transgenic development (plant genetic engineering)
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Transgenic Development (Plant Genetic Engineering). Why do scientists want to change gene instructions?. to produce needed chemicals to carry out useful processes to give an organism desired characteristics. Genetic Engineering. - PowerPoint PPT PresentationTRANSCRIPT
Transgenic Development (Plant Genetic Engineering)
Genetic EngineeringGenetic Engineering
The process of manipulating and transferring instructions carried by genes from one cell to
another
Why do scientists want to change gene instructions?
to produce needed chemicals to carry out useful processes to give an organism desired
characteristics
Isolate desired gene for a new trait from
any organism
Isolate plasmid DNA
Gene inserted into plasmid.
Introduce modified plasmid into bacterium for replication. Grow in culture
to replicate
THE SCIENCE OF GENETIC THE SCIENCE OF GENETIC ENGINEERINGENGINEERING
getting DNA into a cell
getting it stably integrated
getting a plant back from the cell
Plant transformation
Requirement1.a suitable transformation method
2.2.a means of screening for transformantsa means of screening for transformants
3.an efficient regeneration system
4.genes/constructs vectors Promoter/terminator
reporter genes selectable marker genes ‘genes of interest’
Transformation techniqueTransformation technique
Biological.Biological.• Agrobacterium mediated Agrobacterium mediated
transformation.transformation. Mechanical.Mechanical.
• Particle bombardment.Particle bombardment.• Electroporation.Electroporation.• Microinjection.Microinjection.
Chemical.Chemical.• Polyethylene glycol.Polyethylene glycol.
Transformation methods
DNA must be introduced into plant cells
Indirect Agrobacterium tumefaciens
Direct 1. Microprojectile bombardment
2. Electroporation
3. Microinjection
Method depends on plant type, cost, application
Agrobacterium-mediated transformation
Transformation by the help of agrobacterium
Agrobacterium is a ‘natural genetic engineer’
i.e. it transfers some of its DNA to plants
Agrobacterium tumefaciens
Ti plasmid
AgrobacteriumGenomic DNA
Plant cellGenomic DNA
(carries the gene the gene of interestof interest)
+
Ti plasmid with the gene of interest
Gene of interest
Empty plasmid
Restriction
enzyme AA
Restriction
enzyme AA
Agrobacterium
Ti plasmid with the new gene
Plant cell
cell’s DNA
Transgenic plant Cell division
The new gene
+ Transformation
Agrobacterium tumefaciens
A. tumefaciens
binary vector
T-DNA
Success FactorSuccess Factor
Species Genotypes
Explant Agrobacterium strains
Plasmid
Direct gene transfer
Introducing gene directly to the target cell
1. Electroporation
2. Microinjection
3. Particle Bombardment
Explants: cells and protoplasts
Most direct way to introduce foreign DNA into the nucleus
Achieved by electromechanically operated devices
Transformation frequency is high
Electroporation
Duracell
DNA containingthe gene of interest
Plant cell
Protoplast
Electroporation Technique
Power supply
DNA inside the plant cell
The plant cell withthe new gene
Most direct way to introduce foreign DNA into the nucleus
Achieved by electromechanically operated devices that control the insertion of fine glass needles into the nuclei of individuals cells, culture induced embryo, protoplast
Labour intensive and slow
Transformation frequency is very high, typically up to ca. 30%
MicroinjectionMicroinjection
Microprojectile bombardment
• uses a ‘gene gun’
• DNA is coated onto gold (or tungsten) particles (inert)
• gold is propelled by helium into plant cells
• if DNA goes into the nucleus it can be integrated into the plant chromosomes
• cells can be regenerated to whole plants
In the "biolistic" (a cross between biology and ballistics )or "gene gun" method, microscopic gold beads are coated with the gene of interest and shot
into the plant cell with a pulse of helium.
Once inside the cell, the gene comes off the bead and integrates into the cell's genome.
“Gene Gun” TechniqueDNA coated
golden particles
Gene gun
Cell division
A plant cell withthe new gene
Plant cell
Cell’s DNA
Transgenic plant
Model from BioRad: Biorad's Helios Gene
Gun
In Planta Transformation
♣ Meristem transformation
♣ Floral dip method♣ Pollen
transformation
Reason:There are many thousands of cells in a leaf disc or callus clump - only a proportion of these will have taken up the DNA, therefore can get hundreds of
plants back - maybe only 1% will be transformed
Screening technique
Technique which is exploited to screen the transformation product
(transformant Cell)
Screening (selection)
Select at the level of the intact plant Select in culture
• single cell is selection unit• possible to plate up to 1,000,000 cells
on a Petri-dish.• Progressive selection over a number of
phases
Selection StrategiesSelection Strategies PositivePositive Selectable marker geneSelectable marker gene NegativeNegative Selectable marker geneSelectable marker gene VisualVisual Reporter geneReporter gene
Positive selection
Add into medium a toxic compound e.g. Add into medium a toxic compound e.g. antibiotic, herbicideantibiotic, herbicide
Only those cells able to grow in the presence of Only those cells able to grow in the presence of the selective agent give coloniesthe selective agent give colonies
Plate out and pick off growing colonies.Plate out and pick off growing colonies. Possible to select one colony from millions of Possible to select one colony from millions of
plated cells in a days work.plated cells in a days work. Need a strong selection pressure - get escapesNeed a strong selection pressure - get escapes
Only individuals with characters satisfying the Only individuals with characters satisfying the breeders are selected from population to be breeders are selected from population to be used as parents of the next generationused as parents of the next generation
Seed from selected individuals are mixed, then Seed from selected individuals are mixed, then progenies are grown togetherprogenies are grown together
Negative selection
Add in an agent that kills dividing cellsAdd in an agent that kills dividing cells Plate out leave for a suitable time, wash out Plate out leave for a suitable time, wash out
agent then put on growth medium.agent then put on growth medium. All cells growing on selective agent will die All cells growing on selective agent will die
leaving only non-growing cells to now grow.leaving only non-growing cells to now grow. Useful for selecting auxotrophs.Useful for selecting auxotrophs.
The most primitive and least widely used The most primitive and least widely used method which can lead to improvement only in method which can lead to improvement only in exceptional cases exceptional cases
It implies culling out of all poorly developed It implies culling out of all poorly developed and less productive individuals in a population and less productive individuals in a population whose productivity is to be genetically whose productivity is to be genetically improvedimproved
Positive and Visual SelectionPositive and Visual Selection
How do we get plants back from cells?We use tissue culture techniques to regenerate whole plants from single cellsGetting a plant back from a single cell is important so that every cell has the new DNA
Regeneration System
Transformation series of events
Transform individual cells
Callus formation
Auxins
CytokininsRemove from sterile conditions
Gene construct
BamHI
gus-intron nptII T 35S P 35S T 35S LB RB
P SAG12 ipt P 35S T nos
Gene construct
VectorsPromoter/terminator
Reporter genesSelectable marker genes
‘Genes of interest’.
Vectors
Ti-plasmid based vectora. Co-integrative plasmidb. Binary plasmid
Coli-plasmid based vectora. Cloning vectorb. Chimeric Plasmid
Viral vectora. It is normally not stably integrated into the plant cellb. It may be intolerant of changes to the organization of its genomec. Genome may show instability
A vehicle such as plasmid or virus for carrying recombinant DNA into a living cell
Ti plasmid
The binary Ti plasmid system
Binary vector system
Binary vector system
Promoter
1.1. A nucleotide sequence within an operonA nucleotide sequence within an operon2.2. Lying in front of the structural gene or genesLying in front of the structural gene or genes3.3. Serves as a recognition site and point of Serves as a recognition site and point of
attachment for the RNA polymeraseattachment for the RNA polymerase4.4. It is starting point for transcription of the It is starting point for transcription of the
structural genesstructural genes5.5. It contains many elements which are involved It contains many elements which are involved
in producing specific pattern and level of in producing specific pattern and level of expressionexpression
6.6. It can be derived from pathogen, virus, plants It can be derived from pathogen, virus, plants themselves, artificial promoterthemselves, artificial promoter
Types of Promoter Promoter always expressed in most tissue
(constitutive)-. 35 s promoter from CaMV Virus-. Nos, Ocs and Mas Promoter from bacteria-. Actin promoter from monocot-. Ubiquitin promoter from monocot-. Adh1 promoter from monocot-. pEMU promoter from monocot
Tissue specific promoter-. Haesa promoter-. Agl12 promoter
Inducible promoter-. Aux promoter
Artificial promoter-. Mac promoter (Mas and 35 s promoter)
Easy to visualise or assay
- ß-glucuronidase (GUS) (E.coli)
-green fluorescent protein (GFP) (jellyfish)
- luciferase (firefly)
Reporter gene
GUS
Cells that are transformed with GUS will form a blue precipitate when tissue is soaked
in the GUS substrate and incubated at 37oC
this is a destructive assay (cells die)
The UidA gene encoding activity is commonly used. Gives a blue colour from a colourless
substrate (X-glu) for a qualitative assay. Also causes fluorescence from Methyl Umbelliferyl Glucuronide (MUG) for a quantitative assay.
5 -- glucuronidase Genes
very stable enzyme cleaves -D glucuronide linkage simple biochemical reaction
• It must take care to stay in linear range detection sensitivity depends on substrate
used in enzymatic assay (fast)• colorimetric and fluorescent substrates
available
5 - -glucuronidase Genes Advantages
• low background• can require little equipment (spectrophotometer)• stable enzyme at 37ºC
Disadvantages• sensitive assays require expensive substrates or
considerable equipment• stability of the enzyme makes it a poor choice for
reporter in transient transfections (high background = low dynamic range)
Primary applications• typically used in transgenic plants with X-gus
colorimetric reporter
β-Glucorodinase gene
Bombardment of GUS gene
- transient expression
Stable expression of GUS in moss Phloem-limited expression of
GUS
GFP (Green Fluorescent Protein)
GFP glows bright green when irradiated by blue or UV light
This is a non destructive assay so the same cells can be monitored all the way
through It fluoresces green under UV illumination It has been used for selection on its own
Green fluorescent protein (GFP)
Source is bioluminescent jellyfish Aequora victoriaGFP is an intermediate in the bioluminescent
reaction Absorbs UV (~360 nm) and emits visible light.
has been engineered to produce many different colors (green, blue, yellow, red)
These are useful in fluorescent resonance energy transfer experiments
Simply express in target cells and detect with fluorometer or fluorescence microscope
Sensitivity is lowSensitivity is lowGFP is non catalytic, 1 GFP is non catalytic, 1 M concentration in cells is M concentration in cells is
required to exceed auto-fluorescencerequired to exceed auto-fluorescence
Green fluorescent protein (GFP))
Advantages• can detect in living cells• inexpensive (no substrate)
Disadvantages• low sensitivity and dynamic range• equipment requirements
Primary applications• lineage tracer and reporter in transgenic
embryos
GFP
protoplast colony derived from protoplast
mass of callus
regenerated plant
Luciferase luc gene encodes an enzyme that is responsible
for bioluminescence in the firefly. This is one of the few examples of a bioluminescent reaction that only requires enzyme, substrate and ATP.
Rapid and simple biochemical assay. Read in minutes
Two phases to the reaction, flash and glow. These can be used to design different types of assays.• Addition of substrates and ATP causes a flash of light that Addition of substrates and ATP causes a flash of light that
decays after a few seconds when [ATP] dropsdecays after a few seconds when [ATP] drops• after the flash, a stable, less intense “glow” reaction continues after the flash, a stable, less intense “glow” reaction continues
for many hours - AMP is responsible for thisfor many hours - AMP is responsible for this
Luciferase flash reaction is ~20x more sensitive than flash reaction is ~20x more sensitive than
glowglow glow reaction is more stableglow reaction is more stable
• allows use of scintillation counterallows use of scintillation counter• no injection of substrates requiredno injection of substrates required• potential for simple automation in microplate potential for simple automation in microplate
formatformat
Luciferase
AdvantagesAdvantages• large dynamic range up to 7 decades, depending on
instrument and chemistry• rapid, suitable for automation• instability of luciferase at 37 °C (1/2 life of <1hr)• inexpensive • widely used
disadvantages • Equipment requirement • luminometer (very big differences between models)• liquid scintillation counter (photon counter)r)
Gene which confer tolerance to a phytotoxic substance
Most common:
1. antibiotic resistance
kanamycin (geneticin), hygromycin
Kanamycin arrest bacterial cell growth by blocking various steps in protein synthesis
2. herbicide resistance
phosphinothricin (bialapos); glyphosate
Selectable Marker Gene
Effect of Selectable Marker
Transgenic = Has Kan or Bar Gene
Plant grows in presenceof selective compound
Plant dies in presenceof selective compound
Non-transgenic = Lacks Kan or Bar Gene
X
KanamycinKanamycin
Targets 30s ribosomal subunit, causing a Targets 30s ribosomal subunit, causing a frameshift in every translationframeshift in every translation
Bacteriostatic: bacterium is unable to produce Bacteriostatic: bacterium is unable to produce any proteins correctly, leading to a halt in any proteins correctly, leading to a halt in growth and eventually cell deathgrowth and eventually cell death
Kanamycin use/resistance Over-use of kanamycin has led to many wild Over-use of kanamycin has led to many wild
bacteria possessing resistance plasmidsbacteria possessing resistance plasmids As a result of this (as well as a lot of side As a result of this (as well as a lot of side
effects in humans), kanamycin is widely effects in humans), kanamycin is widely used for genetic purposes rather than used for genetic purposes rather than medicinal purposes, especially in medicinal purposes, especially in transgenic plantstransgenic plants
Resistance is often to a family of related Resistance is often to a family of related antibiotics, and can include antibiotic-antibiotics, and can include antibiotic-degrading enzymes or proteins protecting degrading enzymes or proteins protecting the 30s subunitthe 30s subunit
G418-GentamycinG418-Gentamycin
source: aminoglycoside antibiotic related source: aminoglycoside antibiotic related to gentamycinto gentamycin
activity: broad action against prokaryotic activity: broad action against prokaryotic and eukaryotic cellsand eukaryotic cells• inhibits protein synthesis by blocking inhibits protein synthesis by blocking
initiationinitiationresistance - bacterial neo gene (neomycin resistance - bacterial neo gene (neomycin
phosphotransferase, encoded by Tn5 phosphotransferase, encoded by Tn5 encodes resistance to kanamycin, encodes resistance to kanamycin, neomycin, G418neomycin, G418•but also cross protects against bleomycin but also cross protects against bleomycin
and relatives.and relatives.
G418 - Gentamycin Stability:
• 6 months frozen selection conditions:
• E. coli: 5 g/ml• Eukaryotic cells:
300-1000 g/ml. G418 requires careful optimization for cell types and lot to lot variations Kill curves required It requires at least seven days to obtain resistant colonies, two weeks is more typicalIt requires at least seven days to obtain resistant colonies, two weeks is more typical
G418 - Gentamycin
use and availability:use and availability:• perhaps the most widely used selection in perhaps the most widely used selection in
mammalian cellsmammalian cells• vectors very widely availablevectors very widely available
Surv
ivin
g ce
lls
Increasing dose ->
Hygromycin
source: aminoglycoside antibiotic from source: aminoglycoside antibiotic from Streptomyces hygroscopicus. Streptomyces hygroscopicus.
Activity: kills bacteria, fungi and higher Activity: kills bacteria, fungi and higher eukaryotic cells by inhibiting protein eukaryotic cells by inhibiting protein synthesissynthesis• interferes with translocation causing misreading interferes with translocation causing misreading
of mRNAof mRNA resistance: conferred by the bacterial gene resistance: conferred by the bacterial gene
hphhph• no cross resistance with other selective no cross resistance with other selective
antibioticsantibiotics
Hygromycin stability:stability:
• one year at 4 ºC, 1 month at 37 ºC
selection conditions:selection conditions:• E. coli: 50 g/ml• Eukaryotic cell lines:
50 - 1000 50 - 1000 g/ml (must be optimized)g/ml (must be optimized) 10 days- 3 weeks required to generate effect10 days- 3 weeks required to generate effect
use and availability:use and availability:• vectors containing hygromycin resistance gene
are widely available• in use for many years
Glyphosate resistanceGlyphosate resistance
Glyphosate = “Roundup”, “Tumbleweed” = Systemic herbicide
Glyphosate inhibits EPSP synthase (S-enolpyruvlshikimate-3 phosphate – involved in chloroplast amino acid synthesis)
Escherichia coli EPSP synthase = mutant form less sensitive to glyphosate less sensitive to glyphosate
Cloned via Ti plasmid into soybeans, tobacco, petunias
•Increased crop yields of crops treated with herbicides
+ Glyphosate
X
RoundUp Sensitive Plants
X
X
Shikimic acid + Phosphoenol pyruvate
3-Enolpyruvyl shikimic acid-5-phosphate(EPSP)
Plant EPSP synthase
Aromaticamino acids
Without amino acids, plant dies
X
BacterialEPSP synthase
Shikimic acid + Phosphoenol pyruvate
3-enolpyruvyl shikimic acid-5-phosphate(EPSP)
Aromaticamino acids
RoundUp Resistant Plants
+ Glyphosate
With amino acids, plant lives
RoundUp has no effect;enzyme is resistant to herbicide
BialaphosBialaphos Glufosinate – active substance of a broad-
spectrum-herbicide = synthetical copy of the aminoacid phosphinothricin produced by Streptomyces viridochomogenes
Inhibit glutamine-synthetase (important enzyme in nitrogen-cycle of plants) caused plant dies
Herbicide-tolerance is reached by gene-transfer from the bacterium to the plant
The transfered gene encodes for the enzyme phophinothricin-acetyl-transferase degrade glufosinate
Bialaphos*Bialaphos (Phosphinothricin-alanyl-alanine) is an
herbicide that inhibits a key enzyme in the nitrogen assimilation pathway, glutamine
synthetase, leading to accumulation of toxic levels of ammonia in both bacteria and plant cells
Only those cells that have taken up the DNA
can grow on media containing the selection
agent