Download Genetic Transformation

Genetic Transformation
Historical Perspective
• Frederick Griffith 1928 London
– First controlled demonstration of genetic transformation
– Griffith made the observation that nonpathogenic bacteria
(Streptococcus pneumoniae) became pathogenic when mixed
with a virulent strain of heat-killed S. pneumoniae (i.e. injected
mixture killed mice)
– The mechanism of transforming
nonpathogenic bacteria to deadly
bacteria was not known
• In 1944 Oswald Avery demonstrated
that DNA is responsible for conferring
pathogenic properties
What is Genetic Transformation?
• Genetically modification of a cell
– Involves uptake of foreign DNA
– Replication within organism
– Gene expression
DNA
RNA
Protein
• Introduction of foreign DNA: Terms to know
– By viruses: Transduction
– Between bacteria: Conjugation
– In mammalian cells: Transfection
Gene Cloning
• Amplification and isolation of a particular gene sequence
– Requires the generation of recombinant DNA (rDNA)
i.e. combining DNA that does not naturally occur
• Insertion of the gene into a plasmid (circular DNA)
• Transformation of bacteria for replication
• Select for cells that have received the recombinant
DNA
• Select individual colony for scale-up culture and
replication of cloned DNA
Cloning a Gene into a Plasmid
BamH1 sites
Ampicillin-resistance gene
PCR-generated target gene
with BamH1 sites
Plasmid
BamH1 digest
Sticky ends
DNA ligase
Plasmid containing Ampicillin
resistance gene and target gene
Genetic Transformation into E.coli
Ampicillin resistance gene (Ampr)
and target gene on bacterial plasmid
Individual colony is selected and
cultured to amplify recombinant DNA
Plasmid enters some
bacteria
Cell division
Bacterial clones
Only E. coli containing plasmid
survive on Ampicillin plates
Transformation mixture is plated
on to agar plate containing
Ampicillin
Key Steps for Transformation
Protein of interest
Protein for
antibiotic resistance
Plasmid DNA enters the bacterial cell and the genes are expressed.
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Bacterial cell suspension is placed in CaCl2 solution
Cells must be in log phase of growth.
Cells are kept on ice until heat shock treatment
Heat shock at 42 ˚C for one minute
Recover period in LB broth
Cells are spread on appropriate selection plates
Components of Gene Cloning
• Plasmid (to carry rDNA into cell)
• Enzymes:
– Restriction enzymes for cutting vector and insert
– DNA ligase for joining DNA fragments
• Selection process
Plasmids
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Small circular dsDNA separate from bacterial DNA
Plasmids exist in bacteria, yeast, organelles
Single or multiple plasmid copies per cell
Easy to isolate and manipulate
Used as vector for transforming bacteria with foreign
DNA
Foreign DNA is inserted after cutting with restriction
enzymes
Plasmids contain certain genes which offer a competitive
advantage for bacteria (i.e. antibiotic resistance)
Positive Selection: confers growth advantage i.e. able to
grow in presence of antibiotic
Insert gene for expression (<10kb insertion)
Arabinose Operon
• Gene induction
• Arabinose operon
– Three structural genes: araB, araA, and araD encode enzymes
for arabinose metabolism
– Initiator region, araI contains both the operator and promoter
– The araC gene encodes an activator protein, AraC, which binds
to initiator region
Arabinose Operon Regulation
• Activation
– Arabinose binds the activator protein
– AraC/arabinose complex facilitates binding of RNA polymerase
to the promoter which turns on the ara operon.
– Activation also depends on cyclic AMP
• Repression
– Without arabinose, AraC protein binds araI and araO regions
forming a loop and preventing transcription of the ara operon
• Inducible promoter is used to control gene expression
Competent Cells
• Competence is the ability of cells to take up exogenous
DNA from the environment
• Two types of competence:
– Natural competence: Bacteria have cellular machinery to take up
DNA from environment
– Artificial competence: Cells are made competent in the
laboratory allowing them to take up DNA
Preparing Competent Bacteria
• Heat Shock:
– Drives DNA into cells
– Hold cells on ice in presence of CaCl2 to promote permeability of
cells to plasmid DNA
– Cells are heat shocked at 42 ºC for 50 – 60 seconds to allow
circular plasmid DNA to enter cells
• Electroporation:
– Subject cells to electric shock to perforate membrane
– Plasmid DNA enters cells through temporary holes
– Efficient transformation of large plasmids
Plant Transformation
• What is plant transformation
• Objective: To transform the entire organism not
individual cells
• Systemic infection of Arabidopsis
by transformation of female gametes
Genetic Engineering
• Involves:
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Isolating genes
Modifying genes for improved function
Packaging gene for insertion into new organism
Developing transgenes
• Development of organisms that express new traits not
found in nature
– Extended shelf-life (produce)
– Herbicide resistance (Roundup Ready)
– Faster growth rate, larger
• Terms:
– Transgene is a genetically engineered gene added to a species
– Transgenic refers to an organism containing an artificially
introduced transgene (i.e. not through breeding)
Agrobacterium tumefaciens
• Natural tool for plant transformation
• How it works – tumor induction
• Transfer of DNA to plant
Methods of Plant Transformation
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Agrobacterium
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Particle Bombardment
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Easiest and most simple
Cut plant tissue in small pieces, soak in Agrobacterium suspension
Some cells will be transformed by the bacterium
Grow on selection medium (rooting or shooting)
Some plants will not transform with the method
DNA is coated onto gold or tungsten particles
Particles are shot into young plant cells
Low efficiency
Most plants can be transformed
Electroporation
– Electric shock induces transient holes in cell membranes
– DNA enters cells
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Viral transformation
– Use plant virus as vector to introduce DNA
– Not always integrated into plant genome
Applications and Potential
• Genetically Modified
Organisms
• Agriculture
• Health and Medicine
• Biotechnology
• Scientific Research
• Industry and Environment
• Gene therapy
Genetically Modified Organisms
(GMOs)
• GMOs
– Express traits not normally found in nature
– Result of introducing foreign DNA
– Highly controversial
• Safety concerns
• Environmental implications
• Can we blindly trust profit-driven industry?
Agriculture
• Herbicide resistant crops
– Soybean, corn canola, lettuce, strawberry, potato, wheat
• Virus resistance
– Papaya resistance to papaya ringspot virus
• Golden rice
– Engineering rice to produce Vitamin A
• Edible vaccines in development
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Plant containing pathogen protein is ingested
Body produces antibodies against protein
Conferring resistance (ex diarrhea, hepatitis B, measles)
Bananas, potato, tomato
Health and Medicine
• Biotherapeutics
– Antibodies
– Hormone
– Enzymes
• Disease Indications
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Liver disease
Genetic diseases
Kidney disorders
Digestive disorders
Cancer
Infectious disease
Biotechnology
• Chymosin:
– Genetically engineered enzyme
– Used for curdling milk productsin cheese production
– Revolutionized cheese production
• Previously rennin was isolated from newborn calf intestine
(expensive, inhumane)
• Inexpensive, readily available
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Bovine somatotropin (bST):
– increased milk production in cows
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Other examples:
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Insulin
Interleukin
Human growth hormone
Interferon
Scientific Research
• Protein production using genetic transformation
• Objectives:
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Generate antibodies
Assay development
Structure determination
Protein-protein interaction
Industry and Environment
• Bioremediation: Using bioengineered microbes to clean
up pollution and contaminated sites
• Indicator bacteria: Detecting pollution and contamination
in the environment
• Waste management
– Sewage
– Petroleum products
Gene Therapy Overview
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Viral vector is used to deliver genetic material to target cells (ex. liver, lung)
The viral vector then injects the gene for a defective or missing protein
The cell then produces the functional protein and restores the target cell to
a normal state
Viruses used for gene therapy
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Retroviruses
Adenoviruses
Adeno-associated viruses
Herpes simplex viruses
Gene therapy is experimental with poor success in clinical trials
There are no FDA-approved gene therapy products on the market