Lecture 6 Thursday January 24 2008

• tumor
• food
• very similar to what humans do with insulin
• extract and use insulin
• kind of ingenius

tumefacians

• a natural tool for plant transformation
• cytokinins
• Humans independently discovered this phenomenon
• we use it for tissue cultures

tumors

• have a lot of cells
• protein
• bacteria lives in the tumor cell and eats the protein

Ti plasmid

• transfer DNA
• we can utilize the bacteria in tumors to insert DNA
• splice cut and insert

Recombinant DNA technology

• restriction enzyme can cut sequences of DNA at certain sports
• it can then be fused via a certain type of enzyme
• this key enzyme won the nobel prize and
• it can now be bought and sold at will

plasmids

• sticky end
• will form a double strand unless properly controlled
• complementary sequences
• recombinant DNA

modified Ti Plamid

• does not contain the genes for synthesis of particular proteins and plant hormones
• disarmed TDNA - it will not cause a tumor because when the introduced DNA was mixed its growth hormone gene was altered
• transformed plant cells - but this does not cause tumor formations
• a tumefacien transfer this modified T - DNA
• bacteria tries to insert DNA into nucleous

two types of modern bio-technology

• recombinant dna
• tissue culture

tissue culture

• whole plant is regenerated from this transformed cell using tissue culture techniques
• tissue hormone used to regenerate plant
• the origion of the plant is the cell that has been modified - transgenic or GM plant
• it expresses the gene of interest
• modified Ti Plasmid

selection marker gene

• antibiotic resistance genes are used as a selection marker
• this can not grow in the medium that contains antibiotics - e coli
• who ever if this bacteria gets a antibiotic resistance gene and the gene of interest than both can be insert via a plasmid

other genes cn be used as markers

• though antibiotic genes are common there are other methods

selection markers can be removed

• recombinant DNA is used
• gametes
• as long as the selection marker is resessive it will be breed out of existence but the gene of interest will be dominant and persist
• who wants to eat an antibiotic resistant gene?
• some researchers remove the DNA

two major ways of creating GM plants

• Using Agrobacterium tumefaciens

1. particle bombardment

particle bombardment

• tiny metal particles coated with genes with desired characteristics are put into a particle gun and fired directly into the plant cells
• these genes are incorporated into the plant cell's DNA, and the cells are then grown into the full plants
• DNA is not fragile or mysterious it is simple, well understood and durable
• is dried it will become a powder - if water is added it will reform
• gene gun
• shoot the leaves
• by chance the inserted gene will combine with the rest of the DNA

• each event is different and independent
• plant was regenerated
• three individuals transgentic offspring are different
• this is because we cannot control where the gene was inserted
• the transgene's location in the genome is unknown and veries
• the amount of copies of the transgene varies
• expression of the gene may be double

Microinjection

• usually used for making transgenic mice
• mice with new genes added to their cells
• to make transgenic mice, special embryonic stem cells are grown in culture
• and then indiividual cells are injected wih DNA through microscopic glass needles
• remove egg nucleous, replace it with the one you want to propogate
• make a clone
• cannot be used in plants because of their thick cell wall

stem cells

Transgeneic mouse

• very useful to study mammalian gene function and regulation becasue analysis is carried out in a whole organism
• transgenic mice are lo used to model human diseases
• the transgenic mice (with the jelly fish protein) can be reinserted inutero

cloning is not transgeneic

• completely different
• cloning doesn't need recombinant DNA
• removed the entire dna set from the host nucleous
• then the donor's DNA was injected into the host
• the host gave birth to a clone of the donor
• a copy not a recombination