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

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 Strategies Positive Selectable marker gene Negative Selectable marker gene Visual 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 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.

Positive and Visual Selection

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

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)

-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 mass of callus colony derived from protoplast protoplast 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 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 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) 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: antibiotic resistance kanamycin (geneticin), hygromycin Kanamycin arrest bacterial cell growth by blocking various steps in protein synthesis 2. herbicide resistance phosphinothricin (bialapos); glyphosate

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.

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

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

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: selection conditions: 6 months frozen 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

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

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: 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: 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 (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

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

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

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

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