A modular gene targeting system for sequential transgene stacking in plants.
A modular, selection-based method was developed for site-specific integration of transgenes into a genomic locus to create multigene stacks. High-frequency gene targeting was obtained using zinc finger nuclease (ZFN)-mediated double-strand break (DSB) formation at a pre-defined target genomic location using a unique intron directly downstream of a promoter driving a selectable marker gene to facilitate homology between target and donor sequences. In this system, only insertion into the target locus leads to a functional selectable marker, and regeneration from random insertions of the promoterless donor construct are reduced on selection media. A new stack of transgenes can potentially be loaded with each successive cycle of gene targeting by exchanging the selectable marker gene using the intron homology. This system was tested in maize using the pat selectable marker gene, whereby up to 30% of the plants regenerated on Bialaphos-containing medium were observed to have the donor construct integrated into the target locus. Unlike previous gene targeting methods that utilize defective or partial genes for selecting targeted events, the present method exchanges fully functional genes with every cycle of targeting, thereby allowing the recycling of selectable marker genes, hypothetically for multiple generations of gene targeting.
Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA. Electronic address: Skumar9@dow.com.
Sandeep
S
Kumar
Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA.
Diaa
D
AlAbed
Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA.
Andrew
A
Worden
Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA.
Stephen
S
Novak
Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA.
Huixia
H
Wu
Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA.
Carla
C
Ausmus
Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA.
Margaret
M
Beck
Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA.
Heather
H
Robinson
Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA.
Tatyana
T
Minnicks
Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA.
Daren
D
Hemingway
Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA.
Ryan
R
Lee
Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA.
Nicole
N
Skaggs
Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA.
Lizhen
L
Wang
Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA.
Pradeep
P
Marri
Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA.
Manju
M
Gupta
D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D060054:Agrobacterium / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors; D016254:Mutagenesis, Insertional / Q000379:methods*; D030821:Plants, Genetically Modified / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D014170:Transformation, Genetic; D019076:Transgenes; D003313:Zea mays / Q000235:genetics
D000217:Acyltransferases
Corn; Gene targeting; Plants; Transgene stacking; Zinc finger nuclease