Improved FLP Recombinase, FLPe, Efficiently Removes Marker Gene
From Transgene Locus Developed by Cre-lox Mediated Site-specific Gene
Integration in Rice
Presented by
Krysta Haggins
M. Aydm Akbudak, Vibha Srivastava
Marker Genes
http://www.jyi.org/features/ft.php?id=453
Assures that transformation was successful in inserting the gene of interest (GOI)
Two types of markers
Selectable
Scorable or Screening
Green florescent protein (GFP)
Beta glucuronidase (GUS)
http://plants.usda.gov/java/profile?symbol=CASA2&mapType=large&photoID=casa2_001_avd.tif
Common Scorable (Reporter) Genes
Beta glucuronidase (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.
Neal Stewart http://plantsciences.utk.edu/pbg/
GUS positive plants and cells
Neal Stewart http://plantsciences.utk.edu/pbg/
35S:GFP Canola
White light UV light in a darkened room
Neal Stewart http://plantsciences.utk.edu/pbg/
Public concern
Selectable Markers- give antibiotic resistance
Scorable Marker- foreign DNA from other organism
http://www.jyi.org/features/ft.php?id=453
FLP is taken from yeast Saccharomyces cerevisiae
Site-directed recombination
Recombination of sequences from short Flippase Recognition Target (FRT) with the Flippase recombination enzyme (FLP)
DNA Sequence
5'-GAAGTTCCTATTCtctagaaaGTATAGGAACTTC-3'
NPT
FLP Activity
FRT
http://www.umanitoba.ca/afs/plant_science/COURSES/39_768/l08/l08.2.html
Video of Protocol http://www.dnatube.com/video/4224/Cre-Lox-Recombination
Delete marker gene from transgenic rice
Using FLP-FRT recombination system
Plasmid Construct
Target construct= pAK7
Donor construct pRP4
GUS and NPTII
2 FLP recombinases
FLPwt
FLPe
Use Cre-lox
strategy to generate SSI
Use FLP-FRT to excise maker gene
Site specific integration + site specific excision
=
marker free site specific gene integration
8/10 transgenic lines amplified excision footprint
Fig. 5 Efficiency of NPT excision in E lines based on relative copy number using parental lines as a reference
Issues
pAK7 transformation generated an error
Not all lines showed excision and inversion footprints
Alternative Option
Incorporate gene and marker gene into separate plants
Questions and Comments