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

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.

-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 victoriasource is bioluminescent jellyfish Aequora victoria• GFP is an intermediate in the bioluminescent reactionGFP is an intermediate in the bioluminescent reaction

absorbs UV (~360 nm) and emits visible light.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 low

• GFP is non catalytic, 1 GFP is non catalytic, 1 M concentration in cells M concentration in cells is required to exceed autofluorescenceis required to exceed autofluorescence

Green fluorescent protein (GFP))

advantagesadvantages• can detect in living cellscan detect in living cells

kinetics possiblekinetics possible lineage tracing possiblelineage tracing possible FACS analysis possibleFACS analysis possible

• inexpensive (no substrate)inexpensive (no substrate) disadvantagesdisadvantages

• low sensitivity and dynamic rangelow sensitivity and dynamic range• equipment requirementsequipment requirements

primary applicationsprimary applications• lineage tracer and reporter in transgenic lineage tracer and reporter in transgenic

embryosembryos

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

Glow reaction

Flash reaction

Luciferase

Luciferase flash reaction is ~20x more sensitive than glowflash reaction is ~20x more sensitive than glow

• 5 fg of luciferase or subattomolar levels (105 fg of luciferase or subattomolar levels (10-18-18 mol) mol)• substrate must be injected just before reading substrate must be injected just before reading

(equipment requirement)(equipment requirement)• stabilized assay utilized (5’ 1/2 life). This uses CoA stabilized assay utilized (5’ 1/2 life). This uses CoA

(increased cost)(increased cost) 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 formatpotential for simple automation in microplate format

add reagents, read at leisureadd reagents, read at leisure

flash

glow

Luciferase

Luciferase

advantagesadvantages• large dynamic range up to 7 decades, large dynamic range up to 7 decades,

depending on instrument and chemistrydepending on instrument and chemistry• rapid, suitable for automationrapid, suitable for automation• instability of luciferase at 37 °C (1/2 life of instability of luciferase at 37 °C (1/2 life of

<1hr) improves dynamic range of transient <1hr) improves dynamic range of transient assaysassays

at least one vendor has stabilized luciferase by at least one vendor has stabilized luciferase by removing the peroxisome targeting signal - lower removing the peroxisome targeting signal - lower dynamic rangedynamic range

• inexpensive inexpensive • widely usedwidely used

Luciferase

disadvantage is equipment requirementdisadvantage is equipment requirement• luminometer (very big differences between luminometer (very big differences between

models)models) photon counters - very sensitive, saturate rapidly photon counters - very sensitive, saturate rapidly

(~100,000 events/second) 5 decades or so(~100,000 events/second) 5 decades or so induced current - do not saturate but may not be induced current - do not saturate but may not be

as sensitive (5 decades)as sensitive (5 decades) a very few are sensitive and have large linear a very few are sensitive and have large linear

range (6-7 decades)range (6-7 decades)

• liquid scintillation counter (photon counter)liquid scintillation counter (photon counter)

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 to gentamycin

activity: broad action against prokaryotic and eukaryotic cells•inhibits protein synthesis by blocking

initiationresistance - 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:

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 ºCone year at 4 ºC, 1 month at 37 ºC selection conditions:selection conditions:

• E. coli: 50 E. coli: 50 g/mlg/ml• Eukaryotic cell lines:Eukaryotic cell lines:

50 - 1000 50 - 1000 g/ml (must be optimized)g/ml (must be optimized) 10 days- 3 weeks required to generate foci10 days- 3 weeks required to generate foci

use and availability:use and availability:• vectors containing hygromycin resistance vectors containing hygromycin resistance

gene are widely availablegene are widely available• in use for many yearsin use for many years

Glyphosate resistanceGlyphosate resistance

Glyphosate = “Roundup”, “Tumbleweed” = Glyphosate = “Roundup”, “Tumbleweed” = Systemic herbicideSystemic herbicide

Glyphosate inhibits EPSP synthase (S-Glyphosate inhibits EPSP synthase (S-eenolnolppyruvlyruvlsshikimate-3 hikimate-3 pphosphate – hosphate – involved in chloroplast amino acid involved in chloroplast amino acid synthesis)synthesis)

Escherichia coliEscherichia coli EPSP synthase = mutant EPSP synthase = mutant form form less sensitive to glyphosate less sensitive to glyphosate

Cloned via Ti plasmid into soybeans, tobacco, Cloned via Ti plasmid into soybeans, tobacco, petuniaspetunias

• Increased crop yields of crops treated with Increased crop yields of crops treated with herbicidesherbicides

+ 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

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