DNA technology Ch. 10 Cloning genes

Into bacteriaInto plantsInto animals

Recombinant bacteriaCh 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?

TodayhGH 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 1987Factor VIII for hemophiliacs 1993Interferon for chemotherapy 1993EPO for anemia 1992FSH for fertility clinics 1996TPA to prevent blood clots 1996Hepatitis B vaccine

bGH to increase milk yield in cows

Advantages of rbacteria/drugsCleanWorlds supply in one labCheap ?

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 282Transgenic 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

Infection stimulates excision of 30 kb region of Ti called T-DNA insertion into chromosome

ssT-DNA~20kb excised

5. grow explant

= transgenic plant

Agrobacterium tr ns n pl nta ge ic a 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

iron-storage gene from wild soybeans and vitamin E has been enhanced by the use of genes from rice and rock cress. 2008

Resistance gene to potato beetle have been approved in the US and in Canada.

Fungus chitinase gene

Nicotine-free GM tobacco

Osmotin gene to tolerate salt stress

Benefits Increased crop yieldResistance to drought, freezing Decreased use of pesticideDecreased use of herbicides Increased nutrition Increased shelf lifeCan remove allergens

Drawbacks increased seed costspesticide resistant bugsnew allergensharmful to insects?may spread to other plants

Bacillus thuringiensis (Bt) produce a protein crystal toxic to insects.

The toxin genes are on a plasmidThe proteins bind to receptors in insect's

intestine. Humans do not have these receptors…………………….

Bt transgenic corn Ch. 10 pg. 284Corn engineered with gene

that codes for a Bt toxin

More on Bt corn Univ. MN

Non engineered cotton Bt cotton

Effect of insect infestation on 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 deficiencyLeading cause of childhood blindness (500,000 new

cases /year)

Engineer rice to produce genes needed for carotene in endosperm (phytoene synthase and phytoene desaturase)

Tissue specific expression cassette

One serving of golden rice supplies enough beta-carotene to meet 10 percent of the daily requirement for  Vitamin A 

Promoter--- transit sequence--- gene--- -

Do we need legislation for labeling of GM foods?

Should GM genes, plants, animals, be patented?