GMOs PDF

Expression system selection Choice depends on size, character of protein ❑Large proteins (>100 kD)? Choose eukaryote ❑Small proteins (<30 kD)? Choose prokaryote ❑Glycosylation essential? Choose baculovirus or mammalian cell culture ❑High yields, low cost? Choose E. coli ❑Post-translational modificat...

Expression system selection Choice depends on size, character of protein ❑Large proteins (>100 kD)? Choose eukaryote ❑Small proteins (<30 kD)? Choose prokaryote ❑Glycosylation essential? Choose baculovirus or mammalian cell culture ❑High yields, low cost? Choose E. coli ❑Post-translational modifications essential? Choose yeast, baculovirus or other eukaryote Cell culture • Routine laboratory technique for in vitro analysis of life processes • Involves culturing of cells derived from multicellular eukaryotes May be primary or continuous cell lines ❑ Primary – finite life span in culture ❑ Continuous – abnormal; often transformed or immortal Maintaining cells in culture Cells grown, maintained at an appropriate temperature and gas mixture (typically, 37°C, 5% CO2) in a cell incubator. • Cell growth media used will depend on cell type • General medium requirements 1. Bulk ions - Na, K, Ca, Mg, Cl, P, Bicarb or CO2 2. Trace elements - iron, zinc, selenium 3. sugars - glucose is the most common 4. amino acids - 13 essential 5. vitamins - B, etc. 6. choline, inositol 7. serum - has a large number of growth promoting activities • e.g., buffering toxic nutrients; neutralizes trypsin and other proteases; has undefined effects on the interaction between cells and substrate; contains peptide hormones or hormone-like growth factors that promote healthy growth 8. antibiotics - used to control bacterial and fungal contaminants A typical laminar flow hood. Filtered air enters the work space from the back. A typical incubator for cell culture. • internal temperature controlled by a precise thermostat and a heater • many incubators = CO2 incubators; continually flow CO2 -containing air through the incubator Some of the apparatus used for cell culture. 1. plastic culture flask (75 square cm) 2. a small vial in which cells are frozen 3. 10 ml syringe with an attached filter for sterilizing liquids Note the flask contains culture medium which appears red, owing to the presence of the pH indicator, phenol red. The red color indicates the medium is at the correct pH for cell culture. Cultured cells are visualized using an "inverted" microscope, which allows one to peer through the plastic on the bottom of the culture flask and view the cells that are growing on this surface Primary cells most closely represent tissue of origin • taken directly from tissue • processed to establish them under optimized culture conditions • may be manipulated for indefinite subculture through an in vitro process called transformation Primary cells: advantages https://www.sigmaaldrich.com/PH/en/technicaldocuments/technical-article/cell-culture-and-cellculture-analysis/primary-cell-culture/primary-cell-culture • Avoids ethical objections raised against animal experiments ➢Allows experiments on human tissues which could not have been done in vivo • provides more relevant results than cell lines • cost-effective; helps reduce expenditure on animal models required for in vivo studies Table 1 Common Primary Cell Types Epithelial Endothelial Fibroblasts Keratinocytes Melanocytes Neurons Astrocytes Hepatocytes Skeletal Muscle Smooth Muscle Osteoblasts Myocytes Chondrocytes Adipocytes Synoviocytes Hair Cells Blood Cells Stem Cells Primary cells: disadvantages • take more time to grow than other cell lines ➢ have limited growth potential even under optimal growth conditions ➢ eventually senesce and die • cells taken from different donors behave differently in response to pro-inflammatory cytokines • cost of isolation and culture often high ➢ though cheaper than animal models • characteristics may change w/ each subsequent passage if optimum culture conditions not maintained Primary cell culture applications 3D Cell Culture: Since non-transformed, non-immortalized, closely simulate a living model, yield more physiologically significant results and can be used to model 3D tissues A) H&E B) BrdU and C) Filaggrin staining of a 3D skin model using primary human dermal fibroblasts, keratinocytes and melanocytes Drug screening and toxicity testing: used to study cytotoxicity of new drugs (to study the effect and safe dosage) and/or drug carriers (nanoparticles) Vaccine Production: used in production of viruses used to produce vaccines (e.g., against polio, rabies, chicken pox, measles, hepatitis B Genetic Engineering: for commercially important genetically engineered proteins; e.g., monoclonal antibodies, insulin, hormones Virology: Detection, isolation, growth, and development cycles, modes of infection Tissue or Organ Replacement: reconstruction of damaged tissue or replacement of non-functional cells or tissues Stem Cell Therapy: A patient’s own stem cells or those from a donor are grown in vitro; can generate enough cells to regenerate tissue or replace functionally deficient cells Primary vs continuous cell lines Continuous/transformed cell lines Continuous cell lines have undergone immortalization: ➢involve mutations of genes that deregulate cell cycle or enhance proliferation processes e.g., cancerous tissues (HeLa cells) The “workhorse” in most laboratories e.g., MCF-7 (breast cancer MDA-MB-438 (breast cancer) U87 (glioblastoma) A172 (glioma), HeLa (cervical cancer) HL60 (promyelocytic leukemia) A549 (lung cancer) Table 3 Comparison between Primary and Continuous Cell lines Properties Primary cells Cell lines Biological Relevance High Low Lifespan and Proliferation Limited/Finite Unlimited/Infinite Consistency Medium High Genetic Integrity Retains In Vivo Tissue Genetic Makeup Subject to Genetic Drift Ease-of-Use Needs Optimized Culture Conditions Well Established Conditions and Protocols Time & Expense Slower Cell Growth and Faster Cell Growth and Higher Cost Lower Cost Stem cell lines • Stem cells have ability to selfrenew or to differentiate into various cell types in response to appropriate signals ➢ of special interest in medical research, due to their capabilities for tissue repair, replacement, and regeneration • Differentiation triggered by various factors in vivo, some of which can be replicated in in vitro stem cell cultures Cell culture transfection • introduction of foreign DNA into eukaryotic cells • Typically involves opening transient "holes" or gates in cells to allow the entry of extracellular molecules e.g., supercoiled plasmid DNA, siRNA Transfection may be stable or transient Cell culture transfection methods Goals of Transfection 1. 2. Expression of protein product Analysis of effect of DNA transfected on cell function; Usually involves reporter gene assays e.g., activation assays, repression assays Common reporter genes Examples: 1. green fluorescent protein (GFP) • causes cells that express it to glow green under UV light 2. Luciferase enzyme • catalyzes rxn w/ luciferin to produce light 3. the lacZ gene • Encodes β-galactosidase enzyme • enzyme causes bacteria expressing the gene to appear blue when grown on medium containing substrate analog X-gal or IPTG 4. chloramphenicol acetyltransferase (CAT) gene • used in bacteria; confers resistance to chloramphenicol Genetically modified organisms (GMOs) • Also known as transgenic organisms • an organism whose genetic material has been altered using genetic engineering techniques • DNA molecules from different sources are combined into one molecule (chimera) to create a new set of genes • DNA is then transferred into an organism, giving it modified or novel genes Traditional breeding vs. Genetic engineering Domesticated tomato • low sugar content • bigger size • higher yield Wild tomato • high sugar content • smaller size • lower yield Conventional cross of wild tomato vs. domesticated tomato = X domesticated tomato wild tomato hybrid tomato Analogy is mixing up two stacks of books: • 100 pages out of ~ 1.7 million pages contain wild species • However, included in the 100 pages is a gene not only for sweetness, but also a gene for reduced fertility Genetic modification of domesticated tomato = X domesticated tomato sweetness gene from wild tomato ~1 page GM domesticated tomato • Genetically modified tomato will contain ~ 1 page wild tomato / 1.7 million pages • One page exchanged contains ONLY the gene for sweetness Normally blackand-silver zebrafish was turned green or red by inserting various versions of the GFP gene. Called GloFish ☺ A genetically altered cat can make cells that resist a version of the AIDS virus that affects cats, along with GFP protein Genetically engineered tomatoes containing a softening inhibition gene ANDi: 1st transgenic primate ➢ 24 unfertilized rhesus eggs were infected with a GFP virus ➢ Eggs were implanted into 20 surrogate mothers ➢ 5 males were born, two were stillborn ➢ ANDi = only live monkey carrying the GFP gene Transgenic animals as source of recombinant proteins Animals can be used for commercial, large-scale production of recombinant proteins Can generate protein in low-cost, renewable way; e.g., Transgenic sheep has human gene for alpha-1antitrypsin, which it expresses in its milk. Alpha-1-antitrypsin is used to treat human emphysema Schematic representation of the transgenic production process, using the production of rhAT in the milk of transgenic goats as an example CASE STUDY: Bt-modified ORGANISMS Bt = Bacillus thuringiensis • • • spore-forming bacterium; produces protein crystals that are toxic to insects ONLY Bt – modified organisms produce Bt crystals Insect eats Bt crystals, which then binds to receptors in the gut ➢ insects stops eating ➢ crystals cause gut wall to break down ➢ insect dies as spores and gut bacteria proliferate in the body Agricultural GMOs • Eggplant farmers suffer yield losses due to the Eggplant Fruit and Shoot Borer (FSB) • Female moths deposit eggs mostly on eggplant leaves; hatched larvae then feed on leaf tissues, tunnel inside shoots, fruits Non-Bt eggplant • eggplant farmers spray chemical insecticides up to 80x/growing season • Bt talong toxic to FSB, but safe for humans Photos: UPLB IPB Bt Eggplant Project, 2014 Bt Eggplant How to make GMO plants? http://www.isaaa.org/resources/publications/briefs/49/infographic/pdf/B49-MapInfographic-English.pdf PHILIPPINE GMOs ❑ Import, propagation of Bt corn (December 2002) ❑ Development of “golden rice” ➢ Engineered to produce -carotene, niacin, iron, other essential minerals in the seed 12/16: Philippine Supreme Court orders permanent ban on field trials of GM eggplant, temporary halt to approving applications “contained use, import, commercializ’n and propagation” of GMO crops and products The golden rice story • Vitamin A deficiency a major health problem ❑Causes blindness ❑ Influences severity of diarrhea, measles • >100 million children suffer from the problem Improved vitamin A content in widely consumed crops an attractive alternative -Carotene pathway problem in plants IPP Geranylgeranyl diphosphate Phytoene synthase Phytoene Problem: Rice lacks these enzymes Phytoene desaturase ξ-carotene desaturase Lycopene Normal Vitamin A “Deficient” Rice Lycopene-beta-cyclase  -carotene (vitamin A precursor) The Golden Rice Solution -Carotene Pathway Genes Added IPP Geranylgeranyl diphosphate Daffodil gene Phytoene synthase Phytoene Vitamin A Pathway is complete and functional Single bacterial gene; performs both functions Phytoene desaturase ξ-carotene desaturase Lycopene Daffodil gene Golden Rice Lycopene-beta-cyclase  -carotene (vitamin A precursor) 23 July 2021, Los Baños, PHILIPPINES – Filipino farmers will become the first in the world to be able to cultivate a variety of rice enriched with nutrients to help reduce childhood malnutrition, after receiving the green light from regulators. Golden Rice was developed by the Department of Agriculture-Philippine Rice Research Institute (DA-PhilRice) in partnership with the International Rice Research Institute (IRRI) to contain additional levels of beta-carotene, which the body converts into vitamin A. Genetically modified products: benefits 1. Crops • Enhanced taste and quality • Reduced maturation time • Increased nutrients, yields, and stress tolerance • Improved resistance to disease, pests, and herbicides • New products and growing techniques 2. Animals • increased resistance, productivity, hardiness, and feed efficiency • Better yields of meat, eggs, and milk • Improved animal health and diagnostic methods 3. Environment • "Friendly" bioherbicides and bioinsecticides • Conservation of soil, water, and energy • Bioprocessing for forestry products • Better natural waste management • More efficient processing 4. Society • Increased food security for growing populations Genetically modified products: controversies 1. Safety • Potential human health impact ➢ Allergens ➢ transfer of antibiotic resistance markers ➢ unknown effects 2. Potential environmental impact ➢ unintended transfer of transgenes through crosspollination ➢ unknown effects on other organisms (e.g., soil microbes) ➢ loss of flora and fauna biodiversity 3. Access and Intellectual Property • Domination of world food production by a few companies • Increasing dependence on industralized nations by developing countries • Biopiracy—foreign exploitation of natural resources