Targeted mutagenesis in the progeny of maize transgenic plants.
We have demonstrated that targeted mutagenesis can be accomplished in maize plants by excision, activation, and subsequent elimination of an endonuclease in the progeny of genetic crosses. The yeast FLP/FRT site-specific recombination system was used to excise and transiently activate the previously integrated yeast I-SceI homing endonuclease in maize zygotes and/or developing embryos. An artificial I-SceI recognition sequence integrated into genomic DNA was analyzed for mutations to indicate the I-SceI endonuclease activity. Targeted mutagenesis of the I-SceI site occurred in about 1% of analyzed F1 plants. Short deletions centered on the I-SceI-produced double-strand break were the predominant genetic lesions observed in the F1 plants. The I-SceI expression cassette was not detected in the mutant F1 plants and their progeny. However, the original mutations were faithfully transmitted to the next generation indicating that the mutations occurred early during the F1 plant development. The procedure offers simultaneous production of double-strand breaks and delivery of DNA template combined with a large number of progeny plants for future gene targeting experiments.
Yang
Meizhu
M
Pioneer Hi-Bred International, A DuPont Business, Research Center, 7300 NW 62nd Avenue, Johnston, IA 50131-1004, USA.
Djukanovic
Vesna
V
Stagg
Jessica
J
Lenderts
Brian
B
Bidney
Dennis
D
Falco
S Carl
SC
Lyznik
L Alexander
LA
D001483:Base Sequence; D003433:Crosses, Genetic; D053903:DNA Breaks, Double-Stranded; D015342:DNA Probes / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D018390:Gene Targeting; D005800:Genes, Fungal; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016296:Mutagenesis; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D003313:Zea mays / Q000196:embryology / Q000235:genetics* / Q000378:metabolism
D015342:DNA Probes; D004269:DNA, Bacterial; D018744:DNA, Plant; D004274:DNA, Recombinant; D029701:Saccharomyces cerevisiae Proteins; C036483:T-DNA; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific