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Biotechnology
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• What is Biotechnology?
– Use of organisms, especially microbes, to produce
useful products?
• Beer, wine, bread, organic solvents, antibiotics
• By this definition, very very old.
– Use of recombinant DNA techniques to harness the
power of organisms to make useful products.
• Very new technology
• Includes herbicide-resistant plants, human
proteins produced in yeasts, new vaccines.
Biotechnology has several applications:
overview
• Agriculture
– Herbicide resistant plants
– Improved nutritional qualities
• Pharmaceuticals
– Production of human proteins as drugs
– Production of vaccines
• Medical, legal, biological
– Screening for, treatment of genetic disease
– DNA fingerprinting, biological conservation
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Herbicide resistance
• Example: glyphosate resistant plants
– More than 2/3 of US soybeans and cotton
– Glyphosate inhibits EPSP synthase
• Engineered plants have extra copies of gene,
make more enzyme, so are more resistant.
• Steps in engineering:
– Gene from E. coli. Put next to strong promoter
– Cloned into Ti plasmid, plasmid put back into
A. tumefaciens which carries plasmid to plant cell.
– Grow whole plant from engineered plant cell
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Why and why not?
• Use of herbicide-resistant plants means less
herbicide use, no-till farming.
– less erosion and less non-point source pollution.
• Safe to eat? Why not?
– Proteins not automatically destroyed during
digestion; allergies possible. Otherwise, what’s
the problem?
• Environmental concerns
– Toxic pollen? Herbicide resistant weeds?
• Biotech: same only more targeted and
quicker.
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Ag-2: improved nutrition
• Not every food product has complete nutrition
– Corn very low in the amino acid lysine
– Countries relying on rice have low intake of betacarotene
– Some plants have health-improving chemicals
• Transgenic plants can provide relief
– Daffodil gene inserted into rice to make betacarotene, precursor to Vitamin A = golden rice
• Critics say: not enough to make a difference.
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Pharmaceuticals
• Dwarfism, diabetes, cancer can be treated
using human proteins
– Obtained with difficulty
– Insulin from slaughterhouse animals
• Recombinant insulin first from E. coli
– Required combination of cloning, chemical
treatment
– Starting point: mRNA, reverse transcriptase, then
insertion into plasmid vector
– E. coli or yeast cells used.
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Future directions
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• “pharming”: growing of protein drugs in farm
plants and animals
– Cloning into sheep (etc.) with mammary specific
promoter, only expressed in that tissue.
• Released in, collected from milk.
– Using tobacco plants, especially for vaccines
• Tobacco easy to grow, easy to engineer, easy to
harvest
• Years of agricultural experience
– Use of hairy roots
• Cloned genes inserted w/ Ti plasmid
Vaccines
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• Exposing host to antigens found on pathogen
– Whole, live, weakened pathogen
• Strong immunity, but risk of live pathogen
– Whole, dead pathogen
• Nucleic acid not “dead”;
• cancer or toxic reaction
– Subunit vaccine: using a molecule from pathogen
• Host reacts, then protects against later exposure
to entire pathogen
Vaccines-2
• Recombinant vaccines
– Clone gene for surface antigen of pathogen
– Express gene i.e. get antigenic proteins made
• Collect proteins, process into vaccine
• Get proteins into harmless virus
– Express proteins in food
• Because there are food allergies, proteins taken
orally can result in immune reactions
• Eliminates worries about sterilization, storage,
needle-phobia
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Transgenic vaccine
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Medical diagnosis
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• Sickle cell anemia
– Fetal cell samples
– CVS or amniocentesis
– Gene obtained from fetal DNA
• Sickle cell anemia caused by a single nucleotide
base substitution that removes a MstII site.
– Different banding pattern on gel indicates whether
fetus will be a carrier or have disease
(homozygous)
Medical diagnosis -2
• Cystic fibrosis
– Most cases causes by a specific deletion of DNA
– PCR used to make allele-specific oligonucleotides
• This DNA hybridizes to region in normal gene
that is deleted in faulty allele
• Absence of hybridization means deletion is
present, person has the Cf allele.
• Huntington disease
– Because of variable number of trinucleotide
repeats, probably PCR or VNTR-type test looking
for varying lengths of DNA fragments.
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Ethics!
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• Genetic engineering, medical tests opens up
wide range of issues and questions
– Environmental and global economic issues
– Stem cell research and cloning
– Who owns the data? Can someone else patent your
genes? Privacy issues.
• Should your boss, insurance company,
government have access to your data?
– We can tell you that you have the disease, but
• We can’t do anything about it!
Gene Therapy
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• Gene therapy: the attempt to cure an underlying
genetic problem by insertion of a correct copy
of a gene.
– Tantalizingly simple and profound in theory,
maddeningly difficult to actually achieve.
– Easiest targets: access to or retrieval of cells
• Respiratory and blood
• Engineered virus, infects cells, carries in good
gene.
• Engineer cell in tube, return to body.
Recent successes
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• Adenosine Deaminase deficiency
– Defect in nucleoside metabolism especially affects
white blood cells
• X-linked chronic granulomatous disease
– Neutrophils fail to make superoxide
• General scheme: Retrovirus used to replace
gene in bone marrow cells, return cells to
patient
Failures
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• Genes don’t always make into genome
– “cure” is short-lived as DNA disappears
• Viruses carrying genes insert in bad places
– Cause over-expression of genes or DNA deletions
• Cancer
• Immune system becomes sensitized to vector
• Death of patient in clinical trial in 1999.
New development
• Sleeping Beauty transposon system
– First transposon available in vertebrates?
– Originally from inactive fish transposon
• Engineer with gene of interest
– Transposon inserts with gene, getting it into the
chromosome
– Inserts in different places from viral vector
– Much higher rate of gene insertion
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Sleeping Beauty Transposon system
Active transposon
engineered from
an inactive fish
transposon.
Transposon jumps
into chromosome
bringing good
gene with it.
http://www.discoverygenomics.net/sbts.html
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