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Chapter 31

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Chapter 31 l.jpg

Chapter 31

Genetic Engineering and Biotechnology

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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.

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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.

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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

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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?

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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.

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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.).

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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.?

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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.

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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.

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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.

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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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