Transcript + Glyphosate X

Screening technique
Technique which is exploited to screen the transformation product
(transformant Cell)
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 (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 Petridish.
• Progressive selection over a number of phases
Selection Strategies
 Positive
 Negative
 Visual
Selectable marker gene
Selectable marker gene
Reporter gene
Positive selection
 Only individuals with characters satisfying the breeders are
selected from population to be used as parents of the next
generation
 Seed from selected individuals are mixed, then progenies are
grown together
 Add into medium a toxic compound e.g. antibiotic, herbicide
 Only those cells able to grow in the presence of the selective
agent give colonies
 Plate out and pick off growing colonies.
 Possible to select one colony from millions of plated cells in a
days work.
 Need a strong selection pressure - get escapes
Negative selection
 The most primitive and least widely used method which can
lead to improvement only in exceptional cases
 It implies culling out of all poorly developed and less
productive individuals in a population whose productivity is to
be genetically improved
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Add in an agent that kills dividing cells
Plate out leave for a suitable time, wash out agent then put on
growth medium.
All cells growing on selective agent will die leaving only nongrowing cells to now grow.
Useful for selecting auxotrophs.
Positive and Visual Selection
Reporter gene
easy to visualise or assay
- ß-glucuronidase (GUS)
(E.coli)
-green fluorescent protein (GFP)
(jellyfish)
- luciferase
(firefly)
GUS
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.
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)
-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
-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 victoria
• GFP 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 low
• GFP is non catalytic, 1 M concentration in cells is required to
exceed autofluorescence
Green fluorescent protein
(GFP)
 advantages
• can detect in living cells
 kinetics possible
 lineage tracing possible
 FACS analysis possible
• 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
regenerated plant
mass of callus
Luciferase
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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 decays after a
few seconds when [ATP] drops
• after the flash, a stable, less intense “glow” reaction continues for many
hours - AMP is responsible for this
Luciferase
Flash reaction
Glow reaction
Luciferase
 flash reaction is ~20x more sensitive than glow
• 5 fg of luciferase or subattomolar levels (10-18 mol)
• substrate must be injected just before reading (equipment
requirement)
• stabilized assay utilized (5’ 1/2 life). This uses CoA (increased
cost)
 glow reaction is more stable
• allows use of scintillation counter
• no injection of substrates required
• potential for simple automation in microplate format
 add reagents, read at leisure
Luciferase
flash
glow
Luciferase
advantages
• 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)
improves dynamic range of transient assays
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at least one vendor has stabilized luciferase by removing the
peroxisome targeting signal - lower dynamic range
• inexpensive
• widely used
Luciferase
disadvantage is equipment requirement
• luminometer (very big differences between models)
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photon counters - very sensitive, saturate rapidly
(~100,000 events/second) 5 decades or so
induced current - do not saturate but may not be as
sensitive (5 decades)
a very few are sensitive and have large linear range (6-7
decades)
• liquid scintillation counter (photon counter)
Selectable Marker Gene
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
Effect of Selectable Marker
Non-transgenic = Lacks Kan or Bar Gene
Plant dies in presence
of selective compound
X
Transgenic = Has Kan or Bar Gene
Plant grows in presence
of selective compound
Kanamycin
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Targets 30s ribosomal subunit, causing a frameshift in every
translation
Bacteriostatic: bacterium is unable to produce any proteins
correctly, leading to a halt in growth and eventually cell death
Kanamycin use/resistance
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Over-use of kanamycin has led to many wild bacteria
possessing resistance plasmids
As a result of this (as well as a lot of side effects in
humans), kanamycin is widely used for genetic purposes
rather than medicinal purposes, especially in transgenic
plants
Resistance is often to a family of related antibiotics, and
can include antibiotic-degrading enzymes or proteins
protecting the 30s subunit
G418-Gentamycin
 source: aminoglycoside antibiotic related to
gentamycin
 activity: broad action against prokaryotic and
eukaryotic cells
• inhibits protein synthesis by blocking initiation
 resistance - bacterial neo gene (neomycin
phosphotransferase, encoded by Tn5 encodes
resistance to kanamycin, neomycin, G418
• but also cross protects against bleomycin and
relatives.
G418 - Gentamycin
Stability:
• 6 months frozen
selection conditions:
• E. coli: 5 g/ml
• Eukaryotic cells:
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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 typical
Surviving cells
G418 - Gentamycin
Increasing dose ->
use and availability:
• perhaps the most widely used selection in mammalian
cells
• vectors very widely available
Hygromycin
source: aminoglycoside antibiotic from
Streptomyces hygroscopicus.
Activity: kills bacteria, fungi and higher
eukaryotic cells by inhibiting protein synthesis
• interferes with translocation causing misreading of
mRNA
resistance: conferred by the bacterial gene hph
• no cross resistance with other selective antibiotics
Hygromycin
stability:
• one year at 4 ºC, 1 month at 37 ºC
selection conditions:
• E. coli: 50 g/ml
• Eukaryotic cell lines:
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50 - 1000 g/ml (must be optimized)
10 days- 3 weeks required to generate foci
use and availability:
• vectors containing hygromycin resistance gene are
widely available
• in use for many years
Glyphosate resistance
 Glyphosate = “Roundup”, “Tumbleweed” = Systemic
herbicide
 Glyphosate inhibits EPSP synthase (Senolpyruvlshikimate-3 phosphate – involved in
chloroplast amino acid synthesis)
 Escherichia coli EPSP synthase = mutant form  less
sensitive to glyphosate

Cloned via Ti plasmid into soybeans, tobacco, petunias
• Increased crop yields of crops treated with herbicides
RoundUp Sensitive Plants
Shikimic acid + Phosphoenol pyruvate
+ Glyphosate
Plant
EPSP synthase
X
3-Enolpyruvyl shikimic acid-5-phosphate
(EPSP)
Without amino acids,
plant dies
X
X
Aromatic
amino acids
X
RoundUp Resistant Plants
Shikimic acid + Phosphoenol pyruvate
+ Glyphosate
Bacterial
EPSP synthase
RoundUp has no effect;
enzyme is resistant to herbicide
3-enolpyruvyl shikimic acid-5-phosphate
(EPSP)
With amino acids,
plant lives
Aromatic
amino acids
Bialaphos
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Glufosinate – active substance of a broad-spectrumherbicide = synthetical copy of the aminoacid
phosphinothricin produced by Streptomyces
viridochomogenes
Effect: inhibition of the glutamine-synthetase
(important enzyme in nitrogen-cycle of plants)
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
harmless
degradation of 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