Applications and Ethics of Engineering - Lecture Slides | SOCR 330, Study notes of Genetics

Download Applications and Ethics of Engineering - Lecture Slides | SOCR 330 and more Study notes Genetics in PDF only on Docsity! 4/24/2018 1 Genetic Engineered Organisms Genetic Engineering in Agriculture Genetic Testing and Medicine Medical Therapy Ethical Issues in Genetic Engineering 4/24/2018 2 Techniques used to create, replicate, and analyze recombinant DNA molecules build on discoveries made in molecular biology, biochemistry and bacterial genetics in the mid to late 1970’s. The commercial application of recombinant DNA technology began in 1982 when the Food and Drug Administration approved the sale of human insulin produced in bacterial cells. Today, recombinant DNA technology is used to produce medicine, vaccines, industrial chemicals, genetically modified food products and organisms. Golden Rice Deficiency in dietary Vitamin A is estimated to kill 670,000 children under 5 each year 4/24/2018 5 Transgenic Insulin Production: To synthesize recombinant human insulin, synthetic oligonucleotides encoding the insulin A and B chains were inserted at the tail end of a cloned E. coli lacZ gene. The recombinant plasmids were transformed into E. coli hosts, where the β-gal/insulin fusion protein was synthesized and accumulated in the host cells. Fusion proteins were then extracted from the host cells and purified. Insulin chains were released from β-galactosidase by treatment with cyanogen bromide. The insulin subunits were purified and mixed to produce a functional insulin molecule. 4/24/2018 Other Pharmaceuticals Produced by Genetically Engineered Organisms ‘L255 5) Examples of Genetically Engineered Biopharmaceutical Products Available or under Development Gene Product Erythropoitin Interferons Tissue plasminogen activator tPA Human growth hormone Monoclonal antibodies against vascular endothelial growth factor (VEGF) Human clotting factor Vill C1 inhibitor Recombinant human antithrombin Hepatitis B surface protein vaccine Immunoglobulin IgG1 to HSV-2 Recombinant monoclonal antibodies Norwalk virus capsid protein E.coli heat-labile enterotoxin (©2013 Parson Esato, Condition Treated Anemia Multiple sclerosis, cancer Heart attack, stroke Dwarfism Cancers Hemophilia A Hereditary angioedema Hereditary antithrombin deficiency Hepatitis B infections Herpesvirus infections Passive immunization against rabies (also used in diagnosing rabies), cancer, rheumatoid arthritis Norwalk virus infections E.coli infections Host Type E.coli; cultured mammalian cells E.coli; cultured mammalian cells Cultured mammalian cells Cultured mammalian cells Cultured mammalian cells Transgenic sheep, pigs Transgenic rabbits Transgenic goats Cultured yeast cells, bananas Transgenic soybeans Transgenic tobacco, soybeans, cultured mammalian cells Potato (edible vaccine) Potato (edible vaccine) 4/24/2018 7 Edible Vaccines • Inactivated vaccines- prepared from killed virus. • Attenuated vaccines- prepared from live virus without ability to reproduce. • Subunit vaccine-prepared from the surface protein of the virus, allows the host to produce antibodies prior to infection. 4/24/2018 10 Transgenic Animals Animals can be used as Bioreactors to produce pharmaceutical products. • Use of sheep to produce an enzyme α 1-antitrysin to treat a heritable form of emphysema. • Gene cloned into a vector adjacent to a promoter that is only active in milk producing cells. • Transformed vector and microinjected into sheep zygote. • In 2006, recombinant human antithrombin, an anticlotting protein, became the world’s first drug extracted from the milk of farm animals for use in humans. 4/24/2018 11 Traditional Plant Breeding Relied on Selective Breeding Corn was changed by selective breeding over many thousands of years from the wild ancestor Teosinte (Z. canina). 4/24/2018 12 Modern Plant Breeding Incorporates Transgenic Methods to Incorporate Novel Genes into Plants 1. Most transgenic dicot plants are transformed using a bacteria Agrobacterium tumefaciens as the transforming vector. (updated scientific name Rhizobium radiobacter, synonym Agrobacterium radiobacter). Other methods not covered in the text include: 2. The Particle gun that shoots DNA coated pellets into the nuclei of plants. 3. Electroporation- by applying an electric field to pollen, they can take up DNA particles and sometimes insert them into their genome at random. 4/24/2018 EPSP fusion gene into plant chromosome, resulting in high levels of EPSP synthase, making cell resistant to glyphosate Nucleus Cell from plant leaf Calluses with EPSP fusion gene grown in medium containing 7 Agrobacterium transfers plasmid carrying | ei Figure 19-1 part 2 Essentials of Genetics, 6/e (© 2007 Pearson Prentice Hall, Inc. 15 4/24/2018 Calluses with EPSP fusion gene grown in medium containing glyphosate Plants regenerated from calluses are glyphosate- resistant Figure 19-1 part3 Essentials of Genetics, 6/e (©2007 Pearson Prentice Hall, Inc. 16 4/24/2018 Figure 19-5 (a) (b) 17 4/24/2018 20 In 2005, the variety, Golden Rice 2, which produces up to 23 times more beta-carotene than the original golden rice Golden rice was expected to clear final regulatory hurdles and be available to farmers in the Philipines in 2014 or 2015. http://www.goldenrice.org/PDFs/GR_Project.pdf 4/24/2018 21 Where are we today? On November 7, 2013, Pope Francis gave his personal blessing to Golden Rice (GR). Why is this significant? Vitamin A deficiency (VAD) is responsible for 500,000 cases of irreversible blindness and up to 2 million deaths each year. ASPB News The creators of Golden Rice, Profs Peter Beyer and Ingo Portrykus, and the crop protection company Syngenta, who have worked together to make the latest, improved version of Golden Rice available for humanitarian use in developing countries, free of charge Currently, Golden Rice is still under development and evaluation http://www.goldenrice.org/Content4-Info/info.php 4/24/2018 Farmers of the Philippines Say ‘No Way’ to GMO Rice “There are other fixes for vitamin A deficiency 22 4/24/2018 25 Transgenic Atlantic Salmon Over- Expressing a Growth Hormone Normal Transgenic 4/24/2018 26 Transgenic cows that express the lysostaphin gene in milk, which kills the Staphlococus bacteria that cause mastitis. Other examples: EnviroPig GloFish 4/24/2018 27 Cloned Animals Dolly and her surrogate mother Dolly was cloned by taking a fertilized egg, extracting the nucleus, then inserting a nucleus from one of her mothers mammary gland cells. 4/24/2018 30 For amniocentesis, the position of the fetus is first determined by ultrasound, and a needle is then inserted through the abdominal and uterine walls to recover fluid and fetal cells for genetic or biochemical analysis. 4/24/2018 31 A Southern blot diagnosis of sickle-cell anemia, with arrows representing the location of restriction enzyme recognition sites. In the mutant β-globin allele (βs) a point mutation (GAG-- >GTG) has destroyed a restriction enzyme cutting site, resulting in a single large fragment on a Southern blot. In the pedigree, the family has one unaffected homozygous normal daughter (II- 1), an affected son (II-2), and an unaffected carrier fetus (II-3). 4/24/2018 32 Genetic Testing: Allele Specific Oligonucleotides (ASO) ASOs = Alleles that differ by a single nucleotide can be distinguished by synthetic probes known as ASOs. One good example is the single nucleotide change that alters amino acid composition for hemoglobin in humans and causes sickle cell anemia. 4/24/2018 35 In making a DNA microarray, short, single-stranded DNA molecules of known sequence are attached to a glass substrate. Each cluster of identical molecules occupies an area known as a field on the microarray. Each field is about half the width of a human hair. GeneChip® Probe Array Hybridized Probe Cell i t t t t i t. Single stranded, fluorescently SCQY‘D labeled DNA target \ Sy > iy Oligonucleotide probe Each field contains millions of copies of a specific oligonucleotide probe Over 50,000 different probes complementary to genetic information of interest Image of Hybridized Probe Array Chapter 19 Opener Essentials of Genetics, 6/e © 2007 Pearson Prentice Hall, Inc. 4/24/2018 36 4/24/2018 37 The color of dots on an expression DNA microarray represent levels of gene expression. (a) Green dots represent genes expressed only in one cell type (e.g., normal cells), and red dots represent genes expressed only in another cell type (e.g., cancer cells). Intermediate colors represent different levels of expression of the same gene in the two cell types. (b) A small portion of a DNA microarray, showing different levels of hybridization to each field. 4/24/2018 40 Some Vectors Used for Gene Therapy  Retroviruses  Adenoviruses  Adeno-associated viruses  Herpes simplex viruses 4/24/2018 41 Ashanti De Silva, the first person to be treated by gene therapy in 1990. She had “Severe Combined Immunodeficiency (SCID) disease. 4/24/2018 42 To treat SCID using gene therapy, a cloned human ADA gene is transferred into a viral vector, which is then used to infect white blood cells removed from the patient. The transferred ADA gene is incorporated into a chromosome and becomes active. After growth to enhance their numbers, the cells are reimplanted into the patient, where they produce ADA, allowing the development of an immune response.