Transcript Molecular genetics of gene expression

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
=
% light fluoresced
Brightness
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 GFPYFP “Topaz”
514 (94)
527 (60)
Tsien 1998
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)
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