Zinc finger nuclease-mediated transgene deletion.
A transgene, flanked by zinc finger nuclease (ZFN) cleavage sites, was deleted from a stably transformed plant by crossing it with a second plant expressing a corresponding ZFN gene. A target construct, containing a GUS reporter gene flanked by ZFN cleavage sites, a GFP reporter gene and a PAT selectable marker gene, was transformed into tobacco. Basta-resistant plants were regenerated and screened for GUS and GFP expression. A second construct, containing a ZFN gene driven by the constitutive CsVMV promoter and an HPT selectable marker gene, was also transformed into tobacco. Selected T(0) plants were grown to maturity and allowed to self-pollinate. Homozygous target plants, which expressed GUS and GFP, were crossed with homozygous ZFN plants, which expressed the ZFN gene. Numerous GUS-negative plants were observed among the hybrids with one particular cross displaying approximately 35% GUS-negative plants. Evidence for complete deletion of a 4.3 kb sequence comprising the GUS gene was obtained and sequence confirmed. Co-segregation in F(2) progenies of 'truncated' and 'intact' target sequences with expected reporter gene phenotypes were observed. Since ZFNs can be designed to bind and cleave a wide range of DNA sequences, these results constitute a general strategy for creating targeted gene deletions.
Petolino
Joseph F
JF
Dow AgroSciences, 9330 Zionsville Rd., Indianapolis, IN 46268, USA. jfpetolino@dow.com
Worden
Andrew
A
Curlee
Krisi
K
Connell
James
J
Strange Moynahan
Tonya L
TL
Larsen
Cory
C
Russell
Sean
S
D001483:Base Sequence; D015139:Blotting, Southern; D003433:Crosses, Genetic; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D019076:Transgenes / Q000235:genetics*; D016335:Zinc Fingers
D049452:Green Fluorescent Proteins; D004720:Endonucleases; D005966:Glucuronidase
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