Lecture 17 Chapter 9 Marker genes - PowerPoint PPT Presentation

1 / 39
About This Presentation
Title:

Lecture 17 Chapter 9 Marker genes

Description:

2. What are some differences between selectable markers and scorable markers? ... A. victoria GFP YFP 'Topaz' 514 (94) 527 (60) Tsien 1998. A. victoria GFP YFP 'Venus' ... – PowerPoint PPT presentation

Number of Views:2073
Avg rating:3.0/5.0
Slides: 40
Provided by: plantsci8
Category:

less

Transcript and Presenter's Notes

Title: Lecture 17 Chapter 9 Marker genes


1
Lecture 17 Chapter 9 Marker genes
  • Neal Stewart

2
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?

3
Using marker genes helps answers
  • Are my plants transgenic?
  • Is the gene expressed?
  • How is my promoter working?

Negative selectable?
Positive selectable?
4
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

5
Figure 9.2
6
Sometimes escapes occur for kanamycin
resistance markers tissue is redvery stressed
Figure 9.3
7
Barnase kills tapetum cells (and pollen)negative
non-conditional selection useful to engineer
male-sterility
Figure 9.7
8
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.

9
Figure 9.4
GUS positive plants and cells
10
Figure 9.8
11
Firefly luciferase produced in tobacco
Figure 9.9
Brought to you by biotechnologist of the day
David Owwas on the cover of Science
12
35SGFP canola
White light UV light in a darkened room
13
(No Transcript)
14
Pollen-tagged GFPsegregating 11
15
GFP-tagged pollen on a bee leg. Hudson et al 2001
Mol Ecol Notes 1321
16
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!

17
http//www.youtube.com/watch?v90wpvSp4l_0feature
related
18
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
19
Horseweed transformation with GFP
Blue Light with GFP Filter
White Light
20
Blue Light with GFP Filter
White Light
21
Transgenic flower cross section
Transgenic versus wild-type flowers
22
(No Transcript)
23
In planta fluorescence
?ex 395 nm
Relative fluorescence
Wavelength (nm)
24
LIFI-laser induced fluorescence imagingfor
stand-off detection of GFP and other flourescence
25
Journal of Fluorescence 15 697-705
26
(No Transcript)
27
A brief FP history
Patterson Nature Biotechnol. (2004) 22 1524
28
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
29
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
30
Species and FP name Ex max nm (Ext
Coef) Em max nm Reference
(103 M-1 cm-1) (Quantum yield )
31
Excitation scanNontransgenic leaf
fluorescencewhy red fluorescence is better than
green
32
With GFP
Why RFP is better less fluorescence noise in
the red
33
More colors in fluorescent proteins discovered
(mostly from coralsthen improved)
http//www.photobiology.info/Zimmer_files/Fig6.png

34
Orange Fluorescent Protein
GFP
Jennifer Hinds
35
Orange Fluorescent Protein (OFP)
36
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 ERprotected environment allows
more accumulation. Haseloff et al 1997 PNAS 94
2122.
37
ER retention dramatically improves OFP
brightness (monomers)
Mann et al. submitted 160th paper?
3x brighter!
38
Big Orange Fluorescent Proteins
Mann et al. submitted.
39
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
Write a Comment
User Comments (0)
About PowerShow.com