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Applications of Biotechnology Recombinant DNA Products and Organisms Applying Your Knowledge 1. 2. 3. 4. Probe Clone Plasmid Restriction Enzyme A. An enzyme that cleaves DNA at specific sequences is a __________ . B. A sequence of DNA that is complementary to the gene of interest is a _________. C. A small, independently replicating DNA molecule is a ___________. Recombinant DNA Products Pharmaceutical Factor VIII Human Growth Hormone Insulin Interferon Used for Blood Clotting Pituitary Dwarfism Diabetes Cancer Tissue Plasminogen Heart Attack Activator Vaccine Hepatitis B Optimizing the Yield of a Gene Product • • • • Selecting the Vector Selecting the Host Cell Selecting the DNA Delivery Method Constructing the Recombinant DNA Molecule • Maximizing Gene Expression • Collecting the Gene Product Vectors Plasmids Shuttle vectors Viruses Artificial Chromosomes Expression Vectors Selecting the Vector Vector Plasmid Cosmid (from bacterial virus) Virus Advantages Can carry foreign DNA into bacteria and yeast Can carry large DNA fragments Can reach plant and human cells Disadvantages Must remain small in size to increase uptake and avoid damage Technically more difficult to construct than recombinant plasmid Disabled virus may regain diseasecausing properties Selecting the Host Cell Prokaryotic Host E. coli Bacillus Advantages •Genetics is well known •Plasmids and viruses are well known •High reproductive rate •Actively exports proteins • Plasmids and viruses are well known Disadvantages •Can cause disease •Endotoxins from cell wall •Poor protein exporter •Cannot remove introns •Cannot remove introns Selecting the Host Cell Eukaryotic Host yeast mammalian Advantages Disadvantages •Genetics is well known • Non-pathogenic • Proteins of greater complexity can be made •Removes introns •May not be able to modify or export complex mammalian proteins •Avoids toxic bacterial proteins •Allows proper export, protein folding and modification •EXPENSIVE! Applying Your Knowledge 1. Plasmid 2. Cosmid 3. Virus 4. Yeast 5. mammalian cell 6. E. coli A. Best for large sequences of DNA (1-3) B. Best for proper modification of complex proteins (4-6) C. Best for entry into specific cell types (1-3) D. Inexpensive organism that can remove introns (4-6) Virus: Binds to a receptor on the cell surface and injects DNA into the cell (bacteria, plants, animals) Liposome: Sphere of lipids that is compatible with plasma membrane (animal cells, plant protoplasts DNA Delivery Methods Microinjection: Placing DNA in each cell with a thin needle (animal cells, plant protoplasts) Additional DNA Delivery Methods Method How it Works Used For/In Chemicals that open holes in membranes Temporary holes admit DNA Bacterial cells Animal cells Plant protoplasts Electroporation Electric shock causes temporary holes to admit DNA Animal cells Plant protoplasts Particle Bombardment DNA-coated metal pellets are propelled into cells with “gene gun” Plant cells Constructing the Recombinant DNA Molecule Ribosome Binding Site for mRNA Coding Region Promoter - Binding site for RNA polymerase - Initiation of transcription Plasmid Sequences -origin of replication -antibiotic resistance Terminator -Site where transcription ends Expressing the Gene: Production of Protein Goal Increased Yield • To efficiently produce as much protein as possible Possible Methods • Increase plasmid copy number •Increase promoter efficiency •Increase mRNA binding to ribosome Collecting the Gene Product Avoiding Protein Loss using Advantages Disadvantages Protease-deficient Bacteria • less protein breakdown • No cell can be protease-free Fusion to native proteins •proteins not degraded • May be technically difficult •Must remove native protein Accumulation of aggregates •avoids protease activity •Aggregates difficult to extract without breakdown Collecting the Gene Product How it Works Addition of a Signal Sequence •A signal sequence directs a ribosome to attach to rer to complete synthesis of the protein •Protein product is delivered into rer for transport within the cell to the outside Uses • Used to increase export of a protein Pharmaceutical Products Human Growth Hormone Functions: -Increases cell uptake of amino acids -Promotes use of fat Used to Treat: Dwarfism Advantages: Decreased danger of disease Less expensive, available in larger quantities Size 191 Amino Acids Library? cDNA Vector? plasmid Host? bacterial (after removing signal sequence) Pharmaceutical Products Factor VIII Advantages: Easier to obtain Free from contamination Function: Size -Blood Clotting -2332 Amino Acids -26 introns Used to Treat: Hemophilia -25 carbohydrate attachment sites on the protein Library? cDNA Vector? virus Host? mammalian (hamster) Transgenic Animals • Transgenic = containing one or more genes from another species in every cell of the organism • Types 1. “Pharm” Animals 2. Models of Human Diseases 3. Gene Targeted Animals “Pharm” Animals • Animals that produce pharmaceuticals and release them in milk or semen Goats Pigs Product Action tPA = tissue Plasminogen Activator Hemoglobin Breaks up blood clots to treat Heart Attacks Sheep Alpha-1antitrypsin Blood Substitute Hereditary emphysema Models of Human Diseases • Transgenic Mice act as Models of Human Diseases for Research Human Gene Mouse Model For BRCA1 Breast Cancer HD Huntington’s Disease HGH Human Patients Receiving Human Growth Hormone Procedure for Gene Targeting Gene Targeting • Introducing a different form of a gene to replace a host gene “knockout” Inactivation of a gene by gene targeting ---mouse with ADA gene knocked out as a model for SCID “knock in” Introducing a gene with altered function by gene targeting ---mice with human adult hemoglobin human fetal hemoglobin human sickle cell hemoglobin Applying Your Knowledge 1. 2. 3. 4. Knockout Animal “Pharm” Animal Model of Human Disease Knock in Animal A. Animal that secretes medicine in its milk B. Mouse with a human BRCA 1 gene added (no mouse genes altered) C. Mouse with its ADA gene inactivated