Advanced Genetic Manipulation

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Advanced Genetic Manipulation

• Biotechnology 2

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Creating a Transgenic or GM Organism

• Stage I- Purpose
• Stage II- Development
• Stage III- Testing and Approval

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Stage I- Purpose

• First step of scientific method is to identify
  the problem.
• Although the SM is followed the overall goal is
  usually profit.
• Problems may come from individual or team
  brainstorming or as a result of previous
  research.
• Risks, Expenses, and Time required are weighed
  against the potential importance (profits?)

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Selecting Genes

• Genome Mapping is underway for many species
• Lets researchers know where to find useful gene
  segments
• In theory any gene from any organism can be
  removed
• Genes from known allergens avoided for GM foods
• Allergens may or may not be tied to gene
  sequences so scientists have to be careful when
  choosing

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Issues and Problems w/ TGOs

• Transgenic animals are more difficult and more
  expensive to create than TG plants
• TG animal creation usually requires the use of
  specialized reproductive cells
• Germ cells, stem cells, embryos, or haploid cells
• Far fewer animal cells survive the process
• 1-4 will develop into a full-term new born
  animal
• Stem cells have great potential for cloning and
  genetic manipulation but production of new lines
  can only be obtained through the destruction of
  developing embryos

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Stage II- Development

• Find target organism that will be changed
• Locate, Isolate, Extract beneficial gene sequence
• Usually hardest and longest part
• Insert isolated gene into target organism
• Biolistics is easiest, cheapest for plants
• Micromanipulation for animals
• Electroporation and Contact Absorption also
  common
• All methods result in destruction of most cells

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DNA Extraction

• Done with restriction enzymes and (radioactive)
  markers
• Enzymes read DNA segment and cleave (cut) it
  after certain sequences
• Markers indicate size and / or location of
  sequence
• Have a known molecular weight so when analyzed w/
  Gel Electrophoresis gene can be identified

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DNA Insertion- Vectors

• Vectors attach the gene sequence to their own DNA
  and then insert into DNA of target organism
• Bacteriophages (viruses)- inject DNA into cells
• Bacteria (Agrobacterium tumefaciens) cause tumors
  and result in DNA changes to affected cells
• Plasmids- Self-replication DNA loops inside
  protein coats of virus

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Figure 18.5 The lysogenic and lytic reproductive
cycles of phage ?, a temperate phage
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T4 bacteriophage infecting an E. coli cell
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• Review the steps of gene transfer

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Vectors

• Can be inserted into organism several ways
• Into physical wounds
• Placed into contact w/ exposed cells
• Placed into contact w/ dermal tissue
• Some plants (Arabidopis) can be transformed via
  exposure to a liquid solution containing vectors
• Usually a floral dip where developing flowers are
  literally dipped into the solution

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DNA Insertion- Fun Stuff

• Electroporation
• DNA sequence is placed close to cells in a
  solution that is then shocked to merge genetic
  info
• Micromanipulation
• Cell walls (plants)must be removed (also for
  electroporation)
• Biolistics
• Air or gun powder used to fire gold coated
  projectiles coated with target DNA into mass of
  plant cells
• Most effective method for plant cells

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Micromanipulation

• A tiny sharp syringe is used under a microscope
  to inject DNA through the cell membrane
• Most TG animals done this way
• Most exact method, fewest casualty cells
• Either via ennucleation and replacement or by
  simply placing gene sequence into target cell(s)
  and pronucleus

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Stage II- Development

• Test new organism for gene expression
• Marker Genes used
• Very useful in plants (black light
  bioluminescence)
• Markers may not transmit to offspring b/c gene
  sequence of target gene and marker are not linked

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Stage III- Testing and Approval

• Test efficacy of organism at addressing problem
• Original DNA can impact new gene sequence
• Determine if traits will transmit to future
  generations
• Best to cross transgenic w/ natural
• Backcrossing 2nd or 3rd generations might be
  needed when dealing w/ issues of complex heredity

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Field Testing

• Organisms are kept separate in controlled
  environments isolated from natural populations
  until trait transmission is studied
• Field testing ensures the inserted gene will not
  cause dangerous unintended consequences
• Also that they perform the intended function

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Stage III- Testing and Approval

• Gene and new organism registered w/ proper
  federal agency (who have overseen entire process
  anyway)
• USDA
• FDA
• APHIS

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Patenting Genes

• Very controversial since profits and expenses can
  be high
• Disagreement often based on benefits and methods
  used to create and extract/insert the gene