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?

• Is the gene expressed?

• How is my promoter working?

Negative selectable

Positive selectable

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

Figure 9.2

Figure 9.3 Sometimes “escapes” occur– for kanamycin resistance markers tissue is

red—very stressed

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

Pollen-tagged 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 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

White Light

Blue Light with GFP Filter

Horseweedtransformation

with GFP

White Light

Blue Light with GFP Filter

Transgenic flower cross section

Transgenic versus wild-type flowers

In planta fluorescenceR

ela

tive fl

uore

scen

ce

Wavelength (nm)

ex = 395 nm

LIFI-laser induced fluorescence imaging—for stand-off

detection of GFP and other flourescence

Journal of Fluorescence 15: 697-705

Canola LIFS

0

20000

40000

60000

80000

100000

120000

140000

160000

180000

200000

400 450 500 550 600 650 700 750 800

Nanometers (nm)

Inte

nsi

ty

A1

A2

A3

A4

A5

A6

A7

A8

A9

Water Raman Peak

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 Cotton AmCyan

Wheat leaf DsRed Cotton ZsGreen

Rice callus ZsGreen Cotton callus AsRed

Corn callus AmCyan DsRed tobacco

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

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

Excitation scan:Nontransgenic

leaf fluorescence—why red

fluorescence is better than green

Brassica napus leaf fluorescence

0

50000100000

150000

200000

250000300000

350000

400000

425

438

451

464

477

490

503

516

529

542

555

568

581

594

607

620

633

646

Wavelength

CP

S

375 nm excitation 425 nm excitation 475 nm excitation

525 nm excitation 550 nm excitation

Nicotiana tabacum leaf fluorescence

0

50000

100000

150000

200000

250000

300000

350000

400000

425

438

451

464

477

490

503

516

529

542

555

568

581

594

607

620

633

646

Wavelength

CP

S

375 nm excitation 425 nm excitation 475 nm excitation

525 nm excitation 550 nm excitation

With GFP

Brassica napus leaf fluorescence

0

50000

100000

150000

200000

250000

300000

350000

400000

425

438

451

464

477

490

503

516

529

542

555

568

581

594

607

620

633

646

Wavelength

CP

S

375 nm excitation 425 nm excitation 475 nm excitation

525 nm excitation 550 nm excitation GFP 375 nm excitation

Nicotiana tabacum leaf fluorescence

0

50000

100000

150000

200000

250000

300000

350000

400000

425

438

451

464

477

490

503

516

529

542

555

568

581

594

607

620

633

646

Wavelength

CP

S

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

More colors in fluorescent proteins discovered

http://www.photobiology.info/Zimmer_files/Fig6.png

(mostly from corals…then improved)

GFP

Jennifer Hinds

Orange FluorescentProtein

Orange Fluorescent Protein (OFP)

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’

ER retention dramatically improves OFP brightness (monomers)

Mann et al. submitted160th paper?

3x brighter!

Mann et al. submitted.

Big Orange Fluorescent Proteins

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