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Lecture 17 Chapter 9 Marker genes Neal Stewart 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? Using marker genes helps answers • Are my plants transgenic? Negative • Isselectable the gene expressed? • How is my promoter working? Positive selectable Selectable markers • Typically used to recover transgenic plant cells from a sea of non-transgenic cells • Antibiotic resistance markers and herbicide resistance markers are most common Scorable markers (reporter genes) • Can help visualize transient expression • Can help visualize if tissue is stably transgenic • Useful for cellular and ecological studies Figure 9.2 Sometimes “escapes” occur– for kanamycin resistance markers tissue is red—very stressed Figure 9.3 Figure 9.7 Barnase kills tapetum cells (and pollen)— negative non-conditional selection useful to engineer male-sterility 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. Figure 9.4 GUS positive plants and cells Figure 9.8 Figure 9.9 Firefly luciferase produced in tobacco Brought to you by biotechnologist of the day David Ow—was on the cover of Science 35S:GFP canola White light UV light in a darkened room agged GFP—segregating 1:1 GFP-tagged pollen on a bee leg. Hudson et al 2001 Mol Ecol Notes 1:321 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! http://www.youtube.com/watch?v=90wpvSp4l_0&feature=related What is fluorescence? Excitation 475 nm Extinction coefficient Absorption and scattering *Named for Sir George G. Stokes who first described fluorescence in 1852 Stokes shift* x Emission 507 nm Quantum yield = Brightness % light fluoresced Horseweed transformation with GFP Blue Light with GFP Filter White Light Blue Light with GFP Filter White Light Transgenic flower cross section Transgenic versus wild-type flowers Relative fluorescence In planta fluorescence ex = 395 nm Wavelength (nm) LIFI-laser induced fluorescence imaging—for stand-off detection of GFP and other flourescence Journal of Fluorescence 15: 697-705 Canola LIFS 200000 180000 A1 160000 A2 Water Raman Peak 140000 A3 A4 Intensity 120000 A5 100000 A6 80000 A7 60000 A8 A9 40000 20000 0 400 450 500 550 600 Nanom eters (nm ) 650 700 750 800 A brief FP history Patterson Nature Biotechnol. (2004) 22: 1524 Anthozoan FPs in transgenics Wenck et al Plant Cell Rep 2003 22: 244 Soybean ZsGreen Wheat leaf DsRed Rice callus ZsGreen Corn callus AmCyan Cotton AmCyan Cotton ZsGreen Cotton callus AsRed DsRed tobacco Fluorescence Excitation 475 nm Extinction coefficient Absorption and scattering *Named for Sir George G. Stokes who first described fluorescence in 1852 Stokes shift* x Emission 507 nm Quantum yield % fluoresced = Brightness Species and FP name Ex max nm (Ext Coef) Em max nm (103 M-1 cm-1) Reference (Quantum yield %) 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) Discosoma sp. dimer2 552 (69) 579 (29) Campbell et al. 2002, Shaner et al. 2004 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 620 633 646 620 633 646 607 594 581 568 555 542 529 516 503 490 477 464 451 438 425 400000 350000 300000 250000 200000 150000 100000 50000 0 Wavelength 375 nm excitation 425 nm excitation 525 nm excitation 550 nm excitation 475 nm excitation Nicotiana tabacum leaf fluorescence 400000 350000 300000 250000 200000 150000 100000 50000 Wavelength 607 594 581 568 555 542 529 516 503 490 477 464 451 438 0 425 CPS Excitation scan: Nontransgenic leaf fluorescence—why red fluorescence is better than green CPS Brassica napus leaf fluorescence With GFP Brassica napus leaf fluorescence 400000 350000 300000 CPS 250000 200000 150000 100000 50000 646 633 620 607 594 581 568 555 542 529 516 503 490 477 464 451 438 425 0 Wavelength 375 nm excitation 425 nm excitation 475 nm excitation 525 nm excitation 550 nm excitation GFP 375 nm excitation Nicotiana tabacum leaf fluorescence 400000 350000 300000 200000 150000 100000 50000 646 633 620 607 594 581 568 555 542 529 516 503 490 477 464 451 438 0 425 CPS 250000 Why RFP is better– less fluoresc ence “noise” in the red More colors in fluorescent proteins discovered (mostly from corals…then improved) http://www.photobiology.info/Zimmer_files/Fig6.png Relative Brightness (% of EGFP) 250 200 GFP 150 100 50 EBFP EBFP2 Azurite mTagBFP mTurquoise mECFP Cerulean ECFP CyPet TagCFP AmCyan1 mTFP1 (Teal) Midor-Ishi Cyan TurboGFP Azami Green TagGFP AcGFP ZsGreen EGFP Emerald Superfolder GFP mWasabi T-Sapphire TagYFP EYFP Topaz Venus mCitrine Ypet PhiYFP ZsYellow1 mBanana Kusabira Orange mOrange Kusabira Orange2 mOrange2 dTomato dTomato dTomato-Tandem DsRed2 DsRed Ta gRFP Ta gRFP-T DsRed-Express(T1) mTangerine DsRed-Monomer mApple AsRed2 mStrawberry mRuby mRFP1 jRed mCherry HcRed1 dKeima-Tandem mRaspberry HcRed-Tandem mPlum AQ143 300 Brightness of Fluorescent Proteins 445 Jennifer Hinds 489 510 539 Emission Maximum (nm) 584 Orange Fluorescent Protein 0 610 Orange Fluorescent Protein (OFP) An old trick: ER targeting Signal transit 5’ GFP HDEL 3’ peptide Signal peptide directs GFP to endoplasmic reticulum for secretion But HDEL tag sequesters assembled GFP in ER—protected environment allows more accumulation. Haseloff et al 1997 PNAS 94: 2122. ER retention dramatically improves OFP brightness (monomers) Mann et al. submitted 160th paper? 3x brighter! Big Orange Fluorescent Proteins Mann et al. submitted. 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
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