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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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Lecture 17 chapter 9 marker genes

Lecture 17 Chapter 9 Marker genes

Neal Stewart


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)


Figure 9.2 of non-transgenic cells


Sometimes escapes occur for kanamycin resistance markers tissue is red very stressed

Figure 9.3 of non-transgenic cells

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


Figure 9.7 of non-transgenic cells

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


Common reporter genes
Common reporter genes of non-transgenic cells

  • 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 of non-transgenic cells

GUS positive plants and cells


Figure 9.8 of non-transgenic cells


Firefly luciferase produced in tobacco

Figure 9.9 of non-transgenic cells

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 of non-transgenic cells

White light UV light in a darkened room


Pollen-tagged GFP—segregating 1:1 of non-transgenic cells


GFP-tagged pollen on a bee leg. of non-transgenic cells

Hudson et al 2001 Mol Ecol Notes 1:321


Green and other color fluorescent proteins
Green (and other color) fluorescent proteins of non-transgenic cells

  • 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? of non-transgenic cells

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


Horseweed of non-transgenic cells

transformation with GFP

Blue Light with GFP Filter

White Light


Blue Light with GFP Filter of non-transgenic cells

White Light


Transgenic flower cross section of non-transgenic cells

Transgenic versus wild-type flowers


In planta fluorescence of non-transgenic cells

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


Journal of Fluorescence 15: 697-705 detection of GFP and other flourescence


A brief fp history
A brief FP history detection of GFP and other flourescence

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


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 %)


Excitation scan nontransgenic leaf fluorescence why red fluorescence is better than green
Excitation scan: max nm Reference Nontransgenic leaf fluorescence—why red fluorescence is better than green


With gfp
With max nm Reference GFP

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


More colors in fluorescent proteins discovered
More colors in fluorescent proteins discovered max nm Reference

(mostly from corals…then improved)

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


O max nm Reference range

Fluorescent

Protein

GFP

Jennifer Hinds


O range f luorescent p rotein ofp
O max nm Reference range Fluorescent Protein (OFP)


An old trick er targeting
An old trick: ER targeting max nm Reference

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) max nm Reference

Mann et al. submitted

160th paper?

3x brighter!


Big orange fluorescent proteins
Big Orange Fluorescent Proteins max nm Reference

Mann et al. submitted.


Red foliage as output
Red foliage as output max nm Reference

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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