Biotechnology Dolly repairstemcell.files.wordpress.com Potential Uses for Cloned Genes to produce a protein product to endow an organism with a metabolic capability plasminogen activator engineered bacteria that degrade oil spills create more copies of the gene for further study Genetic Engineering Stages 1. Isolate gene cleavage of plasmid 2. Recombinant-DNA 3. Clone * Screen for quality 4. Harvest Gene Protein 1. Isolate Gene Isolate the desired gene from the genome the desired gene will be cut with restriction endonucleases (enzymes) desired gene to be isolated Cleavage cut DNA of host with restriction endonucleases (enzymes) several hundred RE’s exist discovered 1960s produce sticky ends or blunt ends Sticky Ends Escherichia coli Eco RI sticky end Blunt ends Haemophilus aegyptius Hae III GGCC CCGG GG CC CC GG 2. Recombinant DNA splice desired gene into host DNA, DNA ligase seals the strands Vector, generally plasmid of bacteria (prok) or yeast (euk) viral DNA (not for harvesting protein) plasmid with recombinant DNA 3. Clone produce a cell line in which all members have identical copies of a particular gene Screen Choose cells that carry desired gene & eliminate those cells that do not carry desired gene 4. Harvest (or Isolate) harvest protein harvest gene genetic harvesting protein harvesting protein molecules copies of the gene pest resistant gene oil eating bacteria dissolving clot protein human growth hormone Cloning reverse transcriptase plus mRNAs mRNAs Directly from an organism complementary DNA made from mRNA template through reverse transcription (cDNA) Reverse transcriptase can be used to make smaller cDNA libraries These contain only the genes that are transcribed by a particular type of cell recognized by the addition of a RE recognition sequence to it mRNA is degraded by an enzyme DNA polymerase synthesizes the 2nd strand cDNA Genomic Libraries “Book,” a clone containing a foreign DNA Plasmid library (bacterial, yeast) Phage library (virus) Bacterial Artificial Chromosome library Plasmid Library Copies of DNA fragments can be stored in a cloned bacterial plasmid foreign genome Each one of these is considered a "book" recombinant plasmid bacterial clones Phage Library DNA fragments can be stored in a cloned phage foreign genome each phage type is considered a "book" recombinant phage "book" phage clones Bacterial Artificial Chromosome (BAC) Library Copies of multiple DNA fragments can be stored in a bacterial artificial chromosome each clone occupies one well BAC clone plasmid with many genes Gene products Product Made in Use human insulin human growth hormone epidermal growth factor interleukin-2 bovine growth hormone cellulase Taxol hepatitis B vaccine erythropoietin factor VIII tissue plasminogen activator E. coli diabetes E. coli growth defects E. coli burns, ulcers E. coli possibly cancer E. coli improving weight gain E. coli breakdown of cellulose E. coli ovarian cancer S. cerevisiae prevents hepatitis mammalian cells anemia mammalian cells hemophilia mammalian cells heart attacks Other Examples "golden rice" genetically modified rich in beta-carotene prevents blindness news.bbc.co.uk papaya's ring spot disease gene was introduced to control the plague http://www2.dpi.qld.gov.au Human Genome Project Collaborative effort to map and sequence entire human genome Began 1990 4 goals genetic (linkage) mapping physical mapping sequencing analyzing the genomes of other species Genetic Mapping of the Human Genome to locate genetic markers spaced evenly throughout the chromosomes to make it easier to find other loci Physical Mapping of the Human Genome cutting chromosomes into identifiable fragments then determining their order on the chromosome Sequencing the Human Genome determining the exact nucleotide pairs haploid set of human chromosomes contains approximately 3 billion nucleotide pairs Genbank Database where DNA sequences have been deposited publicly available via the Internet final draft, 2004 (over 99% of genome was determined) remain a few 100 gaps of unknown sequences that require special methods to figure out Analyzing Gene Expression Analyze genomes of other important species for genetic engineering Stem Cells unspecialized blastula cells pluripotent adult stem cells gives rise to specific types of cells bone marrow blood cells Applications Medical animal and plant application gold rice salinity resistant gene Environmental Diagnosis Human Gene therapy Pharmaceutical products biofuel oil cleaning bacteria Forensic evidence The Innocence Project conviction of guilty Genomes of other species and H. sapiens Bacteria 1995 1997 S. cerevisiae 6,200 1996 60,000 2002 13,700 22,000 25,000 21,000 2000 2001 2004 2003 Plants 1,700 4,400 Fungi H. influenzae E. coli Oryza sativa (rice) Animals D. melanogaster Mus musculus Rattus norvegius H. sapiens Ethical Issues Should we engineer new genotypes for individuals with anomalies? diabetes, CF, immune deficiencies, MD, stunted growth, sickle-cell disease myopia, altering personalities, increase length of life Should we engineer human germ cells? If they are carrying abnormal genes eugenics - deliberate effort to control the genetic makeup of human populations color of eyes color of skin color of hair We have technology to test for diseases for which there is no cure and sometimes no treatment. (Ex. Huntington’s disease, breast cancer) Would you want to be tested? Who should have right to examine someone’s genetic info? How should that info be used? Should a person’s genome be a factor in determining eligibility for a job or insurance The End