1. Technique which is exploited to screen the transformation product
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

2. 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

3. Selection Strategies Positive Selectable marker gene
Negative Selectable marker gene
Visual Reporter gene

4. 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

5. 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
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 non-growing cells to now grow.
Useful for selecting auxotrophs.

6. Positive and Visual Selection

7. easy to visualise or assay
Reporter gene
easy to visualise or assay
- ß-glucuronidase (GUS) (E.coli)
green fluorescent protein (GFP) (jellyfish)
luciferase (firefly)

8. this is a destructive assay (cells die)
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)

9. -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

10. -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

11. β-Glucorodinase gene Bombardment of GUS gene - transient expression
Stable expression of GUS in moss
Phloem-limited expression of GUS

12. 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

13. 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

14. 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

15. GFP mass of callus colony derived from protoplast protoplast
regenerated plant

16. 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 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

17. Luciferase
Flash reaction
Glow reaction

18. 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

19. Luciferase
flash
glow

20. 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
at least one vendor has stabilized luciferase by removing the peroxisome targeting signal - lower dynamic range
inexpensive
widely used

21. Luciferase disadvantage is equipment requirement
luminometer (very big differences between models)
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)

22. Selectable Marker Gene
Gene which confer tolerance to a phytotoxic substance
Most common:
antibiotic resistance
kanamycin (geneticin), hygromycin
Kanamycin arrest bacterial cell growth by blocking various steps in protein synthesis
2. herbicide resistance
phosphinothricin (bialapos); glyphosate

23. X 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
This slide shows the effect of the selectable marker.

24. Kanamycin
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 is a chemical compound which targets the 30s ribosomal subunit in prokaryotes, binding in such a way as to cause a frameshift in every translation. This has a "bacteriostatic" effect: the bacterium is unable to produce any proteins correctly, leading to a halt in growth and eventually cell death. 24

25. Kanamycin use/resistance
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
Over-use of kanamycin has led to many wild bacteria possessing resistance, which is encoded in 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 comes in three variaties: antibiotic-degrading enzymes, reduced membrane permeability, or proteins protecting the 30s subunit. 25

26. 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.

27. G418 - Gentamycin Stability: selection conditions: 6 months frozen
E. coli: 5 g/ml
Eukaryotic cells:
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

28. G418 - Gentamycin use and availability:
Surviving cells
Increasing dose ->
use and availability:
perhaps the most widely used selection in mammalian cells
vectors very widely available

29. 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

30. Hygromycin stability: selection conditions: use and availability:
one year at 4 ºC, 1 month at 37 ºC
selection conditions:
E. coli: 50 g/ml
Eukaryotic cell lines:
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

31. Glyphosate 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
Cloned via Ti plasmid into soybeans, tobacco, petunias
Increased crop yields of crops treated with herbicides

32. 3-Enolpyruvyl shikimic acid-5-phosphate
RoundUp Sensitive Plants
Shikimic acid + Phosphoenol pyruvate
3-Enolpyruvyl shikimic acid-5-phosphate
(EPSP)
Plant
EPSP synthase
Aromatic
amino acids
+ Glyphosate X X
Without amino acids, plant dies
This slide shows the actual biochemical pathway that we discussed in the previous slide. EPSP synthase synthesizes 3-enolpyruvly shikimic acid-5-phosphate. This is the essential precursor to aromatic amino acids. When plants are sprayed with a glyphosate-containing herbicide, such as RoundUp, this important precursor is not synthesized, and consequently the plant is starved of aromatic amino acids. The result is plant death. X X

33. RoundUp Resistant Plants
Shikimic acid + Phosphoenol pyruvate
+ Glyphosate
RoundUp has no effect;
enzyme is resistant to herbicide
Bacterial
EPSP synthase
3-enolpyruvyl shikimic acid-5-phosphate
(EPSP)
With amino acids, plant lives
RoundUp Resistant plants have a very simple solution. An engineered version of EPSP synthase, one that was discovered in a bacteria, is introduced into the plant. This enzyme can not be bound by glphosate. Therefore, if a field is sprayed with the herbicide, the introduced version of the gene produces a functional enzyme. The 3-enolpyruvl shikimic acid-5-phosphate precursor is synthesized normally, and the plant produces enough aromatic amino acids to survive.
Aromatic
amino acids

34. Bialaphos
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

35. 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

36. Only those cells that have taken up the DNA can grow on media containing the selection agent