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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

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discussion questions
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
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

Selectable markers Scorable markers (reporter genes)
slide7

Figure 9.7

Barnase kills tapetum cells (and pollen)—negative non-conditional selection useful to engineer male-sterility
common reporter genes
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.
slide9

Figure 9.4

GUS positive plants and cells

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

35s gfp canola
35S:GFP canola

White light UV light in a darkened room

slide15

GFP-tagged pollen on a bee leg.

Hudson et al 2001 Mol Ecol Notes 1:321

green and other color fluorescent proteins
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!
what is fluorescence
What is fluorescence?

Emission 507 nm

Excitation 475 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

slide19

Horseweed

transformation with GFP

Blue Light with GFP Filter

White Light

slide21

Transgenic flower cross section

Transgenic versus wild-type flowers

slide23

In planta fluorescence

ex = 395 nm

Relative fluorescence

Wavelength (nm)

lifi laser induced fluorescence imaging for stand off detection of gfp and other flourescence
LIFI-laser induced fluorescence imaging—for stand-off detection of GFP and other flourescence
a brief fp history
A brief FP history

Patterson Nature Biotechnol. (2004) 22: 1524

anthozoan fps in transgenics wenck et al plant cell rep 2003 22 244
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

fluorescence
Fluorescence

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

slide30

Species and FP name Ex max nm (Ext Coef) Em max nm Reference

Zoanthus sp. ZsGreen

Aequorea victoria GFP

395 (27)

497 (36)

506 (63)

504 (79)

Tsien 1998

Matz et al. 1999

Zoanthus sp. ZsYellow

A. victoria GFP S65T

528 (20)

489 (55)

510 (64)

538 (20)

Matz et al. 1999

Tsien 1998

A. victoria EGFP

Anemonia majano AmCyan

488 (56)

458 (40)

508 (60)

486 (24)

Tsien 1998

Matz et al. 1999

Heteractis crispa t-HcRed1

A. victoria GFP “Emerald”

590 (160)

487 (58)

509 (68)

637 (4)

Tsien 1998

Fradkov et al. 2002

A. victoria GFPYFP “Topaz”

Discosoma sp. DsRed

558 (75)

514 (94)

527 (60)

583 (79)

Tsien 1998

Matz et al. 1999

Discosoma sp. mRFP1

A. victoria GFPYFP “Venus”

584 (50)

515 (92)

607 (25)

528 (57)

Campbell et al. 2002, Shaner et al. 2004

Nagai et al. 2002

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

Discosoma sp. mStrawberry

574 (90)

596 (29)

Discosoma sp. mCherry

587 (72)

610 (22)

Shaner et al. 2004

(103 M-1 cm-1)(Quantum yield %)

with gfp
With GFP

Why RFP is better– less fluorescence “noise” in the red

more colors in fluorescent proteins discovered
More colors in fluorescent proteins discovered

(mostly from corals…then improved)

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

slide34

Orange

Fluorescent

Protein

GFP

Jennifer Hinds

an old trick er targeting
An old trick: ER targeting

Signal transit

peptide

5’

3’

GFP

HDEL

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
ER retention dramatically improves OFP brightness (monomers)

Mann et al. submitted

160th paper?

3x brighter!

big orange fluorescent proteins
Big Orange Fluorescent Proteins

Mann et al. submitted.

red foliage as output
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