GENETIC ENGINEERING

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BY SANA RUBABCMS No. 11286

CONTENTS1) INTRODUCTION2) BASICS OF GENETIC ENGINEERING3) HISTORY OF GENETIC ENGINEERING4) Methods of preparation5) Applications6) Future perspective7) Upgradation QC tests

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3CONTENTS

2) BASICS OF GENETIC ENGINEERING

3) HISTORY

1) INTRODUCTION

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INTRODUCTIONGenetic engineering is a part of biotechnology.

BiotechnologyApplication and industrial use of organisms for human welfare is called biotechnology.

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INTRODUCTION continuation..

Biotechnology is a huge topic. Its hard to define its exact boundaries.

Some European scientists divide the field into :

1) Red biotechnology2) Green biotechnology3) Blue4) White Biotechnology .

5Book : Biotechnology & Genetic engineering (Kathy wilson peacock) 2010,Edi:1 : Page No. 4 (Chapter 1)

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05/02/2023 6Book : Biotechnology & Genetic engineering

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INTRODUCTION continuation..

• 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 or organism.

• Patterns of inheritance from parent to offspring, and gene distribution, variation and change in populations.

7Book : Genetics and the Organism: Introduction

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8U S National Library of Medicine

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CONTENTS1) INTRODUCTION

2) BASICS OF GENETIC ENGINEERING

3) HISTORY OF GENETIC ENGINEERING

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BASICS OF GENETIC ENGINEERING

• Different terms used for genetic engineering :

1)Gene manipulation2)Gene cloning3)Recombinant DNA

technology4)Genetic modification5)New genetics

10An Introduction to Genetic Engineering (Desmond S. T. Nicholl) Edi :3rd 2008Chapter 2 . Page 3

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BASICS OF GENETIC ENGINEERING CONTINUATION..

Direct manipulation of an organism's 

genome using biotechnology .

First isolating and copying the

genetic material of interest

using molecular cloning methods

Generate a DNA sequence

New DNA  inserted in the

host genome

An Introduction to Genetic Engineering (Desmond S. T. Nicholl) Edi :3rd 2008 Chapter 2.

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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.

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13CONTENTS1) INTRODUCTION2) BASICS OF GENETIC

ENGINEERING3)HISTORY OF GENETIC

ENGINEERING

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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.

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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.

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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 organism's genome.

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.

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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 1984,

Cystic fibrosis gene cloned and sequenced

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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 DNA  molecules. 

Trials for gene therapy begin in 90s.

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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

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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

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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 activator.

With the discovery of microRNA in 1993

came the possibility of using RNA interference to silence an organisms endogenous genes

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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.

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• His work cloned

frogs laid the

foundations for

somatic cell

nuclear

transfer, the

application of

which led to

Dolly the

sheep.

John Gurdon

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“The Father of Cloning”

Hans Spermann

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“The Father of Genetics”

Gregor Mendel

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In 1973 created a transgenic mouse by introducing foreign DNA into its embryo, making it the world’s first transgenic animal.Rudolf Jaenisch 

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Human Genome Project

• 1990-2003 The human genome worked out

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 databases

• improve tools for data analysis• transfer related technologies to the private sector.

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Methods of preparation of Genetic Engineering

Presented By Kashaf Ur Rehman

Cms # 8882

Basic steps in genetic engineering

• 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

Methods

Plasmid method Vector method Biolistic method

Plasmid Method

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.

Vector method

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.

Biolistic Method

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 cells.

Insulin

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 levels.

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.

Steps

1. A small piece of circular DNA called a plasmid is extracted from the bacteria or yeast cell.

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.

General Applications

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.

rDNA products

• Interleukin-2For treatment of cancer• Factor VIIINeeded by hemophiliacs for blood clotting• ErythropoietinFor treatment of anemia• Tumor necrosis factorFor treatment of tumors• Tissue plasminogen activatorUse to dissolve blood clots

Gene Therapy

A normal gene inserted to compensate for the defective gene. Abnormal gene replaced with a normal one Abnormal gene repaired through selective reverse mutation.

References

• http://mrlloyder.weebly.com/• http://www.dailymail.co.uk/sciencetech/article 1205442/Theres-fishy-pictures-

Either-genetic-engineerings-got-way-hand-someones-Photoshop-.html#ixzz0wPbrrIrJ

• genetics.thetech.org/about-genetics/making-medicines • www.fda.gov › ... › Genetic Engineering • https://www.ncbi.nlm.nih.gov/book s/NBK215771/

Hybridoma

Hareem Zahra10628

1. Upgradation in genetic engineering

2. Qc test3. Future

Prospective

1. Upgradation in genetic engineering

Bubble boy diseaseADA 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 infections.

• 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 this 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 system 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 their 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 many angles.

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 content 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 radiation.

6. Bacterial endotoxins should be less than 80 IU per 100 IU of insulin.

Quality Control parameter for Injectable Insulin preparation 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

Welcome to the Future

Gene ChipsGene 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 genes.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 drugBy 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 DEET. 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 changes.

Genetic Engineering Alters Mosquitoes’ Sense of Smell

Designer BabiesThe 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 or characteristics. 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 womb.

• Three-parent babyThree-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.

Pros 

Cons

Reduces risk of genetic diseases

Only the rich can afford it

Reduces risk of inherited medical conditions

Could create a gap in society

Keep pace with others doing it Loss of Individuality

Better chance the child will succeed in life

Baby has no choice in the matter

Increased life spanCan give a child genes that the parents do not carry

Genes often have more than one use

Prevent next generation of family from getting characteristics/diseases 

Other children in family could be affected by parent's decision

Transgenic PigsIf we could eliminate the pig proteins that humans don’t have and 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-to-non-human primate transplant models. The use of these modified, or transgenic, pigs as organ

donors 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 already greatly improved the survival of xenografts in non-human primates. Today, a pig kidney may last for months, and a heart can survive for multiple years. However, the primate recipients are still dependent on immunosuppressive medications to control the immune rejection.

Xenograft

Xenograft 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 xenotransplantation model.

• Komal Raja• 11967• Article presentation