Lecture 17 Chapter 9 Marker genes

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Lecture 17 Chapter 9 Marker genes

• Neal Stewart

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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?

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Using marker genes helps answers

• Are my plants transgenic?
• Is the gene expressed?
• How is my promoter working?

Negative selectable?
Positive selectable?
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Selectable markers Scorable markers (reporte
r 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

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Figure 9.2
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Sometimes escapes occur for kanamycin
resistance markers tissue is redvery stressed
Figure 9.3
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Barnase kills tapetum cells (and pollen)negative
non-conditional selection useful to engineer
male-sterility
Figure 9.7
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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.

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Figure 9.4
GUS positive plants and cells
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Figure 9.8
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Firefly luciferase produced in tobacco
Figure 9.9
Brought to you by biotechnologist of the day
David Owwas on the cover of Science
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35SGFP canola
White light UV light in a darkened room
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Pollen-tagged GFPsegregating 11
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GFP-tagged pollen on a bee leg. Hudson et al 2001
Mol Ecol Notes 1321
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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!

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http//www.youtube.com/watch?v90wpvSp4l_0feature
related
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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
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Horseweed transformation with GFP
Blue Light with GFP Filter
White Light
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Blue Light with GFP Filter
White Light
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Transgenic flower cross section
Transgenic versus wild-type flowers
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In planta fluorescence
?ex 395 nm
Relative fluorescence
Wavelength (nm)
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LIFI-laser induced fluorescence imagingfor
stand-off detection of GFP and other flourescence
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Journal of Fluorescence 15 697-705
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A brief FP history
Patterson Nature Biotechnol. (2004) 22 1524
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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
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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
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Species and FP name Ex max nm (Ext
Coef) Em max nm Reference
(103 M-1 cm-1) (Quantum yield )
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Excitation scanNontransgenic leaf
fluorescencewhy red fluorescence is better than
green
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With GFP
Why RFP is better less fluorescence noise in
the red
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More colors in fluorescent proteins discovered
(mostly from coralsthen improved)
http//www.photobiology.info/Zimmer_files/Fig6.png

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Orange Fluorescent Protein
GFP
Jennifer Hinds
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Orange Fluorescent Protein (OFP)
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An old trick ER targeting
Signal transit peptide
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GFP
HDEL
Signal peptide directs GFP to endoplasmic
reticulum for secretion But HDEL tag sequesters
assembled GFP in ERprotected environment allows
more accumulation. Haseloff et al 1997 PNAS 94
2122.
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ER retention dramatically improves OFP
brightness (monomers)
Mann et al. submitted 160th paper?
3x brighter!
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Big Orange Fluorescent Proteins
Mann et al. submitted.
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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