Improved FLP Recombinase, FLPe, Efficiently Removes Marker … · Improved FLP Recombinase, FLPe,...

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