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Chapter 31

Chapter 31. Genetic Engineering and Biotechnology. Principles Underlying Genetic Engineering.

Roberta
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Chapter 31

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  1. Chapter 31 Genetic Engineering and Biotechnology

  2. Principles Underlying Genetic Engineering • Biotechnology: The use of living organisms to carry out defined chemical processes for industrial or commercial application. The modern def. includes invitro genetic techniques; manipulation of DNA. • Genetic Engineering: Techniques including the isolation, manipulation, and sequencing of DNA, as well as control of DNA expression, resulting in genetically modified organisms.

  3. Principles Underlying Genetic Engineering (cont.) • Premise of genetic engineering is based on molecular cloning: a DNA fragment from any organism can be put into and theoretically made to function in any other organism.

  4. Developments Essential for the Development of Genetic Engineering • 1. DNA chemistry • 2. DNA enzymology • 3. DNA replication • 4. Plasmids and conjugation • 5. Temperate bacteriophage • 6. Transformation • 7. RNA chemistry and enzymology • 8. Reverse transcription • 9. Reglation • 10. Translation • 11. Protein chemistry • 12. Protein excretion and posttranslational mod. • 13. The genetic code

  5. Hosts for Cloning Vectors • What are the ideal characteristics of a host that you’d use for obtaining large amounts of cloned DNA? What are some actual examples of this type of host? • What are the disadvantages of these hosts? • What are the advantages of using euk. cells as hosts? • What is transfection of mammalian cells? Why don’t we call it transformation? • What are 3 common ways of transfected euk. cells?

  6. Detecting Clones • Possibilities: Make gene libraries from total genomic DNA or clone a DNA fragment made by PCR. • 2 situations: 1. Gene of interest is expressed: detect by complementation, selection, or antibody – how is this done and what are the limitations? 2. Gene of interest is not expressed and you must look for the DNA itself: nucleic acid probes.

  7. Specialized Vectors • Shuttle vector: cloning vector that can stably replicate in 2 diff. organisms. • Expression vector: can be used not only to clone the desired gene, but also contains the necessary regulatory sequences so that expression of the gene can be subjected to experimental manipulation. • Promoters are important in sufficient transcription in expression vectors. What are ex. of promoters that have been used in E. coli? • Expression vectors must also contain proper ribosome-binding sites (Shine-Dalgarno sequence in prok.).

  8. Specialized Vectors (cont.) • Euk. vectors: Yeasts containing plasmids, primate DNA virus SV40, mammalian vectors utilizing adenovirus and vaccinia virus, integrating vectors: developed so that a cloned gene can be stably maintained and expressed in an organism or tissue (low copy #). Retroviruses can also be used to introduce genes into mammalian cells since these viruses integrate into the host chromosome. • Reporter genes: are incorporated into vectors because they encode proteins that are simple to detect. What are some ex.?

  9. Expression of Mammalian Genes in Bacteria (the Problem of Introns) • Use mRNA instead of DNA since mRNA has introns already removed. The mRNA is used to make cDNA (complementary DNA copy) using what enzyme? Can take advantage of poly-A tails on euk. mRNA to purify mRNA (use poly-T column). • Can also use RT-PCR to synthesize large amounts of cDNA without having to clone it. • Use reverse translation (protein  mRNA  cDNA) – can make a probe for the actual gene or make a synthetic gene. Can make modified genes that have posttranslational processing built in.

  10. Production of Mammalian Products & Vaccines by GMOs • Microbially produced Human Insulin hormone is identical in all respects to insulin purified from human pancreas. • Recombinant vaccines: suspensions of killed or modified pathogenic microorganisms or specific fractions isolated from the microorganisms that cause immunity when injected into an animal (subunit vaccine), ex. Hepatitis B, measles, rabies.

  11. Genetic Engineering in Plant Agriculture • Plant cloning vectors, ex. Ti plasmid from a gram neg. plant pathogen, Agrobacteriumtumefaciens. • Genetic improvement of plants: herbicide, insect, and microbial disease resistance; improved product quality. • Other uses: ex. edible vaccines.

  12. Genetic Engineering in Animal and Human Genetics • Transgenic animals are used to improve livestock, for research, and to produce pharmaceuticals, etc. • DNA Fingerprinting: used to ID individuals. • Sequencing of the Human Genome: primary goal = to understand human genetic diseases. • Gene Therapy: Nonfunctional or dysfunctional gene is augmented or replaced by a functional gene, including using viruses as vectors.

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