DNA technology Ch. 10
 Cloning genes
 Into bacteria
 Into plants
 Into animals
Recombinant bacteria
 Ch 10 p. 281
Human Growth Hormone (hGH) cloned into bacteria
(1980s)
 Pituitary dwarfism mutation in hGH gene
 hGH protein is produced by pituitary gland
 Pre-1985 hGH from cadaver brains
Drawbacks?
 Today
 hGH cloned
26 inches tall
Cloning steps:
 1. isolate hGH mRNA from
normal human
 2. reverse transcribe to cDNA
 How is cDNA different from DNA?
 3. ligate hGH cDNA into plasmid vector
 4. transform bacteria  grow bacteria and they
express hGH
 5. purify protein
Other cloned drugs made by bacteria
Human insulin for diabetics
Factor VIII for hemophiliacs
Interferon for chemotherapy
EPO for anemia
FSH for fertility clinics
TPA to prevent blood clots
Hepatitis B vaccine
bGH to increase milk yield in cows
1987
1993
1993
1992
1996
1996
Advantages of rbacteria/drugs
 Clean
 Worlds supply in one lab
 Cheap ?
 When rBST injected into cows  digestive systems are more efficient at
converting feed to milk. 8 gallons of milk/day/cow  9
 120 studies have found no differences in milk from rBST-supplemented
cows. National Institutes of Health, the Congressional Office of Technology
Assessment and the drug-regulatory agencies of Britain, Canada and the
European Union, Department of Health and Human Services
 Traces of BST are found in milk from all cows, supplemented or not.
rBST is broken down in the human gut, does not enter blood stream
 Even if injected into the human bloodstream, has no biological activity.
 What do you think?
 Affect of high human growth hormone in childhood
 ….. Pituitary tumor
Cloning into plants (GM) pg 282
 Transgenic plants
 Plants acquire new genetic trait by direct introduction of
gene
 We have been modifying plant genes by breeding for
1000’s of years
Getting a gene into a plant embryo
A natural system:
Agrobacterium tumefaciens bacteria infects plants  crown
gall disease (tumor) at wound sites
Agrobacterium tumefaciens cells attached to a plant cell. From
Genome News Network and Martha Hawes.
1. Engineer Ti plasmid


Remove tumor inducing genes
Add gene of interest
 2. transform agrobacterium with Ti plasmid
Ti plasmid is 200kb
 3. wound plant and infect with agrobacterium
 4. gene of interest transferred to plant cell
ssT-DNA
~20kb excised
Infection stimulates excision of 30 kb region of Ti called
T-DNA  insertion into chromosome
 5. grow explant
 = transgenic plant
Agrobacterium transgenic plant Virigina Tech
 Why is this referred to as horizontal gene transfer?
 How could this be used to introduce engineered genes
into a plant cell?
Last year, 12 million farmers in 23 countries grew 282.4
million acres (114.3 million hectares) of GM crops
genetically modified foods in US do not require labeling to
notify consumers.
GM plants 2007
genetically modified plants
Fungus chitinase gene
Resistance gene to
potato beetle have
been approved in the
US and in Canada.
Osmotin gene to tolerate salt stress
Nicotine-free GM tobacco
iron-storage gene from wild soybeans and vitamin E has been
enhanced by the use of genes from rice and rock cress. 2008
Benefits
 Increased crop yield
 Resistance to drought, freezing
 Decreased use of pesticide
 Decreased use of herbicides
 Increased nutrition
 Increased shelf life
 Can remove allergens
 Drawbacks
increased seed costs
pesticide resistant bugs
new allergens
harmful to insects?
may spread to other plants
 Bacillus thuringiensis (Bt) produce a protein crystal toxic to
insects.
 The toxin genes are on a plasmid
 The proteins bind to receptors in insect's intestine.
Humans do not have these receptors…………………….
Bt transgenic corn Ch. 10 pg. 284
 Corn engineered with gene that
codes for a Bt toxin
More on Bt corn Univ. MN
Effect of insect infestation on cotton
Non engineered cotton
Bt cotton
http://cls.casa.colostate.edu/TransgenicCrops/what.html
Mustard plant genetically engineered to remove selenium from soil
Can then grind and use in selenium deficient soils
Golden Rice
Oryza sativa 2012!
 Vitamin A deficiency
 Leading cause of childhood blindness (500,000 new cases /year)
 Engineer rice to produce genes needed for b
carotene in endosperm

(phytoene synthase and phytoene desaturase)
 Tissue specific expression cassette
Promoter--- transit sequence--- gene--- -
One serving of golden rice supplies enough beta-carotene to
meet 10 percent of the daily requirement for Vitamin A
 Do we need legislation for labeling of GM foods?
 Should GM genes, plants, animals, be patented?