1. Lecture 17 Chapter 9 Marker genes
Neal Stewart

2. 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?

3. Using marker genes helps answers
Are my plants transgenic?
Is the gene expressed?
How is my promoter working?
Negative selectable
Positive selectable

4. 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

5. Figure 9.2

6. Figure 9.3
Sometimes “escapes” occur– for kanamycin resistance markers tissue is red—very stressed

7. Figure 9.7
Barnase kills tapetum cells (and pollen)—negative non-conditional selection useful to engineer male-sterility

8. 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.

9. Figure 9.4
GUS positive plants and cells

10. Figure 9.8

11. Firefly luciferase produced in tobacco
Figure 9.9
Firefly luciferase produced in tobacco
Brought to you by biotechnologist of the day David Ow—was on the cover of Science

12. 35S:GFP canola
White light UV light in a darkened room

14. Pollen-tagged GFP—segregating 1:1

15. GFP-tagged pollen on a bee leg.
Hudson et al 2001 Mol Ecol Notes 1:321

16. 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!

18. What is fluorescence? x = Brightness Excitation 475 nm Emission 507 nm
Stokes shift* x
= Brightness
Quantum yield
% light fluoresced
Extinction coefficient
Absorption and scattering
*Named for Sir George G. Stokes who first described fluorescence in 1852

19. transformation with GFP
Horseweed
transformation with GFP
Blue Light with GFP Filter
White Light

20. Blue Light with GFP Filter
White Light

21. Transgenic flower cross section
Transgenic versus wild-type flowers

23. In planta fluorescence
ex = 395 nm
Relative fluorescence
Wavelength (nm)

24. LIFI-laser induced fluorescence imaging—for stand-off detection of GFP and other flourescence

25. Journal of Fluorescence 15: 697-705

27. A brief FP history
Patterson Nature Biotechnol. (2004) 22: 1524

28. 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

29. Fluorescence x = Brightness Emission 507 nm Excitation 475 nm
Stokes shift* x
= Brightness
Quantum yield
% fluoresced
Extinction coefficient
Absorption and scattering
*Named for Sir George G. Stokes who first described fluorescence in 1852

30. A. victoria GFP “Emerald” 487 (58) 509 (68)
Species and FP name Ex max nm (Ext Coef) Em max nm Reference
(103 M-1 cm-1) (Quantum yield %)
Aequorea victoria GFP
395 (27)
504 (79)
Tsien 1998
A. victoria GFP S65T
489 (55)
510 (64)
A. victoria EGFP
488 (56)
508 (60)
A. victoria GFP “Emerald”
487 (58)
509 (68)
A. victoria GFPYFP “Topaz”
514 (94)
527 (60)
A. victoria GFPYFP “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)
Anemonia majano AmCyan
458 (40)
486 (24)
Heteractis crispa t-HcRed1
590 (160)
637 (4)
Fradkov et al. 2002
Discosoma sp. DsRed
558 (75)
583 (79)
Discosoma sp. mRFP1
584 (50)
607 (25)
Campbell et al. 2002, Shaner et al. 2004
Discosoma sp. dimer2
552 (69)
579 (29)
Discosoma sp. mOrange
548 (71)
562 (69)
Shaner et al. 2004
Discosoma sp. dTomato
554 (69)
581 (69)
Discosoma sp. tdTomato
554 (138)
Discosoma sp. mStrawberry
574 (90)
596 (29)
Discosoma sp. mCherry
587 (72)
610 (22)

31. Excitation scan: Nontransgenic leaf fluorescence—why red fluorescence is better than green

32. With GFP
Why RFP is better– less fluorescence “noise” in the red

33. More colors in fluorescent proteins discovered
(mostly from corals…then improved)

34. Orange
Fluorescent
Protein
GFP
Jennifer Hinds

35. Orange Fluorescent Protein (OFP)

36. An old trick: ER targeting
Signal transit
peptide 5’
GFP
HDEL 3’
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.

37. ER retention dramatically improves OFP brightness (monomers)
Mann et al. submitted
160th paper?
3x brighter!

38. Big Orange Fluorescent Proteins
Mann et al. submitted.

39. 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