhi said, “While the effect of Teicoplanin was compared with other
important drugs in use, Teicoplanin was found to be 10-20 fold more effective than the chief drugs
being used against SARS-CoV-2, such as Lopinavir and Hydroxychloroquine in our laboratory
conditions. Teicoplanin is an FDA-approved glycopeptide antibiotic, which is regularly used for
treating Gram-positive bacterial Infections with low toxicity profile in humans."

Simultaneous study on Teicoplanin by Intensive Care COVID-19 Study Group of

Sapienza University

Recently, there has been a clinical study carried out with Teicoplanin as reported by Intensive
Care COVID-19 Study Group of Sapienza University. They recruited a cohort of 21 patients
affected by severe COVID-19 symptoms such as lung involvement, who were hospitalized in
intensive care units (ICUs) of a hospital in Italy, Rome, and complementarily treated with
Teicoplanin. On ICU admission, the patients received Teicoplanin in doses of 6 mg/kg every 24 h.
The median duration of Teicoplanin therapy was 10 days (range 7–12 days). The ICU mortality
rate was only 14.3% (3/21 patients). None of the patients had any adverse effects related to
Teicoplanin. In a nutshell, all studies support each other that Teicoplanin might be a potential
therapeutic option against COVID-19. However, a more detailed clinical investigation is required
on a large cohort, in different stages mild, moderate and critically ill patients to conclude the
definite role of Teicoplanin against COVID-19.
National News on Rajyasabha TV
IIT Delhi study was very well discussed on Rajsabha TV by Prof N.K. Ganguly former ICMR DG
and Prof Sarman Singh AIIMS Bhopal Director.
Link is attached here
https://www.youtube.com/watch?v=RE1LJFSWiEs
https://www.youtube.com/watch?v=RE1LJFSWiEs
https://home.iitd.ac.in/news-teicoplanin.php
https://home.iitd.ac.in/news-teicoplanin.php
 Therapeutic Proteins
Human genes are cloned into bacteria so
they can produce things of humans need.

For example:
Insulin: Recombinant insulin to prevent
Diabetes which is cheaper without side
effects because it is human insulin.
Blood clotting factors: Recombinant
Coagulation Factor IX for the control and
prevention of bleeding episodes.
Clot bluster: Streptokinase to prevent Stroke.
A recombinant vaccine is a vaccine produced
through recombinant DNA technology. This
involves inserting the DNA encoding an antigen
(such as a bacterial/viral surface protein) that
stimulates an immune response into
mammalian cells by expressing the antigen in
these cells.
The use of genetically modified yeast cells to
produce a vaccine against the hepatitis B (Liver
cancer) and human papilloma virus (Cervical
cancer) has been a major success story.

RECOMBIVAX HB® [Hepatitis B Vaccine] Gardasil® by Merck is the first

is used for prevention of infection caused vaccine for cervical cancers
by all known subtypes of hepatitis B virus.
 Transgenic Plants

Agrobacterium tumefaciens
has been used for genetically
engineering plants
A: Agrobacterium tumefaciens
B: Agrobacterium genome
C: Ti Plasmid :
a: T-DNA
b: Vir genes
c: Replication origin
d: Opines catabolism genes

Nitrogen fixation
 Potato for Vitamin Deficiency
• Vitamin A is essential for vision, immunity, organ development, growth, and
reproductive health. And Vitamin A deficiency is the leading cause of
preventable blindness in children.

• Vitamin E protects against oxidative stress and inflammation, conditions

associated with damage to nerves, muscles, vision, and the immune system.

Transformation of potato with a bacterial β-

carotene in a tuber-specific manner results in a
“golden” potato (GP) tuber phenotype resulting in
accumulation of Vitamin A and xanthophylls.

A serving of the yellow-orange lab-engineered

potato has the potential to provide as much as
42% of a child’s recommended daily intake of
vitamin A and 34% of a child’s recommended
intake of vitamin E.
FDA Regulations

FDA a governmental organization which ensures the safety of all domestic and imported foods
and food ingredients.

The manufacturer of any new food that is not Generally Recognized As Safe (GRAS) must
obtain FDA approval before marketing its product.

Adding substances to foods also requires FDA approval.

According to both the FDA and Monsanto, there is no detectable difference between milk from
treated and untreated cows and no way to distinguish between the two.

milk from r BGH treated cows was deemed safe for human consumption by FDA in 1993.

distributors of milk from untreated cows label their milk as “

hormone free”

In humans, studies indicate milk from cows treated with rBGH may
contain elevated levels of insulin-like growth factor-1 (IFG-1), which
can increase the risk of breast cancer , colon cancer and other
types of cancer.
 Artificially copying a piece of DNA from one
organism and joining this copy of DNA into the
DNA of another organism
• Human genes can be inserted into a bacterium
• Human genes can be inserted into cells from other animals
• Bacterium genes can be inserted into plant cells
• Genetic engineering means that DNA from different
organisms can be combined
• Bacteria can be engineered to produce human proteins
• Human genes can be inserted into other animals
• DNA codes for the proteins
that determine our traits.
DNA codes for proteins
• Proteins perform and regulate
all our bodily functions.
“genetic engineering” is a techniques in which DNA may
be cut, rejoined, its sequence determined, or the sequence
of a segment altered to suit an intended use.

1. Isolation of Gene

2. Plasmid & Vectors

3. Cutting

4. Ligation and Insertion

5. Transformation

6. Expression
A. Isolation of a specific gene B. Isolation of plasmid from
from donor e.g. human a bacterial cell
 Cells are broken open
 Isolate DNA
 Probes (Primers) to amply gene
of interest.

Forward Reverse
Primer Primer

Specific DAN Fragment can be amplified by PCR

 PCR, polymerase chain reaction, is an in-vitro technique for
amplification of a region of DNA whose sequence is known or which
lies between two regions of known sequence

Reaction Components PCR Cycle is comprised of 3 steps:

• DNA template: Isolated DNA to • Denaturation of DNA at 950C
be amplified • Primer hybridization ( annealing)
• Primers: 2 sets of primers with at 40-500C
complementary sequences • DNA synthesis ( Primer extension)
• Enzyme: Thermostable polymerases at 720C
(Taq)
(Isolated from Thermus Aquaticus) Kary Mullis awarded Nobel Prize
• dNTPs: Mixture of A,T,G,C in 1993 for PCR.
• Mg2+ & buffers
 Plasmids are circular pieces of
DNA found naturally in bacteria.
Antibiotic resistance gene
 Plasmids replicate separately from
the genome of the organism.
 Plasmids can be engineered to be
useful cloning vectors.

Plasmid vectors can be designed:

 Incorporating Antibiotic resistance
gene
 Multiple cloning sites
 Strong or weak promoters for
Multiple cloning sites
driving expression of a protein
• Bacterial restriction enzymes cut DNA
molecules at specific DNA sequences called
restriction sites
• A restriction enzyme usually makes many
cuts, yielding restriction fragments
• The most useful restriction enzymes cut
DNA in a staggered way, producing
fragments with “sticky ends.”
 Restricti 2. Cutting
Restricti on
Restric
tion
on site
ezym Restriction site Restriction
site
es Donor DNA site

¨ Restriction
enzymes act as
molecular
scissors and cut
DNA at specific
sites called Plasmid
Restriction
restriction sites

enzymes
29
© Biology Support Service 2007
 4. Ligation
 Ligation is the process of joining two
pieces of DNA from different
sources together through the
formation of covalent bond.
 DNA ligase is the enzyme used to
catalyze this reaction.
 Sticky ends can join with
complementary sticky ends of other
fragments
 DNA ligation requires ATP.
Ligation and Insertion
Ligation –rejoining
cut fragments of
DNA and forming
artificial recombinant
molecules
 After creating your new plasmid construct that contains gene of insert , that
construct need to insert it into a bacterial host cell so that it can be replicated.
 The process of introducing the foreign DNA into the bacterial cell is called
transformation.
 To insert the new construct, small holes are made in bacterial cells by chemical
treatment or by electroporation.
 The transformed bacteria cells are grown on selective media (containing antibiotic) to
select for cells that took up plasmid.
 Expression is getting the organism with the recombinant DNA to
produce the desired protein.
 Bacterial cell reproduces by Binary Fisson
 Bacterial cell produces the polypeptide
 When the protein is produced in large amounts it is isolated and
purified
Nuclear composition in cell
 Packaging of DNA into Chromosomes

Challenges of Packaging DNA

• How to get 2 meters of DNA into nucleus of 6 um
• Packaging accomplished with help of proteins
• Must be compacted in manner that still allows for it to
be accessed by enzymes that govern replication,
transcription, and repair
 Packaging of DNA into Chromosomes
DNA is packaged into a set of chromosomes
• DNA divided into set of chromosomes
• Chromosome= single DNA molecule and proteins
associated with it
5 levels of Chromosomal Packaging
 Genome and
Chromosomes

Human Chromosomes
• Human DNA 3.2 x 109 bases
distributed over 23 pairs of
chromosomes
• Each human cell contains 46
chromosomes.
• 22 homologous chromosomes AND
2 sex chromosomes (XX in females;
XY in males)
 Packaging of DNA into Chromosomes
Chromosomes Contain Long Strings
of Genes
• Most important function of
chromosomes = carry genes
• Gene= segment of DNA containing
information for making protein.
• Chromosome number varies
• The female Indian muntjac
number of chromosomes 6.
• The male has 7 chromosomes.
• The similar Reeves's Muntjac
(Muntiacus reevesi), has number
46 chromosomes.
What is the Human Genome Project?

3 billion bases
30,000 genes

http://www.genome.gov/
• An organism’s genome consists of all its genes.
• The Human Genome Project is a multinational research
project to determine the sequence of all 3 x 109 base
pairs and hence all human genes roughly 30000 genes.
The code is universal
§ Since all living
organisms…
u use the same DNA
u use the same code
book
u read their genes
the same way
Essentials of Genetics and outcome of genetic engineering
• Why does a commercial dairy cow produce four times as
much milk as most other mammals?
• Why do we look like our cousins?
• Why do roses come in so many different colors?
The answers to these and other questions about the
diversity of living things involve processes that occur at the
level of genes.

Genetics is concerned with genes that are constructed of

DNA, the basic building blocks of life.

Genetic engineering is concerned with altering the DNA

within particular plants or animals in order to create more
healthy or altered products.
What is a Gene?

A gene is a sequence of DNA on a chromosome that codes

for protein.

• The gene is the basic unit of heredity.

• A gene is a stretch of DNA which codes for a
specific protein.
Why would altering DNA affect our
characteristics/traits?

• DNA codes for the proteins that determine our traits.

• Proteins perform, regulate, or influence all our bodily
functions.
What are Genes?
• Genes are at the very heart of life.
• Genes constitute the blueprint of an organism.
• They decide all the properties and all the capabilities of
an organism.
• We are defined by our genes and how they interact with
the environment.
• In computer terms genes are the master program of life.
• In biological terms this master program is called the
hereditary substance, the chromosomes.
• It is constituted by chains of so called DNA molecules
that carry the "code words" or instructions of the master
program.

• We are just a vehicle for the reproduction of DNA.

What is genetic engineering?
• Genetic engineering is deliberate addition, deletion, or
intentional mutation of an organism’s DNA sequence to
produce a desired result.

• Genetic engineering means manipulation or alteration of

DNA sequences.

• Genes, mostly from other, often totally unrelated species

are inserted in the genetic "master program".

• Genes from e.g. fish, scorpions, bacteria and viruses have

been inserted into food plants in genetic engineering
projects.
• Questions and concerns?

• How can apply our understanding of DNA to manipulate

specific genes to produce desired traits?

• How can we use genetic engineering practice to address

current problems facing humanity?

• What are moral and ethical problems related to its

implementation?

Genetic engineering is so new and astonishing that people

are still trying to figure out the pros and cons. (advantages
and disadvantages).
Genetic engineering

Genetic

Genes

DNA
 What is DNA?
Deoxyribonucleic acid (DNA) is a biomolecule that contains
the complete genetic information for an organism. Every
cell of living organisms and many viruses, contains DNA.
Origin: The Chemical Nature of Nucleic Acids
•Friedrich Miescher, discovered DNA in 1869.
•He extracted a white substance from the nuclei of human cells
and fish sperm.
•The proportion of nitrogen and phosphorus in the substance
was different from that in any other known constituent of cells,
which convinced Miescher that he had discovered a new
biological substance.
•He called this substance “nuclein,” because it seemed to be
specifically associated with the nucleus.
• Because It was slightly acidic, it came to be called nucleic acid.
In the 1920s, the basic structure of nucleic acids was
determined by the scientist P. A. Levene, who found that
DNA contains three main components.
Three main components of Nucleic acid are:
(1) phosphate (PO4) groups;
(2) five-carbon sugars; and
(3) nitrogen-containing bases

called purines (adenine, A, and guanine, G) and

pyrimidines (thymine, T, and cytosine, C; RNA contains
uracil, U, instead of T).

From the roughly equal proportions of these components,

Levene concluded correctly that DNA and RNA molecules
are made of repeating units of the three components.

Each unit, consisting of a sugar attached to a phosphate

group and a base, is called a nucleotide.
The reaction between the phosphate group of
one nucleotide and the hydroxyl group of
another is a dehydration synthesis, eliminating a
water molecule and forming a covalent bond
that links the two groups.

•The linkage is called a phosphodiester bond

because the phosphate group is now linked to
the two sugars by means of a pair of ester
(P—O—C) bonds.

•The two-unit polymer resulting from this

reaction still has a free 5ʹ phosphate group at
one end and a free 3ʹ hydroxyl group at the
other, so it can link to other nucleotides.

•In this way, many thousands of nucleotides can

join together in long chains.
Erwin Chargaff showed that nucleotide composition of DNA molecules
varied in complex ways, depending on the source of the DNA.

Chargaff observed an important regularity in double stranded DNA:

the amount of adenine present in DNA always equals the amount of
thymine, and the amount of guanine always equals the amount of
cytosine.
Chargaff’s rules:
1. The proportion of A always equals that of T, and the proportion of
G always equals that of C: i.e A = T, and G = C.
2. It follows that there is always an equal proportion of purines (A
and G) and pyrimidines (C and T).
The Three- Dimensional Structure of DNA
➠DNA is the molecule that stored the hereditary
information,
➠how such a seemingly simple molecule could carry
out such a complex function?
Rosalind Elsie Franklin (25 July 1920 – 16 April 1958)
§an English chemist and X-ray crystallographer who
made contributions to the understanding of the fine
molecular structures of DNA, RNA, viruses.
§her data and research were key in determining the
structure and formulating Crick and James Watson's
1953 model regarding the structure of DNA.
§Unfortunately she died in 1958, ovarian cancer.
§Watson, Crick and Wilkins shared the Nobel Prize in
Physiology or Medicine in 1962. Watson suggested
that Franklin would have ideally been awarded a
Nobel Prize in Chemistry.
• Rosalind Franklin carried out an X-ray
diffraction analysis of DNA.

• In X-ray diffraction, a molecule is bombarded

with a beam of X rays.

• When individual rays encounter atoms, their

path is bent or diffracted, and the diffraction
pattern is recorded on photographic film.

• When carefully analyzed, they yield

information about the three-dimensional
structure of a molecule.

• The diffraction patterns she obtained

suggested that the DNA molecule had the
shape of a helix, or corkscrew, with a a
complete helical turn every 3.4 Nanometers.
James Watson and Francis Crick, at Cambridge
University, worked out structure for the DNA
molecule

DNA is a double helix. In a DNA duplex

molecule, only two base-pairs are possible:
James Watson and Francis Crick deduced adenine (A) can pair with thymine(T), and
the structure of DNA in 1953 from guanine (G) can pair with cytosine (C). An A-T
Chargaff’s rules and Franklin’s diffraction base-pair has two hydrogen bonds, while a G-C
studies. base-pair has three.
Punnett square is a tool to follow inheritance of the alleles for different types of genes .

A Punnett square is a table that lists the different kinds of sperm or eggs parents can
produce relative to the gene or genes in question and then predicts the possible outcomes
of a cross , or mating, between these parents.
Suppose letters F and f to represent the functional and dysfunctional allele.

every offspring of two carriers has a chance of being affected

 importance of genes in determining the value of quantitative traits.

e.g. farmers who wish to

increase their dairy herd’ s milk
production

(a)Change the herd’ s

environment by changing the
way the cows are reared,
housed, and fed; or
(b)change the herd genetically
by choosing only the offspring
of the best milk producers for
the next-generation herd.

The technique of controlling

the reproduction of individual
organisms to influence the
phenotype of the next
generation is known as
artificial selection.
 physical Analogy consider a pair of shoes and pair of chromosomes because
the two shoes are similar in size, shape, and style, but are not exactly similar.

If 23 students are asked to take off their shoes and place them in a row across
the front of the classroom, and they arrange their shoes so that the left shoe is
on the left, and the right shoe is on the right, the students could then separate
all of the left shoes from the right shoes, just as meiosis separates homologous
chromosomes.

The students could continue making different combinations of left and right
shoes for a very long time, because there are 223(over 8 million) possible ways
to line up these pairs of shoes.

Same is true with chromosomes

223(over 8 million) genetically different gametes are possible.

1 x 8 million x 1 x 8 million = 64 trillion

Together our parents could have made over 64 trillion genetically different
children, and we are only one of the possibilities.
 Artificially copying a piece of DNA from one
organism and joining this copy of DNA into the
DNA of another organism
Conventional therapy Includes
Small molecule
Antibodies
Chemotherapy
Radiation therapy etc

New approaches includes

Stem cell therapy
Genetic therapy

Genetic Therapies

By Gene Transfer, Genome Editing, Gene Addition

Genetic therapies aim to treat or cure conditions by correcting problems in the DNA.

As we know DNA, including specific genes, contains instructions for making proteins that are
essential for good health.

mutations, or changes in the DNA, can lead to proteins that do not work properly or that are
missing altogether.

These changes can cause genetics disorders such as cystic fibrosis, alpha-1 antitrypsin
deficiency, thalassemia, hemophilia, and sickle cell disease.
Genetic therapies
Ø Genetic therapies are approaches that treat genetic disorders by providing new DNA to certain cells or
correcting the DNA.
Ø Gene transfer approaches, also called gene addition, restore the missing function of a faulty or missing
gene by adding a new gene to affected cells. The new gene may be a normal version of the faulty gene or
a different gene that bypasses the problem and improves the way the cell works.
Ø Genome editing is a newer approach that allows precise correction or other targeted changes to the DNA
in cells to restore a cell’s function.

Ø Genome editing can do the following:

• Remove a stretch of DNA that causes a disease
• Turn off a gene to prevent it from making a harmful protein
• Turn on a gene or instruct a cell to make more of a needed protein
• Correct a mutated gene

Ø Gene transfer or genome editing treatments can directly modify the cells in your body, or your cells can
be collected and treated outside of your body and then returned to you.

Ø For example, a doctor can remove immune system cells or bone marrow cells from your body, modify
their DNA, and then re-introduce the cells to your body.

Ø The only genetic therapies that are currently approved by the U.S. Food and Drug Administration (FDA)
are for a rare inherited eye condition, as well as certain types of cancer.

Ø Genetic therapies that are in development could treat or cure other inherited disorders; treat other
cancers; or treat infections, including HIV, SARS-CoV or other diseases.
Gene transfer
Gene transfer introduces an additional gene into specific cells. This gene may stay as an extra piece of DNA in the cell or
be inserted into the cell’s own chromosomes and thus become part of the cell’s own DNA.

A molecular package called a vector carries the gene to the cell nucleus, which is the central part of the cell where DNA is
packaged in chromosomes. Vectors are created in the laboratory, often from viruses that have been modified to remove
viral genes that cause disease and to carry a treatment gene.

Once the gene is inside the nucleus, the cell will start to make the critical protein needed for the cell to work properly.
The new proteins make up for missing or faulty proteins and are meant to improve health for people who receive genetic
therapies.

Gene transfer. These two panels represent a cell with a faulty gene, caused by a mutation, before and after successful gene transfer. The faulty gene in this example makes
proteins that are faulty, as shown at left. After gene transfer, the cell makes normal functional proteins from the addition of the treatment gene. Medical Illustration
Copyright © 2019 Nucleus Medical Media, All rights reserved. external link
 Genome editing
v Genome editing introduces components that function together into cells. One component is a protein that cuts DNA,
similar to a pair of molecular scissors. Another component is a guide molecule that can stick to DNA at specific sites.
When the guide molecule sticks to an area of faulty DNA, the scissors protein attaches to the guide molecule, and
cuts out the faulty DNA.
v After the target DNA is cut, several things can happen. The cell may leave behind a gap, return the DNA to its original
state, or fill in this gap with the corrected DNA. The cell can fill in the corrected DNA if it has a template DNA to direct
the cell to rebuild a healthier version of the DNA that was removed. Therefore, sometimes a small piece of template
DNA is introduced as a third component. This DNA is a corrected version of the faulty DNA, and it is used to rebuild
the DNA correctly after it is cut open.

Genome editing. These two panels represent a cell with a faulty gene before and after successful genome editing. In this case, genome editing repairs the gene itself, rather
than adding an extra gene. After genome editing, the repaired gene allows the cell to make normal functional proteins. Medical Illustration Copyright © 2019 Nucleus
Medical Media, All rights reserved

https://vimeo.com/458994196
In the future, genetic therapies may be used to prevent, treat, or cure certain inherited
disorders, such as cystic fibrosis, alpha-1 antitrypsin deficiency, hemophilia, beta
thalassemia, and sickle cell disease.

They also may be used to treat cancers or infections, including HIV.

Genetic therapies that are currently approved by the FDA are available for people who
have Leber congenital amaurosis, a rare inherited condition that leads to blindness.

CAR T-cell therapy is FDA approved for people who have blood cancers, such as acute
lymphoblastic leukemia (ALL) and diffuse large B-cell lymphoma.
 Genome Editing
• The “guide RNA” is attached
to Cas9, a bacterial enzyme
that will cut the DNA sequence
at the desired site in the
genome.

• Once the genome is broken,

the guide RNA/Cas9 disappear,
which can disable or knock
out a particular gene.

• One can paste a new gene into

the cut at a desired location
and changing the defective
gene with correct one in
genome.
 Gene Therapy
 Gene therapy is the insertion of
correct genes into an individual's
cells and tissues to treat a
disease.
 A vector delivers the therapeutic
gene into a patient’s target cell
 The target cells become infected
with the viral vector.
 The vector’s genetic material is
inserted into the target cell
 Functional proteins are created
from the therapeutic gene
causing the cell to return to a
normal state.
Delivering desired Genes
 Acute Lymphoblastic Leukemia

•Adult acute lymphoblastic leukemia (ALL) is a type of cancer in which the bone marrow
makes too many lymphocytes (a type of white blood cell).

•Leukemia may affect red blood cells, white blood cells, and platelets.

•Previous chemotherapy and exposure to radiation may increase the risk of developing ALL.

•Signs and symptoms of adult ALL include fever, feeling tired, and easy bruising or bleeding.

•Tests that examine the blood and bone marrow are used to diagnose adult ALL.

•Certain factors affect prognosis (chance of recovery) and treatment options

Adult acute lymphoblastic leukemia (ALL; also called acute lymphocytic leukemia) is a cancer
of the blood and bone marrow.

This type of cancer usually gets worse quickly if it is not treated.

 Uses of gene therapy
 Replace missing or defective genes;
 Deliver genes that speed the destruction of cancer cells;
 Supply genes that cause cancer cells to revert back to
normal cells;
 Deliver bacterial or viral genes as a form of vaccination;
 Provide genes that promote or impede the growth of
new tissue; and;
 Deliver genes that stimulate the healing of damaged
tissue.
 Gene Silencing
• RNA interference, or RNAi, a molecular
mechanism that defends plants, fungi,
and animals against viruses made of
RNA.
• When a RNA virus takes over a host
cell, it needs to copy itself and the
copying process creates double
strands of RNA.
• The RNAi defense mechanism
recognizes these double-stranded
RNAs as foreign and degrades them.
• So a gene targeted by RNAi can’t
produce the protein.
• Thus, RNAi-targeted gene as being Useful for silencing bad or defective genes
“knocked down” or “silenced.”
Genetic Engineers Can Modify Foods

Whether you realize it or not, you have been eating genetically modified foods now.

• Improving yield of crop plants is the driving force behind majority of genetic engineering.

• Yield can be increased when plants are engineered to be resistant to pesticides and
herbicides, drought, and freezing.

• People believe that improving farmers’ yields may help decrease world hunger problems

• Crop plants are genetically modified to increase their shelf life, yield, and nutritive value.

• The first genetically engineered fresh produce was tomatoes, in store in 1994.

• These tomatoes were engineered to soften and ripen more slowly.

• It taste better and increases selftime in grocery without overripe and mushy.

• An enzyme called pectinase mediates the ripening process in tomatoes.

• This enzyme breaks down pectin, a naturally occurring substance found in plant cells.

• When pectinase is active, it helps break down the pectin and tomato softens.
• In tomatoes, engineers insert a gene
that produces an mRNA transcript
complementary to the mRNA
produced by transcription of a
pectinase gene.

• In dsDNA, the strand that codes for a

protein is called the sense, and its
complement is called the antisense.

• When antisense version of the

pectinase gene is transcribed, it
produces an mRNA that is
complementary to the mRNA from the
normally transcribed pectinase gene.

• When GE antisense gene base pairs

with its naturally occurring pectinase
complement, ripening is slowed.

• Thus, less of the pectinase enzyme is

produced and ripening occurs more
slowly
 due to uncontrolled population increase, it will demand an increase in yield of
crop plants in order to feed all the world’ s people.
• Genetic engineers are able to increase the nutritive value of crops.

• engineers have increased amount of β-carotene in rice, a staple food for world’ s people.

• this Golden rice will help decrease blindness because cells require β-carotene in order to
synthesize vitamin A, required for vision.

When a gene from one organism is

incorporated into the genome of another
organism, a transgenic organism is produced.

A transgenic organism is commonly referred to

as a genetically modified organism or GMO .

Golden Rice has been genetically

engineered to produce more β-carotene
How Are Crops Genetically Modified?
To modify crop plants, the gene must be able to gain access to the plant cell, which means it
must be able to move through the plant’ s rigid, outer cell wall.

In nature, Agrobacterium tumefaciens bacterium infects plants and causes tumors called galls.
The tumors are induced by a plasmid, called Ti plasmid (for T umor i nducing).

Moving genes into other agricultural crops such as corn, barley, and rice are done by using a
device called a gene gun .

A gene gun shoots tungsten-coated pellets covered with foreign DNA into plant cells .

A small percentage of these DNA genes may be incorporated into the plant’ s genome.
 To help farmer’ s reliance on pesticides, agribusiness companies have engineered
plants that are genetically resistant to pests.

For example, corn plants have been engineered to kill the corn borer.
(a) corn borer damages corn
and decreases yields.

(b) gene present in the

bacterium Bacillus
thuringiensis produces a
protein that is toxic to the
corn borer.

When this gene is inserted into

corn DNA, the plant produces
the protein that kills the corn
borer, thereby providing
resistance to the pest.

Scientists transferred a gene from the soil bacterium Bacillus thuringiensis (Bt) into corn.
The Bt gene encodes proteins that are lethal to corn borers but not to humans

close to one-half of all corn currently grown in the United States is engineered with this gene
Some examples of Genetic engineering

Normally, tomatoes are picked while green

and transported many miles before being
sprayed with ethylene to ripen them.
This prevents damage and perishing on the
journey.

The Flavr Savr tomato is a genetically

engineered tomato which has a gene
inserted to extend shelf-life by slowing
down the rotting process.

The Flavr Savr tomato was the first GM Is it better to spray

fruit to be sold in the World. tomatoes with
ethylene than
genetically engineer
them?
Some examples of Genetic engineering

Genetically engineered rice which contains a gene

from carrots (or other vegetable) which causes the
rice to contain the building blocks for vitamin A
production in the body.

Vitamin A deficiency causes blindness and death.

125 million children suffer from vitamin A
deficiency. Most of these children live in
developing countries where rice is the staple
Some people think that GM food.
crops like this one promote the
use of GM foods to people that Too much vitamin A causes other health
are not in the position to say no. problems.
Some examples of Genetic engineering
The ‘Protato’ contains 60% more protein
per gram than a ‘normal’ potato, it also
has a larger yield. Could the Protato face the
same opposition as Amflora?
A gene was used from the grain
Amaranth which codes for storage
protein.
Amflora
In tests with rats and rabbits there Potato created for the starch industry.
have been no side effects and no
Used antibiotic resistance marker gene.
allergic affects.
Fear that the genes could escape into the
environment.
It was proposed that the waste potato was
fed to livestock.
This caused outrage from some European
countries, why?
Some examples of Genetic engineering
Originally created in an attempt to show Zebra fish with a gene inserted from
levels of pollutions in rivers. jellyfish or coral to make them
Native to India and Bangladesh. None have fluoresce.
survived in American rivers.

The first genetically

engineered organism to be How does this fish
sold as a pet. benefit us? Other fluorescent organisms

They can reproduce, but it is

illegal to do so!
Some examples of Genetic engineering
Spider silk is stronger than steel, lightweight, and very
elastic.
It holds it’s strength between -40°C and 220°C.
Spider silk could be used to manufacture;
• replacement ligaments
•wound covering (it has antiseptic properties and
vitamin K which helps with blood clotting!)
Goats which produce spider silk in their milk! •optical communications
The gene transferred from a spider causes the •bullet proof clothing
goats to produce an extra protein in their milk
which can be extracted and spun into spider silk •waterproof clothing!!
thread.

About 75% of spider goats are

euthanised as there are strict
Spiders cannot be controls meaning that they
farmed as they are cannot leave the facility where
cannibalistic – they are created.
they eat each other! Why create a life to destroy it?
Some examples of Genetic engineering

A gene which controls the growth

hormone from one breed of
This fish has not been consumed by salmon is inserted into the DNA
humans yet – is it safe? of another.
This causes it to grow much quicker
What affect could the than ‘normal’ salmon.
AquAdvantage salmon
have on wild salmon if it
escaped? Could this gene be
transferred to humans
if we eat it?
What could happen if
this occurred?

AquAdvantage
salmon
Normal salmon
Some examples of Genetic engineering

Cabbage which has been genetically engineered

to include the gene for Scorpion venom.

This reduces the use of chemical pesticides

sprayed on crops.

The venom is poisonous to caterpillars – it

acts as a pesticide.
The toxin has been altered - it does not kill
human cells

What if the toxin mutates and

alters again?
What affect will the toxin have
on the biodiversity of the area?
 Some examples of Genetic engineering

Bananas, carrots, potatoes and lettuce

have all been genetically engineering to
deliver vaccines for diseases. The virus’ genes are
transferred to the
The banana has been the most
banana cells and
successful in testing.
become a permanent
part of that banana’s
genetic code.
When the gene has been inserted into the banana, it’s
cells produces virus proteins (not the infectious part).
When you eat the banana you ingest these proteins
and the body produces antibodies against them – this
is exactly how injected vaccines work!

Is there a risk of people taking

the vaccine without realising they
are doing so?
Genetic Engineers Can Modify Humans
The genetic modifications may one day include replacing defective or nonfunctional alleles
of a gene with a functional copy of the gene.

If this happens, it might be possible for physicians to diagnose genetic defects in early
embryos and fix them, allowing the embryo to develop into a disease-free adult.
How could you clone a human?
• Step 1: An egg is removed from a female human
• Eggs are haploid: 23 chromosomes.
• The nucleus of the egg is removed and is thrown away.

• Step 2: A body cell is removed from another person. 23

• The nucleus of the body cell is removed
• Body cells are diploid: 46 chromosomes. EGG CELL
• Step 3:
• The nucleus of the diploid body cell is put into the egg.
• This egg no longer needs to be fertilized since it has all 46
chromosomes.

46

46 Body Cell

EGG CELL
• Step 4: The egg is then
charged with electricity to
start mitosis.
• Step 5: Its then put into a
surrogate mother so it can
grow.

• Its going to be genetically

identical to the parent of the
body cell.
• But it will be a baby.

• Any Plants and animals can be

cloned.
 The Nobel Assembly at Karolinska Institutet
has today decided to award
the 2023 Nobel Prize in Physiology or Medicine
jointly to
Katalin Karikó and Drew Weissman
for their discoveries concerning nucleoside
base modifications that enabled the
development of effective mRNA vaccines
against COVID-19

Billions of people around the world have received the Pfizer or Moderna COVID-19
vaccines. The rapid development of these vaccines changed the course of the
pandemic, providing protection against the SARS-CoV-2 virus.
But these vaccines would not have been possible it if weren’t for the pioneering work
of this year’s winners of the Nobel prize in physiology or medicine decades earlier.

Dr Katalin Karikó and Dr Drew Weissman, researchers from the University of

Pennsylvania, have been given the prestigious award for their discoveries into
mRNA biology. The pair were the first to discover a way of modifying mRNA that
allowed it to successfully be delivered to cells and replicated by them.

Their discovery was not only integral to COVID-19 vaccine development, but may
also lead to the development of many other therapies – such as vaccines for cancer.
Life’s work
Karikó is a Hungarian biochemist and Weissman an American physician scientist.
The two began working together in 1985 when Karikó was a postdoctoral
researcher at the University of Pennsylvania, where Weissman was already working
as an immunologist. They had a shared interest in how mRNA could be used to
make new therapies.

Messenger RNA (better known as mRNA) is an essential molecule to life. It’s made
in the body from our very own DNA in a process called translation. DNA is our
special encoded handbook of instructions for manufacturing proteins, which are
the building blocks for material in the body.

Our mRNA copies and carries these genetic instructions from our DNA to our cells.
The cells then make whatever protein they’ve been instructed to, such as
haemoglobin which helps red blood cells carry oxygen around the body.

Karikó and Weissman thought that if it was possible to commandeer this process,
mRNA could be used to instruct cells to essentially make their own cures. But at the
time they started working together, attempts by other researchers to do this had
been unsuccessful.
COVID vaccines

When their research was first published, it didn’t garner much attention.
But in 2011, two biotech companies – Moderna and BioNTech – took
notice and began research into mRNA medicines.

It’s no wonder why. Traditional vaccine production methods are time

consuming, expensive and don’t work for every vaccine. But Karikó and
Weissman’s work showed that synthetic mRNA could be made at a large
scale.

Researchers had already been working on developing mRNA vaccines

before the pandemic, such as a vaccine for Ebola that didn’t receive
much commercial interest. But in 2020, when COVID-19 began
spreading around the globe, vaccines were needed quickly to offer
protection.

Using the foundational work of Karikó and Weissman, scientists

developed a mRNA sequence which mimicked the spike protein (which
allows the virus to enter our cells). This produced a harmless COVID
particle which our cells then replicated, allowing our bodies to protect us
from severe COVID infections when it encountered the real virus.
How do COVID19 vaccines
stimulate an immune response?

• Vaccine makes coronavirus spike protein in cells, which is presented to immune system

• Immune cells detect spike protein and make antibodies specific for spike protein

• During infection, antibodies bind spike protein on real virus and protect against infection and/or disease

• Antibodies speed up elimination of the coronavirus from the body

Vaccination Coronavirus Vaccine antibody Coronavirus Strong antibody Virus

spike protein response to spike infection response eradicated

Y YY Y
YY
Y Y
days weeks 1 week days
YY

RNA
Y
Are genetic engineers doing more good than harm? This chart lists
some of the pros and cons of genetic engineering
Protesters at a World Trade Organization
meeting in Seattle. These people are
concerned about how GMOs
may affect humans and the environment
References:
•Lehninger g Principles of Biochemistry. 5th Edition. 2008. David L. Nelson and Michael M.Cox.
•Molecular Cell Biology. 5th Edition. 2004. Lodish, Berk, Matsudaira, Kaiser, Krieger, Scott,Zipursky and
Darnell.
•Biology: Science for Life, 4/E Colleen M. Belk, University of Minnesota, Virginia Borden Maier, ISBN-10:
0321767829 • ISBN-13: 9780321767820 ©2013
•An Introduction to Genetic Engineering, Third edition,2008, By Desmond S. T. Nicholl
•Biology, by Raven and Johnson, sixth edition 2002
•Nickell, J. and Fischer, J.F. (1999). Crime Science: Methods of Forensic Detection. Kentucky: The University
Press of Kentucky.
•Riciu , A. (2011, November 26). Pituitary Dwarfism Causes, Symptoms, Diagnosis and Treatment. Retrieved
from http://www.doctortipster.com/6928-pituitary-dwarfism-causes-symptoms-diagnosis-and-
treatment.html
•Acknowledgement:
•Internet sources, teaching slides on slideshare, scitable and others ppts
•Students: Nitika, Praveen and Kirtika

Today:
We learnt about genetic engineering implications
Home assignment:
Advantages and disadvantages of genetic engineering

Next class:
Cancer biology