1. Biotechnology 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.

2. 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

3. Herbicide resistance Steps in engineering:
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

4. 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.

5. 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 beta-carotene
Some plants have health-improving chemicals
Transgenic plants can provide relief
Daffodil gene inserted into rice to make beta-carotene, precursor to Vitamin A = golden rice
Critics say: not enough to make a difference.

6. 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.

7. Future directions
“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

8. Vaccines 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

9. 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

10. Transgenic vaccine

11. Medical diagnosis 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)

12. Medical diagnosis -2 Cystic fibrosis Huntington disease
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.

13. Ethics!
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!

14. Gene Therapy
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.

15. Recent successes 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

16. Failures 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.

17. 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

18. Sleeping Beauty Transposon system
Active transposon engineered from an inactive fish transposon.
Transposon jumps into chromosome bringing good gene with it.