the study of changes in organisms caused by modification of gene expression rather than alteration of the genetic code itself
In biology, histones are highly alkaline proteins found in eukaryotic cell nuclei that package and order the DNA into structural units called nucleosomes. They are the chief protein components of chromatin, acting as spools around which DNA winds, and playing a role in gene regulation.
Friedrich Miescher isolates DNA for the first time. In 1869
DNA proved to be the hereditary material Griffith's experiment, reported in 1928 by Frederick Griffith, was the first experiment suggesting that bacteria are capable of transferring genetic information through a process known as transformation. Griffith's findings were followed by research in the late 1930s and early 40s that isolated DNA as the material that communicated this genetic information.
1961 DNA can be split on heating and stuck back together again on cooling (reannealing) DNA hybridisation possible 1962 The first restriction endonucleases discoveredMolecular scissors 1966 The genetic code worked outDiscovered to be a universal code 1967 DNA ligase discoveredMolecular glueFragments of DNA can be stuck together
In 1907 a bacterium that caused plant tumors, Agrobacterium tumefaciens, was discovered and in the early 1970s the tumor inducing agent was found to be a DNA plasmid called the Ti plasmid By removing the genes in the plasmid that caused the tumor and adding in novel genes researchers were able to infect plants with A. tumefaciens and let the bacteria insert their chosen DNA into the genomes of the plants
1973 DNA cloning carried out on bacteriaGene identifiedCut with restriction enzymeSpliced into a plasmid using ligasePlasmid reintroduced into a bacterium Gene copied whenever the bacterium divides Non-bacterial gene can be expressed in the bacterium 1982 Transgenic mice and fruit flies produced
By removing the genes in the plasmid that caused the tumor and adding in novel genes researchers were able to infect plants with A. tumefaciens and let the bacteria insert their chosen DNA into the genomes of the plants the action or process of introducing DNA or chromosomes into bacteria or other cells using a pulse of electricity to open the pores in the cell membranes briefly.
A knockout rat is a genetically engineered ratwith a single gene turned off through a targeted mutation (gene trapping) used for academic and pharmaceutical research. Knockout ratscan mimic human diseases and are important tools for studying gene function (functional genomics) and for drug discovery and development.
Genetic engineering has been used to produce proteins derived from humans and other sources in organisms that normally cannot synthesise these proteins. Bacteria synthesising human insulin were developed in 1979, being used as a treatment for the first time in 1982. In 1988 the first human antibodies were produced in plants.[In 1997avidin, an egg protein, was expressed in a plant with the intention of extracting, purifying and selling it. The first transgenic livestock were produced in 1985, by micro injecting foreign DNA into rabbits, sheep and pigs eggs. The first animal to synthesise transgenic proteins in their milk were mice, engineered to produce human tissue plasminogen activator. This technology has now been applied to other sheep, pigs, cows and other livestock. With the discovery of microRNA in 1993 came the possibility of using RNA interference to silence an organisms endogenous genes. Craig C. Mello and Andrew Fire discovered a silencing effect in 1998 through injection of double stranded RNA into C. Elegans . Using genetic engineering the microRNA can be expressed long term, permanently silencing the target genes. In 2002 stable gene silencing was induced in mammalian cells,[and in 2005 this was accomplished in a whole mouse.[In 2007 papers were released where insect and nematode genes that formed microRNA were put into plants, resulting in gene silencing of the pest when they ingested the transgenic plant.
SCID involves defective antibody response due to either direct involvement with B lymphocytes or through improper B lymphocyte activation due to non-functional T-helper cells. Consequently, both "arms" (B cells and T cells) of the adaptive immune system are impaired due to a defect in one of several possible genes. SCID is the most severe form of primary immunodeficiencies, and there are now at least nine different known genes in which mutations lead to a form of SCID
PEG-ADA; polyethylene glycol-coated bovine ADA derived from cows
Leukaemia is a cancer of the white blood cells. The term lymphoblastic means that the cancer affects a type of white blood cell called a lymphocyte, which are mostly used to fight viral infections. ‘Acute’ refers to the rapid nature at which this particular cancer progresses.
Size-exclusion chromatography (SEC), also known as molecular sieve chromatography, is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel-filtration chromatography, versus the name gel permeation chromatography, which is used when an organic solvent is used as a mobile phase. SEC is a widely used polymer characterization method because of its ability to provide good molar mass distribution (Mw) results for polymers.
HIV. "Physicians might edit a patient's immune cells to delete the CCR5 gene, conferring the resistance to HIV carried . Some forms of genetic blindness. Inactivating a certain variant of a gene in the retinal cells of the eye could stop some types of inherited, progressive blindness in their
Familial hypercholesterolemia. An inherited condition, familial hypercholesterolemia can lead to extremely high levels of "bad" cholesterol and heart attacks at a young age. Editing liver cells could fix this inherited disorder
Sickle-cell anemia. Editing blood stem cells could cure this disease, which affects about 100,000 Americans and can cause lifelong pain and even organ damage.
Hemophilia. Another blood disorder could be cured by editing blood stem cells, hemophilia causes frequent bruising, pain, and excessive bleeding because of low or no levels of the proteins needed to create clots
Unlike the kind of embryo editing that could lead to permanent changes in the human race, these edits would be made in babies, children, or adults. That means, they pose no unique ethical issues because they affect only a patient's own [ordinary] cells."
genetic engineering, future perspectives and QC validation
BY SANA RUBAB
CMS No. 11286
2) BASICS OF GENETIC ENGINEERING
3) HISTORY OF GENETIC ENGINEERING
4) Methods of preparation
6) Future perspective
7) Upgradation QC tests
2) BASICS OF GENETIC ENGINEERING
Genetic engineering is a part
Application and industrial
use of organisms for human
welfare is called
Biotechnology is a huge topic.
Its hard to define its exact
Some European scientists divide the
field into :
1) Red biotechnology
2) Green biotechnology
4) White Biotechnology
Book : Biotechnology & Genetic engineering (Kathy wilson peacock)
2010,Edi:1 : Page No. 4 (Chapter 1)
Book : Biotechnology & Genetic engineering
• Genetics – science of genes, heredity
and variation in living organisms.
• Genetics deals with the molecular
structure and function of genes, and
gene behavior in context of a cell
• Patterns of inheritance from parent to
offspring, and gene distribution,
variation and change in populations.
Book : Genetics and the Organism: Introduction
U S National Library of Medicine
• Different terms used for
genetic engineering :
1) Gene manipulation
2) Gene cloning
3) Recombinant DNA technology
4) Genetic modification
5) New genetics
An Introduction to Genetic Engineering (Desmond S. T. Nicholl) Edi :3rd 2008
Chapter 2 . Page 3
GENETIC ENGINEERING CONTINUATION..
Direct manipulation of an
genome using biotechnology .
First isolating and
genetic material of
inserted in the host
An Introduction to Genetic Engineering (Desmond S. T. Nicholl)
Edi :3rd 2008 Chapter 2.
How genes are cloned???
• The procedure consists of inserting
a gene from one organism, often
referred to as ``foreign DNA`` into
the genetic material of a carrier
called a vector.
• Examples of vectors include
bacterias, yeast cells and viruses.
• After the gene is inserted, vector
is places in laboratory conditions
that prompt it to multiply,
resulting in the gene being copied
many times over. 1210/12/2016
Genetic inheritance was first
discovered by Gregor Mendel in 1865
following experiments crossing peas.
Although largely ignored for 34 years he
provided the first evidence of hereditary
segregation and independent assortment.
In 1889 Hugo de Vries came up with the name
"(pan)gene" for after postulating that particles
are responsible for inheritance of characteristics.
Term "genetics" was coined by William
Bateson in 1905.
In 1928 Frederick Griffith proved the
existence of a "transforming principle" involved
in inheritance, which Avery, MacLeod and
McCarty later (1944) identified as DNA.
Edward Lawrie Tatum and George Wells
Beadle developed the central dogma that genes
code for proteins in 1941.
The double helix structure of DNA was
identified by James Watson and Francis
Crick in 1953.
In 1970 Hamilton Smiths lab
discovered restriction enzymes that allowed
DNA to be cut at specific places and separated
out on an electrophoresis gel.
• This enabled scientists to isolate genes from an
DNA ligases,. that join broken DNA together,
had been discovered earlier in 1967 and by
combining the two enzymes it was possible to
"cut and paste" DNA sequences to
create recombinant DNA.
Plasmids, discovered in 1952, became
important tools for transferring information
between cells and replicating DNA sequences.
Polymerase chain reaction (PCR), developed
by Kary Mullis in 1983, allowed small sections
of DNA to be amplified and aided identification
and isolation of genetic material
The Discovery of DNA Fingerprinting was
done by Dr. Alec Jeffreys, . In September
Cystic fibrosis gene cloned
Transformationusing electroporation was
developed in the late 1980s, increasing the
efficiency and bacterial range
In 1972 Paul Berg utilised restriction enzymes
and DNA ligases to create the first recombinant
Trials for gene therapy begin in 90s.
• Herbert Boyer and Stanley N. Cohen took
Bergs work a step further and introduced
recombinant DNA into an bacterial cell.
In 1981 the laboratories of Frank Ruddle,
Frank Constantini and Elizabeth Lacy
injected purified DNA into a single-cell mouse
embryo and showed transmission of the genetic
material to subsequent generations.
On June 19, 2013 the leaders of three research teams who
originated the technology, Robert T. Fraley of
Monsanto; Marc VanMontagu of Ghent University
in Belgium and founder of Plant Genetic Systems
and CropDesign ; and Mary-Dell Chilton of Washington
University in St. Louis and Syngenta were awarded with
the World Food Prize
The first recorded knockout mouse was created
by Mario R. Capecchi, Martin
Evans and Oliver Smithies in 1989. They are
used to study gene function and make useful
models of human diseases.
In 1992 onco-mice with tumor suppressor
genes knocked out were generated.
Creating Knockout rats are much harder and has
only been possible since 2003
Bacteria synthesising human insulin were
developed in 1979, being used as a treatment for
the first time in 1982
In 1988 the first human antibodies were
produced in plants.
The first animal to synthesise transgenic
proteins in their milk were mice, engineered
to produce human tissue plasminogen
With the discovery of microRNA in 1993
came the possibility of using RNA
interference to silence an organisms
In 1976 Genentech, the first
genetic engineering company,
was founded by Herbert Boyer
and Robert Swanson and
a year later the company
produced a human protein
(somatostatin) in E.coli.
• His work cloned
frogs laid the
the application of
which led to Dolly
“The Father of
“The Father of
In 1973 created
DNA into its
making it the
Human Genome Project
• 1990-2003 The human genome
Goals Of The Human Genome Project
• identify all the approximately
20,000-25,000 genes in human DNA
• determine the sequences of the 3
billion chemical base pairs that
make up human DNA
• store this information in
• improve tools for data analysis
• transfer related technologies to
the private sector.
Methods of preparation of
Kashaf Ur Rehman
Cms # 8882
Basic steps in genetic
• Isolate the gene
• Insert it in a host using a vector
• Produce as many copies of the host as possible
• Separate and purify the product of the gene
This method is the most commonly used method in genetic engineering. In this
method uses small circular pieces of a DNA molecule called plasmids. This method is
mainly used for altering microorganisms such as bacteria.
This method uses vectors, which are small carrier molecules, which are normally
viruses. Viruses are made of a protein capsule and have their DNA inside, they
attach onto a cell then inserts its DNA or RNA into the host cell, then it detaches
itself. The DNA, now inside the host cell, will start replicating itself by using the
genetic information of the host cell, which means the gene that was inserted will
now be part of the host cell.
The vector method is better than the plasmid method because the plasmid method
offers genetic variation because the newly formed plasmids are made with random
pieces of DNA, while the vector method uses a specific gene to get a specific result.
This method is also called the gene gun method, This method is mainly used for the
engineering of the plants.
DNA can become "sticky" under certain conditions allowing it to adhere to abiotic
particles such as metals. They normally use tungsten, gold or silver. These metals are
extremely small particles, which are now coated with DNA.
The particles are placed inside the gene gun and a partial vacuum is created
between the target tissue and the gun.
The particles are then fired at the target and the DNA is effectively introduced to the
To Explain the process of genetic engineering we have taken the example of insulin,
a protein that helps regulate the sugar levels in our blood.
Normally insulin is produced in the pancreas, but in people with type
1 diabetes there is a problem with insulin production.
People with diabetes therefore have to inject insulin to control their blood sugar
Genetic engineering has been used to produce a type of insulin, very similar to our
own, from yeast and bacteria like E. coli.
This genetically modified insulin, ‘Humulin’ was licensed for human use in 1982.
1. A small piece of circular DNA called a plasmid is extracted from the bacteria or
2. A small section is then cut out of the circular plasmid by restriction
enzymes, ‘molecular scissors’.
3. The gene for human insulin is inserted into the gap in the plasmid. This plasmid is
now genetically modified.
4. The genetically modified plasmid is introduced into a new bacteria or yeast cell.
5. This cell then divides rapidly and starts making insulin.
6. To create large amounts of the cells, the genetically modified bacteria or yeast are
grown in large fermentation vessels that contain all the nutrients they need. The
more the cells divide, the more insulin is produced.
7. When fermentation is complete, the mixture is filtered to release the insulin.
8. The insulin is then purified and packaged into bottles and insulin pens for
distribution to patients with diabetes.
Genetic engineering has applications in medicine, research, industry and agriculture
and can be used on a wide range of plants, animals and microorganisms.
• In medicine, genetic engineering has been used to mass-produce insulin, human
growth hormones, human albumin, monoclonal antibodies, antihemophilic
factors, vaccines, and many other drugs.
• Industrial applications include transforming microorganisms such as bacteria or
yeast, with a gene coding for a useful protein.
• Genetic engineering is also used in agriculture to create genetically-modified crops.
For treatment of cancer
• Factor VIII
Needed by hemophiliacs for blood clotting
For treatment of anemia
• Tumor necrosis factor
For treatment of tumors
• Tissue plasminogen activator
Use to dissolve blood clots
A normal gene inserted to compensate for the defective gene.
Abnormal gene replaced with a normal one
Abnormal gene repaired through selective reverse mutation.
Bubble boy disease
ADA deficiency is an inherited condition that occurs in fewer than one in 100,000 live births
worldwide. Individuals with ADA deficiency inherit defective ADA genes and are unable to produce
the enzyme adenosine deaminase in their cells. The enzyme adenosine deaminase is needed to break
down metabolic byproducts that become toxic to T-cell lymphocytes, and is essential to the proper
functioning of the immune system . T-cell lymphocytes, white blood cells, are not able to remove the
byproducts in the absence of ADA.
Without ADA, the toxins derived from the metabolic byproducts kill the T cells shortly after they are
produced in the bone marrow. Instead of having a normal life span of a few months, T cells of
individuals with ADA deficiency live only a few days. Consequently, their numbers are greatly reduced,
and the body's entire immune system is weakened. ADA deficiency is the first known cause of a
condition known as severe combined immunodeficiency (SCID).
Prior to present-day treatments, most ADA-deficient SCID victims died from infections before reaching
the age of two. Although SCID is usually diagnosed in the first year of life, approximately one-fifth of
ADA deficient patients have delayed onset SCID, which is only diagnosed later in childhood.
David Vetter from Texas also had SCID
and had to live in a sterile environment
for most of his life during the 1970/80s.
He was known to the media as 'the boy
in the plastic bubble' and wore a special
'spacesuit' to protect him from
• Treatment of SCID
The treatment of choice for ADA deficiency is
bone marrow transplantation from a matched
sibling donor. Successful bone marrow
transplants can relieve ADA deficiency.
Unfortunately, only 20–30% of patients with
ADA deficiency have a matched sibling donor.
Another treatment involves injecting the
patient with PEG-ADA. The PEG coating helps
keep the ADA from being prematurely
degraded. Supplying the missing enzyme in
this way helps some patients fight infections,
while others are helped very little
Acute lymphoblastic leukemia
Progress has also been made in people with acute lymphoblastic leukemia.
In acute lymphoblastic leukemia, a subset of lymphocytes called B-cells become cancerous. Scientists
therefore decided to modify another type of immune cell, the T-cell to attack only B-cells. They did
by reprogramming T-cells to attack all cells with a protein called CD19 on their surface. CD19 is a
molecule that is only found on the surface of B-cells. When these reprogrammed T-cells were
reintroduced back into acute lymphoblastic leukemia patients they attacked and destroyed all
cancerous and normal B-cells in the body. With all of the body’s B-cells destroyed the immune
could make new, normal, non-cancerous B cells over the next few months.
“In some patients it took just a few weeks for all of their cancer cells to be removed.
Although it is early days, the results from this trial are very promising, with 10 out of 13 individuals
treated ending up in remission from the cancer. In some patients it took just a few weeks for all of
cancer cells to be removed.
QC Test for Insulin
Insulin is a peptide hormone, produced by beta cells of the pancreas and is
central to regulating carbohydrate and fat metabolism in the In diabetes
treatment. Mostly referred as “HUMULIN
At every step, someone or something makes sure that the insulin production is
going smoothly Everything from the water to the air to the operators is held to
the highest standards of cleanliness. It’s that clean. Sterility is also vital during
growth, as one rogue pathogen could ruin a batch.
Quality control of Insulin After every one of the many steps of purification,
scientists check and double-check the insulin’s purity. Even the packaging
process is super-tightly regulated, as each vial of insulin is photographed from
Injectable Insulin Preparation
Injectable insulin preparation are sterile preparation of .They contain NLT 90% and NMT the equivalent of
110% of the amount of insulin stated on the label.
1. pH It should be 6.9 to 7.8 otherwise as per specific monograph.
2. Insulin in the supernatant should be NMT 2.5% of the total insulin content, and insulin
of supernatant liquid(s) is determined by chromatographic method
3. Impurities with molecular masses greater than that of insulin It is
examined by size-exclusion chromatography.
4. Related proteins It is examined by liquid chromatography.
5. Total Zinc It is determined by atomic absorption spectroscopy. Prepare test and reference solutions
and measure absorbance at 213.9 nm using a zinc hollow cathode lamp or air-acetylene flame as source of
6. Bacterial endotoxins should be less than 80 IU per 100 IU of insulin.
Quality Control parameter for Injectable
7)Assay examine by liquid chromatography. Mostly, it is carried out by High
Performance Liquid Chromatography (HPLC).
8) Sterility test
(9) Particulate Matter Testing
10) Package Integrity Tests
Gene chips are medical sensors about the size of a
matchbox, and feature a tiny "DNA microarray". Every
square on this grid contains a particular DNA snippet.
When a sample of patient DNA comes into contact with
the gene chip this causes some of its squares to
illuminate, so revealing the level of activation of particular
Use in diagnosis
Different types of cancer have been classified on the basis
of the organs in which the tumors develop. Now, with the
evolution of microarray technology, it will be possible for
the researchers to further classify the types of cancer on
the basis of the patterns of gene activity in the tumor cells.
Example: characterizing acute lymphoblastic leukemia.
Also breast cancer. • Use in prognosis • Example:
assessing the likelihood of metastasis in medulloblastoma
(brain tumor in children)
Use in toxicological research
Microarray technology provides a robust platform for the
research of the impact of toxins on the cells and their
passing on to the progeny. Toxicogenomics establishes
correlation between responses to toxicants and the
changes in the genetic profiles of the cells exposed to
such toxicants. The microarray permits researchers to
examine thousands of different genes in the same
experiment and thus to obtain a good understanding of
the relative levels of expression between different genes
in an organism
Use in selection of drug
By examining the gene chip under a microscope a
patient's genetic suitability for certain drugs can thereby
be assessed. Experimental gene chips are already
available from suppliers for trail.
Gene Chips continued…
In one of the first successful attempts at genetically
engineering mosquitoes, researchers have altered the
way the insects respond to odors.
scientists announced the completion of the full genome
sequence of Aedes aegypti, the mosquito that transmits
dengue and yellow fever. They have altered the sense of
smell of mosquitoes for humans and the insect repellant
zinc-finger nucleases to are used specifically to mutate
the orco gene in Aedes aegypti. They injected the
targeted zinc-finger nucleases into mosquito embryos,
waited for them to mature, identified mutant individuals,
and generated mutant strains that allowed them to study
the role of orco in mosquito biology. The engineered
mosquitoes showed diminished activity in neurons linked
to odor-sensing. Then, behavioral tests revealed more
Genetic Engineering Alters Mosquitoes’
Sense of Smell
The colloquial term "designer baby" refers to a baby whose genetic makeup has been artificially selected by
genetic engineering combined with in vitro fertilization to ensure the presence or absence of particular genes
In simpler terms, using biotechnology to choose what type of baby you want. Latest research is making
designer babies a reality now, using technology developed originally for use in animals.
• What traits could be changed in a designer baby?
1)Gender 2) Appearance 3)Intelligence 4) Disease 5)Personality
• Trait selection
• Embryo screening involves a process called pre-implantation genetic diagnosis (PGD). Embryos are created
by in-vitro fertilization and grown to the eight-cell stage, at which point one or two cells are removed.
Scientists then examine the DNA of these cells for defects, and only normal embryos are replaced in the
• Three-parent baby
Three-parent babies are human offspring with three genetic parents, created through a specialized form of In
vitro fertilisation in which the future baby's mitochondrial DNA comes from a third party. The procedure is
intended to prevent mitochondrial diseases including muscular dystrophy and some heart and liver
conditions. It is the subject of considerable controversy in the field of bioethics.
Reduces risk of genetic diseases Only the rich can afford it
Reduces risk of inherited medical
Could create a gap in society
Keep pace with others doing it Loss of Individuality
Better chance the child will succeed
Baby has no choice in the matter
Increased life span
Can give a child genes that the
parents do not carry
Genes often have more than one
Prevent next generation of family
Other children in family could be
affected by parent's decision
If we could eliminate the pig proteins that humans don’t have
introduce necessary human proteins in the pigs via genetic
engineering, then the chances of rejection could be minimized.
The creation of such genetically modified pigs could solve the
problem of organ availability. In 2002, scientists reported the
generation of a cloned, genetically modified pig lacking a sugar
molecule that normally stimulates a strong immune response.
Since then, pigs containing many human genes that can help the
pig immune cells interact successfully with human immune cells
have been generated and their organs have been tested in pig-
non-human primate transplant models.
The use of these modified, or transgenic, pigs as organ
can help prevent the recipient’s immune system from
immediately rejecting the xenograft. The transplantation of
hearts and the kidneys from these transgenic pigs has
greatly improved the survival of xenografts in non-human
primates. Today, a pig kidney may last for months, and a
can survive for multiple years. However, the primate
are still dependent on immunosuppressive medications to
control the immune rejection.
Another exciting strategy in xenotransplantation is the
tolerance approach, which tricks the recipient’s immune
system into recognizing pig molecules as self by
administering pig bone marrow cells to the recipient prior
to organ transplantation. Donor bone marrow contains
progenitor immune cells that can subsequently develop
into mature immune cells in the recipient’s body. These
donor immune cells in the recipient will not attack the
transplanted organ, because they recognize it as self. This
method was first used successfully in human-to-human
transplantation clinical .
Patients who received donor bone marrow with a kidney
transplant became tolerant, meaning that no
immunosuppression drugs were required for the
transplanted kidney to function even one year after the
kidney transplant. Scientists are currently studying
tolerance in a well-established pig to non-human primate