Genetic Engineering Objectives: 1. Define genetic engineering; 2. Outline the processes involved in genetic engineering 3. Discuss the applications of recombinant DNA Genetic Engineering Direct alteration and modification of an organism's genetic composition by artificial means and/or biotechnology. In genetic engineering, recombination can also refer to artificial and deliberate recombination of pieces of DNA, from different organisms, creating what is called recombinant DNA. 2 Genetic Engineering Genetic engineering involves the use of molecular techniques to modify the traits of a target organism. The modification of traits may involve: I. introduction of new traits into an organism II. enhancement of a present trait by increasing the expression of the desired gene III. enhancement of a present trait by disrupting the inhibition of the desired genes’ expression. 3 Genetic Engineering Technique A general outline of recombinant DNA may be given as follows: 1. Identify an organism that contains a desirable gene. 2. Extract the entire DNA from that organism. 3. Cut or cleavage of DNA by restriction enzymes (REs) selection of an appropriate vector or vehicle which would propagate the recombinant DNA ( eg. circular plasmid in bacteria with a foreign gene of interest) 5 5. Ligation (join together) of the gene of interest (eg. from animal) with the vector ( cut bacterial plasmid) 6. Transfer of the recombinant plasmid into a host cell (that would carry out replication to make huge copies of the recombined plasmid) 7. Selection process to screen which cells actually contain the gene of interest 8. Sequencing of the gene to find out the primary structure of the 6 ✗ ✗ ✗ ✗ ✗ When a gene from one organism is transferred to different organism, it is called transformation: The organisms that have DNA transferred to them are called transgenic organisms. trans: means different, genic: refers to genes Genetic engineering has given rise to a new technological field called biotechnology (technology of life). Processes Used in Recombinant DNA Technology There are three methods by recombinant DNA is made namely: ✗ Transformation ✗ Vectorless gene transfer ✗ Transduction. 8 Transformation Using a Vector ✗ Vectors are organisms that are normally harmless but may help spread infection by transferring the genetic material from one host to another. ✗ A vector is an entity that transmits a disease or parasite from one animal or plant to another. 9 Types of Vector ✗ Plasmid is small circular DNA molecules that replicate separately from the bacterial chromosome ✗ Viruses certain viruses are often used as vectors because they can deliver the new gene by infecting the cell. The viruses are modified so they can't cause disease when used in people. 10 Transduction ✗ is the process of gene transfer where viruses act as the vector, or gene transportation vehicle. For this process a special type of virus is required, which infects bacterial cells. These viruses are known as “bacteriophages.” 11 Vectorless gene transfer ✗ This process is similar to transformation, but it does not involve vectors. The types of vectorless gene transfer include electroporation, protoplast fusion, microinjection, and use of a particle gun. 12 electroporation ✗ The application of an electric current to a living surface (as the skin or a cell membrane) in order to open pores or channels through which something (as a drug or DNA) may pass. ✗ In molecular biology, the electroporation process is commonly used for cell transfection/transformation, the non-viral DNA transfer, of bacteria, yeast, and plant protoplasts. Electroporation is also highly effective for the introduction of foreign genes in tissue culture cells, especially mammalian cells. 13 14 protoplast fusion ✗ Protoplast fusion is a physical phenomenon. During fusion, two or more protoplasts come in contact and adhere with one another either spontaneously or in presence of fusion inducing chemicals. After adhesion, membranes of protoplasts fuse in some localized areas and, eventually, the cytoplasm of the two protoplasts intermingle. 15 16 microinjection ✗ Microinjection is a direct method to introduce DNA into either cytoplasm or nucleus. It is a microsurgical procedure conducted on a single cell, using a glass needle (i.e., a fine, glass microcapillary pipette), a precision positioning device (a micromanipulator) to control the movement of the micropipette, and a microinjector. 17 18 particle gun ✗ Biolistic gun or gene gun method initially developed to transfer the gene in plant cell directly. It is a method involving the transfer of genes to the plants without the use of any vectors 19 20 Role of Genetic Engineering to Human Life 21 Insulin The pancreas, among other functions, produces a crucial hormone called insulin. ✗ This peptide hormone (protein) ensures that glucose is taken up by the cells for cellular respiration. ✗ If the pancreas is defective then the blood sugar levels get dangerously high causing many physiological effects (Diabetes mellitus). ✗ Using very similar technique as HGH production previously mentioned, scientists were able to use E. coli to bioengineer synthetic insulin in 1977. ✗ Other transgenic organisms used to produce insulin today are yeast (Saccharomyces cerevisiae) and a plant called safflower (Carthamus tinctorius). 22 23 Golden Rice The World Heath Organization estimates that between 1 and 2 million children die each year from vitamin A deficiency. ✗ Golden rice is a genetically modified food that is fortified with beta carotene, which the human body converts into vitamin A. ✗ This transgenic organism is the result of mixing genes from a bacterium and from daffodils into the rice genome. ✗ It is not currently used due to regulatory issues. 24 25 1. Transgenic (GMO) animals: genes inserted into animals so they produce what humans need. ✗ Why?: A way to improve the food supply: A. Transgenic cows: gene inserted to increase milk production. B. Spider goat: gene from spider inserted into goat. ✗ Goats makes silk of the spider web in their milk. ✗ Flexible, stronger than steel. Used in bullet proof jackets. C. Glow-in-the-dark cats ✗ Scientist used a virus to insert DNA from jellyfish ✗ The gene made the cat produce a fluorescent protein in its fur. ✗ Cat owners might find a glow-in-the-dark kitty to be fairly useful—you’ll never trip over the cat at night again—but the Mayo Clinic scientists who created this glowing cat had a bigger goal in mind: fighting AIDS. 2. Transgenic bacteria: gene inserted into bacteria so they produce things humans need. ✗ For example: insulin and clotting factors in blood are now made by bacteria. 3. Transgenic plants: plants are given genes so they meet human needs. ✗ BT Corn -was developed to incorporate the production of a toxin (i.e. ✗ Btendotoxin) from Bacillus thuringensis in corn plants that results in the death of pests that feed on these plants like the corn borer larvae but is non-toxic to humans, mammals, fish and birds. Flavr-Savr tomato -The trait modified in this tomato is its ripening process. A gene for an enzyme that causes the degradation of pectin in the cell walls (i.e. polygalacturonase) normally softens the fruit as it ripens. In Flavr Savr tomatoes, an inhibitor (i.e. antisense RNA) disrupts the expression of this gene, thereby delaying the softening of the fruit and extending the time it may be kept in storage and transported to markets. B. Venomous cabbage ✗ A cabbage genetically modified to include the “poison gene” from scorpion tails is currently in development. While it sounds more than mildly terrifying, the scientists in Beijing who developed this say the poison the cabbage produces isn’t harmful to humans yet will kill caterpillars when they nibble the leaves, therefore requiring fewer pesticides. C. Banana Vaccine ✗ Biotechnologists at the Boyce Thompson Institute for Plant Research at Cornell University in New York State, are genetically engineering a banana to produce an antigen found in the outer coat of the hepatitis B virus. Banana vaccines would be ideal for developing countries because they would cost just a few cents per dose, compared to the $100 to $200 per dose for traditional vaccines ✗ As the plant grows, it produces the virus proteins — but not the disease part of the virus. ✗ When people eat a bite, their immune systems creates antibodies to fight the disease — just like a traditional vaccine ✗ A virus is often used to deliver DNA. ✗ In the movie “I Am Legend,” A healthy gene was inserted into a virus. ✗ The virus invaded the cancer cells and inserts the healthy gene to cure cancer. ✗ Worked at first but the virus mutated and became deadly. ✗ This is being attempted in real life. CONTROVERSY Some questions being asked: 1. Have they been tested enough to prove they are safe? 1. Is there any side effect once consumed? 2. Will it shorten our lifespan? 3. Will it alter our DNA? 34 Entire organisms can be cloned. ✗ A clone is a genetically identical copy of a gene or of an organism. ✗ Cloning occurs in nature. ✗ bacteria (binary fission) ✗ some plants (from roots) ✗ some simple animals (budding, regeneration) • Mammals can be cloned through a process called nuclear transfer. – nucleus is removed from an egg cell – nucleus of a cell from the animal to be cloned is implanted in the egg Somatic Cell Nuclear Transfer (SCNT) CONTROVERSY Some questions being asked: 1. Can we clone a human? 2. Does cloning violate human rights? 3. Can clones become selfsufficient? 4. Can a clone possess unknown personality different from the original? 38 Gene therapy is the replacement of faulty genes. ✗ Gene therapy replaces defective or missing genes, or adds new genes, to treat a disease. • Several experimental techniques are used for gene therapy. – genetically engineered viruses used to “infect” a patient’s cells – insert gene to stimulate immune system to attack cancer cells – insert “suicide” genes into cancer cells that activate a drug – Inactivating, or “knocking out,” a mutated gene that is functioning improperly – Introducing a new gene into the body to help fight a disease Fig. 20-22 Cloned gene 1 Insert RNA version of normal allele into retrovirus. Viral RNA 2 Retrovirus capsid Let retrovirus infect bone marrow cells that have been removed from the patient and cultured. 3 Viral DNA carrying the normal allele inserts into chromosome. Bone marrow cell from patient 4 Inject engineered cells into patient. Bone marrow Examples ✗ Gene therapy is being used in many ways. For example, to: ✗ 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; ✗ Deliver genes that stimulate the healing of damaged tissue. Reasons for Gene Therapy ✗ To alter genes to correct genetic defects ✗ prevent or cure genetic diseases. ✗ Not to be confused with Genetic engineering which changes the genes to make the organism better than normal. ✗ Possibility to cure deadly diseases like cancer Parkinsons ✗ Patients with Parkinson's disease gradually lose cells in the brain that produce the signaling molecule dopamine. As the disease advances, patients lose the ability to control their movements. ✗ A small group of patients with advanced Parkinson's disease were treated with a retroviral vector to introduce three genes into cells in a small area of the brain. These genes gave cells that don't normally make dopamine the ability to do so. After treatment, all of the patients in the trial had improved muscle control. Controversies of Gene Therapy ✗ Pros: ✗ Could wipe out genetic diseases ✗ Prevent genetic disorders in future generations for families who know they have it in their bloodline ✗ Cons: ✗ Still a dangerous and difficult process ✗ When used in unborn children, not sure of effects ■ Child doesn’t have a choice Gel electrophoresis: a technique used to compare DNA from two or more organisms. It is one indirect method of rapidly analyzing and comparing genomes. Why compare DNA: 1. Find your baby’s daddy 2. Who committed a crime. 3. How closely species are related. How is electrophoresis done? A. The DNA is cut into fragments with a restriction enzyme. B. The cut DNA is then put into the wells of a machine filled with gel. ✗ The gel is spongy and the DNA squeezes through the pores. Fig. 20-9 TECHNIQUE C. The machine is plugged in and the fragments get separated based on their size. ✗ The smaller fragments move further than the large. Why does DNA move? ✗ DNA has a negative charge. ✗ When the machine is plugged it, its moves towards the positive pole created by the electricity Mixture of DNA molecules of different sizes Power source – Cathode Anode + Gel 1 Power source – + Longer molecules 2 RESULTS Shorter molecules Separation of DNA based on size of fragments. ✗ Your DNA is so unique its considered to be a DNA fingerprint. ✗ Gel electrophoresis will separate your DNA differently from anyone else. Final result of electrophoresis Long duration assignment ✗ Watch the movie Gattaca and: ✗ By Group: Write a term paper or a scientific report on it. ✗ Individual: Answer the set of questions given by the teacher. ✗ Deadline: February 23, 2024 51 Group activity for next meeting ✗ Search online databases for Applications of Genetic Engineering and Recombinant DNA in different field and report it to the class. ✗ ✗ ✗ ✗ Agriculture Forensic Investigation Medicine Industry 52
