lecture 17 chapter 9 marker genes neal stewart. discussion questions 1. why use marker genes? 2....
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Lecture 17 Chapter 9 Marker genes
Neal Stewart
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Discussion questions
1. Why use marker genes?
2. What are some differences between selectable markers and scorable markers?
3. Discuss the relative merits of GUS and GFP as reporters. Does the profile of experimentation
using these reporter genes overlap directly or partially?
4. What are the advantages, if any, for the use of the manA gene over the nptII gene as a selectable marker for food and feed crops, and would the use of the manA gene overcome public concern over the use of the nptII gene? Conversely, what are the disadvantages?
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Using marker genes helps answers
• Are my plants transgenic?
• Is the gene expressed?
• How is my promoter working?
Negative selectable
Positive selectable
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Selectable markers Scorable markers(reporter genes)
• Typically used to recover transgenic plant cells from a sea of non-transgenic cells
• Antibiotic resistance markers and herbicide resistance markers are most common
• Can help visualize transient expression
• Can help visualize if tissue is stably transgenic
• Useful for cellular and ecological studies
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Figure 9.2
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Figure 9.3 Sometimes “escapes” occur– for kanamycin resistance markers tissue is
red—very stressed
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Figure 9.7
Barnase kills tapetum cells (and pollen)—negative non-conditional selection useful to
engineer male-sterility
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Common reporter genes
• Beta glururonidase (GUS) uidA protein from Escherichia coli– needs the substrate X-gluc for blue color
• Luciferase proteins from bacteria and firefly yields light when substrate luciferin is present.
• Green fluorescent protein (GFP) from jellyfish is an example of an autofluorescent protein that changes color when excited by certain wavelengths of light.
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Figure 9.4
GUS positive plants and cells
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Figure 9.8
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Figure 9.9
Firefly luciferase produced in tobacco
Brought to you by biotechnologist of the day David Ow—was on the cover of Science
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35S:GFP canola
White light UV light in a darkened room
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Pollen-tagged GFP—segregating 1:1
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GFP-tagged pollen on a bee leg.Hudson et al 2001 Mol Ecol Notes 1:321
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Green (and other color) fluorescent proteins
• FP properties
• Detection and measurement
• Anthozoan FPs
• Why red is better than green
• Why orange is best of all!
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http://www.youtube.com/watch?v=90wpvSp4l_0&feature=related
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What is fluorescence?Excitation 475 nm Emission 507 nm
Extinction coefficientAbsorption and scattering
Quantum yield % light fluoresced
x = Brightness
Stokes shift*
*Named for Sir George G. Stokes who first described fluorescence in 1852
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White Light
Blue Light with GFP Filter
Horseweedtransformation
with GFP
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White Light
Blue Light with GFP Filter
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Transgenic flower cross section
Transgenic versus wild-type flowers
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In planta fluorescenceR
ela
tive fl
uore
scen
ce
Wavelength (nm)
ex = 395 nm
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LIFI-laser induced fluorescence imaging—for stand-off
detection of GFP and other flourescence
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Journal of Fluorescence 15: 697-705
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Canola LIFS
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Nanometers (nm)
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Water Raman Peak
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A brief FP history
Patterson Nature Biotechnol. (2004) 22: 1524
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Anthozoan FPs in transgenics Wenck et al Plant Cell Rep 2003 22: 244
Soybean ZsGreen Cotton AmCyan
Wheat leaf DsRed Cotton ZsGreen
Rice callus ZsGreen Cotton callus AsRed
Corn callus AmCyan DsRed tobacco
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FluorescenceExcitation 475 nm
Emission 507 nm
Extinction coefficientAbsorption and scattering
Quantum yield % fluoresced
x = Brightness
Stokes shift*
*Named for Sir George G. Stokes who first described fluorescence in 1852
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Aequorea victoria GFP 395 (27) 504 (79) Tsien 1998
A. victoria GFP S65T 489 (55) 510 (64) Tsien 1998
A. victoria EGFP 488 (56) 508 (60) Tsien 1998
A. victoria GFP “Emerald” 487 (58) 509 (68) Tsien 1998
A. victoria GFPYFP “Topaz” 514 (94) 527 (60) Tsien 1998
A. victoria GFPYFP “Venus” 515 (92) 528 (57) Nagai et al. 2002
Zoanthus sp. ZsGreen 497 (36) 506 (63) Matz et al. 1999
Zoanthus sp. ZsYellow 528 (20) 538 (20) Matz et al. 1999
Anemonia majano AmCyan 458 (40) 486 (24) Matz et al. 1999
Heteractis crispa t-HcRed1 590 (160) 637 (4) Fradkov et al. 2002
Discosoma sp. DsRed 558 (75) 583 (79) Matz et al. 1999
Discosoma sp. mRFP1 584 (50) 607 (25) Campbell et al. 2002, Shaner et al. 2004
Discosoma sp. dimer2 552 (69) 579 (29) Campbell et al. 2002, Shaner et al. 2004
Discosoma sp. mOrange 548 (71) 562 (69) Shaner et al. 2004
Discosoma sp. dTomato 554 (69) 581 (69) Shaner et al. 2004
Discosoma sp. tdTomato 554 (138) 581 (69) Shaner et al. 2004
(103 M-1 cm-1) (Quantum yield %)
Species and FP name Ex max nm (Ext Coef) Em max nm Reference
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Excitation scan:Nontransgenic
leaf fluorescence—why red
fluorescence is better than green
Brassica napus leaf fluorescence
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525 nm excitation 550 nm excitation
Nicotiana tabacum leaf fluorescence
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525 nm excitation 550 nm excitation
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With GFP
Brassica napus leaf fluorescence
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375 nm excitation 425 nm excitation 475 nm excitation
525 nm excitation 550 nm excitation GFP 375 nm excitation
Nicotiana tabacum leaf fluorescence
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375 nm excitation 425 nm excitation 475 nm excitation
525 nm excitation 550 nm excitation GFP 375 nm excitation
Why RFP is better– less fluorescence “noise” in the red
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More colors in fluorescent proteins discovered
http://www.photobiology.info/Zimmer_files/Fig6.png
(mostly from corals…then improved)
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GFP
Jennifer Hinds
Orange FluorescentProtein
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Orange Fluorescent Protein (OFP)
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An old trick: ER targeting
GFP HDEL
Signal transit
peptide
Signal peptide directs GFP to endoplasmic reticulum for secretionBut HDEL tag sequesters assembled GFP in ER—protected environmentallows more accumulation.
Haseloff et al 1997 PNAS 94: 2122.
5’ 3’
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ER retention dramatically improves OFP brightness (monomers)
Mann et al. submitted160th paper?
3x brighter!
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Mann et al. submitted.
Big Orange Fluorescent Proteins
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Red foliage as output
Arabidopsis MYB transcription factor PAP1 regulates the expression of anthocyanin biosynthesis genes: overexpression of PAP1 results in a red-plant phenotype