Reason:

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

8. 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. Flash reaction Glow reaction Luciferase

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. Plant dies in presence of selective compound Plant grows in presence of selective compound Effect of Selectable Marker Non-transgenic = Lacks Kan or Bar Gene X Transgenic = Has Kan or Bar Gene

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

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

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: • 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 typical

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

29. Hygromycin • source: aminoglycoside antibiotic from Streptomyceshygroscopicus. • 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: • one year at 4 ºC, 1 month at 37 ºC • selection conditions: • E. coli: 50 g/ml • Eukaryotic cell lines: • 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

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. Shikimic acid + Phosphoenol pyruvate Plant EPSP synthase 3-Enolpyruvyl shikimic acid-5-phosphate (EPSP) Aromatic amino acids RoundUp Sensitive Plants + Glyphosate X X Without amino acids, plant dies 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 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