Genetic Engineering 1

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Genetic Engineering Changing The Living World!! By PRANAV BHASKAR M.Sc SEM-I DAVV, INDORE

What Is Genetic Engineering? : 

What Is Genetic Engineering? Directly altering/ manipulating structure and characteristic of the gene so as to manipulate biological processes and/ or organisms for the benefit of humankind Also known as recombinant DNA technology or gene manipulation or gene cloning, means altering the genes in a living organism to produce a Genetically Modified Organism (GMO) with a new genotype Various kinds of genetic modification are possible: inserting a foreign gene from one species into another, forming a transgenic organism; altering an existing gene so that its product is changed; or changing gene expression so that it is translated more often or not at all

BASIC STEPS OF GENE MANIPULATION : 

BASIC STEPS OF GENE MANIPULATION A fragment of DNA, containing the gene to be cloned, is inserted into a circular DNA molecule called a vector, to produce a recombinant DNA or (rDNA) rDNA contains transformation cassette

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Transformation Cassettes Contains

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2. The vector transports the gene into a host cell, which is usually a bacterium 3. Within the host cell the vector multiplies, producing the numerous identical copies not only of itself but also of the gene it carries 4. When the host cell divides, copies of the rDNA are passed to the progeny 5. After several cell divisions, a colony identical host cells (clone) is produced. Each cell in the clone contains one or more copy of rDNA BASIC STEPS OF GENE MANIPULATION

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The process of genetic engineering

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The outcomes of genetic engineering

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TOOLS OF GENETIC ENGINEERING

1. Resctriction enzymes : 

1. Resctriction enzymes DNA cutting enzymes (molecular scissors) Restriction endonucleases cuts DNA at a specific site defined by a sequence of bases in the DNA (recog.site) forming “sticky ends” Palindromic sites Eg. BamHI cuts 5' GGATCC 3'3' CCTAGG5’ Eg.2 HaeIII cuts 5'GGCC3'3'CCGG5' several hundred endonucleases have been extracted from bacteria and many are used in recombinant DNA research. eg EcoR1,Hind III, HaeIII, TaqA1, Sau3A

2. VECTORS : 

2. VECTORS Carrier of DNA (can transfers the rDNA into the host cell) Within host cells, vectors can replicate producing many identical copies of the DNA segment (CLONES) Host cells pass on the recombinant DNA mol to their progeny Cloned DNA segments recovered from host cells for purification and analysis should be small ( <10kb), should have an origin of replication and could replicate in the host cell It may be plasmid, viral genome or yeast chromosome

Plasmids : 

Plasmids Molecules of DNA that are found in bacteria Act as a system to transfer genetic material to other bacteria, allowing those to express the transmitted genes. small (a few thousand base pairs) & circular usually carry only one or a few genes have a single origin of replication can survive in normally toxic concentrations of antibiotics Eg. pBR322, Ti plasmid, pUC19, BAC, etc.

Ti Plasmid : 

Ti Plasmid Ti plasmid is the part of genome of Agrobacterium tumefaciens (causes Crown Gall disease in Dicot plants) 206,479 bases long Has 196 genes, coding 195 proteins Are natural gene vector It carries the cry1Ac gene from the bacteria Bacillus thuringiensis to the plant cell

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p standing for plasmid, B & R for Bolivar and Rodriguez plasmid ds-DNA 4361 BP contains a replicon region, the ampR gene and the tetR gene has single cleavage site for Pst I, EcoR I, Hind III, Sal I and Cla I high copy number (20 – 30) can insert dna fragment of size 1-5kb

Bacterial Artificial Chromosome (BAC) : 

Bacterial Artificial Chromosome (BAC) Developed by Mel Simon Maintained in E. coli as large single copy plasmids Contain inserts of < 300 kb Contain F-plasmid origin of replication F-plasmid gene controls plasmid replication and plasmid copy number

2. VIRAL DNA : 

2. VIRAL DNA gene(s) of interest are incorporated into the viral genome the cloned gene can be introduced into cells at higher frequency than by simple transformation as viruses infect cells with high efficiency some viral vectors are specialized for producing high levels of proteins of interest Eg. Bacterial M13 based vectors (insert of 1-4kb) λ (lambda) phage based vectors (insert of 5-25kb)

YEAST ARTIFICIAL CHROMOSOME (YAC) : 

YEAST ARTIFICIAL CHROMOSOME (YAC) Used for cloning large DNA (200-2000kb) Contains centromeres Contains telomeres protect the ends of the chromosome from nuclease attack Contains ARS act as origin of replication. Bacterial ColE1 ori and a selectable marker (Ampicillin resistant) Contains Selectable Markers TRP1 for tryptophan biosynthesis URA3 for uracil biosynthesis

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METHODS OF INSERTING “GENE OF INTEREST” INTO THE HOST CELL

DNA can be inserted into cell by: : 

DNA can be inserted into cell by: Transformation – Naturally competent cells – Treat cells (E.coli, yeast, mammal cells) to make competent; soak E.coli in CaCl, mix with DNA, mild heat shock

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DNA can be inserted into cell by: • Electroporation – Cells with cell wall need to be converted to protoplasts

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DNA can be inserted into cell by: Gene gun – DNA is coated on tiny gold beads and propelled into the cells

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DNA can be inserted into cells by: Microinjection – Glass pipette punctures the cell membrane & inserts the DNA

Applications of Genetic Engineering : 

Applications of Genetic Engineering

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To repair a genetic defect. To enhance a natural effect e.g. growth. To increase crop resistance to disease or climate. To test and screen for genetically inherited diseases. To cure disease by altering the genes. To select human genes – embryo selection (designer babies)

Transgenic organisms : 

Transgenic organisms Organisms that contain genetic information from other species Q: How does one do this? A: Take a gene from one organism and place it in another This idea has sparked the new booming industry of biotechnology

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Genetic Engineering & The Production Of Transgenic Organism GMO

Fun With Fireflies : 

Fun With Fireflies There is an enzyme that makes fireflies glow Luciferase Could we take a gene out of an animal and put it in something else? Could we get things that don’t glow, to glow

Glowing Tobacco Plant : 

Glowing Tobacco Plant Put luciferase gene in a tobacco plant and you can get a glowing tobacco plant

Glow - Fish : 

Glow - Fish

Glow - Mice : 

Glow - Mice

Transgenic Microorganisms : 

Transgenic Microorganisms Before: Diabetics had to use insulin from cadavers Now: We make bacteria that produce human proteins such as insulin, growth hormone, clotting factor Future: Bacteria may produce substances to fight cancer, make raw materials for plastic and fibers

Recombinant Bacteria : 

Recombinant Bacteria Remove bacterial DNA (plasmid). Cut the Bacterial DNA with “restriction enzymes”. Cut the DNA from another organism with “restriction enzymes”. Combine the cut pieces of DNA together with another enzyme and insert them into bacteria. Reproduce the recombinant bacteria. The foreign genes will be expressed in the bacteria.

Benefits of Recombinant Bacteria : 

Benefits of Recombinant Bacteria Bacteria can make human insulin or human growth hormone. Bacteria can be engineered to “eat” oil spills.

Transgenic Animals : 

Transgenic Animals There are currently no transgenic animals that are approved for human consumption. But there are a lot of experimental studies being done

Genetically modified organisms are called transgenic organisms. : 

Genetically modified organisms are called transgenic organisms. TRANSGENIC ANIMALS Mice – used to study human immune system Chickens – more resistant to infections Cows – increase milk supply and leaner meat 4. Goats, sheep and pigs – produce human proteins in their milk

Human DNA in a Goat Cell This goat contains a human gene that codes for a blood clotting agent. The blood clotting agent can be harvested in the goat’s milk. . Transgenic Goat

Spider Web Goats? : 

Spider Web Goats? Take the gene for making spider web silk

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Put it in a goat Then milk it Extract the spider web silk in large quantities… And we could have…

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The best bullet proof vest ever! Strongest steel cables And much more…

Transgenic Plants : 

Transgenic Plants Already here, already controversial Here are some facts and myths There are only six categories of trangenic plants

Soy Beans : 

Soy Beans 50 percent of soy beans today are genetically modified

Corn : 

Corn 25 percent of corn is genetically modified

Bt - COTTON : 

Bt - COTTON Bt cotton – pest and insect resistant cotton Containing cry1Ac gene form Bacillus thuringiensis

Flavr - savr : 

Flavr - savr First genetically modified fruit With increased shelf life (upto 4-7 weeks)

Golden Rice : 

Golden Rice Rice genetically modified to have added vitamins Not marketed to the public yet Suggested by Dr. Ingo Potrykus

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The Golden Rice Story Vitamin A deficiency is a major health problem Causes blindness Influences severity of diarrhea, measles >100 million children suffer from the problem For many countries, the infrastructure doesn’t exist to deliver vitamin pills Improved vitamin A content in widely consumed crops an attractive alternative

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-Carotene Pathway Problem in Plants

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The Golden Rice Solution Daffodil gene Single bacterial gene; performs both functions Daffodil gene -Carotene Pathway Genes Added

Insect Resistant : 

Insect Resistant The bulk of both soy beans and corn that is genetically modified is modified to have a natural insecticide

Herbicide resistant : 

Herbicide resistant Others resist weed killing chemicals

Transgenic Plant Myths : 

Transgenic Plant Myths The fish tomato and the fish berry Genetically modify plants to have a fish gene that makes them able to live in colder temperatures Experimentally tested, but never worked

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ANIMAL CLONING

Clone : 

Clone A member of a population of genetically identical cells produced by a single cell

How to get a clone in 4 easy steps : 

How to get a clone in 4 easy steps 1. Remove the nucleus of an egg

How to get a clone in 4 easy steps : 

How to get a clone in 4 easy steps 2. Fuse egg with a cell taken from another organism 3. Place in the uterus of a foster mother 4. Foster mother gives birth to cloned baby

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Donor Nucleus These two cells are fused using an electric shock. Fused Cell The fused cell begins dividing normally. Embryo The embryo is placed in the uterus of a foster mother. The embryo develops normally into a lamb—Dolly Egg Cell An egg cell is taken from an adult female sheep. The nucleus of the egg cell is removed.

Dolly : 

Dolly First time this was done in a mammal was in 1997 A gigantic scientific breakthrough at the time

We have also cloned : 

We have also cloned Cows Pigs Mice Cats Dogs Horse Monkey

The cutest clone : 

The cutest clone First cloned cat “CC”

Cloned Mice : 

Nucleus Donor Egg Donor Surrogate Mother Cloned babies Cloned Mice

Female gives birth to her own dam twin! : 

Female gives birth to her own dam twin! Dam = female horse

The worlds first cloned dog : 

The worlds first cloned dog

The latest clones : 

The latest clones Will be used to study stem cells And animal to human transplants

Breaking News in the Cloning World! : 

Breaking News in the Cloning World! Scientists just announced today that for the first time, they have successfully cloned a rhesus monkey embryo

Somatic Cell Therapy : 

Somatic Cell Therapy This is when a gene is introduced into a patient to help them recover from a disease. It could be used to help those suffering from cystic fibrosis. Only the patient is affected and so there are few ethical concerns.

Germ Line Therapy : 

Germ Line Therapy Changes are made to genes that will affect subsequent generations. We do not know the consequences of this. It will affect what it means to be human. We take charge of our own evolution.

Hope through Gene Therapy : 

Chapter 13 74 Hope through Gene Therapy

Pros and Cons of Cloning : 

Pros and Cons of Cloning Pros Saving endangered species Transgenic animals for human consumption Organ and tissue transplants Cons Cloned animals have genetic defects Health problems

Should we reproductively clone humans? : 

Should we reproductively clone humans? No! It took 188 tries on Dolly Success rate of .4 on the horse Success rate of 1.6 on the dog How many humans would die before this worked?

Ethics Involving Genetic Engineering : 

Ethics Involving Genetic Engineering Ethical issues concerning human beings and animals - Are we playing God? - Religions beliefs - Creates new viruses - Designer Babies Genetic Engineering and Plants - Effects on our Environment - Long run effects on environment

For further studies you can refer to: : 

For further studies you can refer to: Gene Cloning and DNA Analysis: An Introduction, 5th edition – T. A. Brown Genes & Genomes: A Changing Perspective – Maxine Singer & Paul Berg

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Especially to Dr. jyoti Bhojwani Ma’m for her encouragement, guidance and support. And my colleagues for being so gentle and patient audience.

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