Zinc finger nuclease-mediated transgene deletion

• Published in: Plant molecular biology 2010-08, Vol.73 (6), p.617-628
• Main Authors: Petolino, Joseph F, Worden, Andrew, Curlee, Krisi, Connell, James, Strange Moynahan, Tonya L, Larsen, Cory, Russell, Sean
• Format: Article
• Language: English
• Subjects: Base Sequence; Biochemistry; Biomedical and Life Sciences; Blotting, Southern; Cell growth; Crosses, Genetic; Endonucleases - genetics; Endonucleases - metabolism; Gene expression; Glucuronidase - genetics; Glucuronidase - metabolism; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Hybrids; Life Sciences; Marker gene removal; molbio; Molecular Sequence Data; Mutagenesis; Nicotiana - genetics; Nicotiana - metabolism; Plant biology; Plant Pathology; Plant Sciences; Plants, Genetically Modified - genetics; Plants, Genetically Modified - metabolism; Proteins; Sequence Deletion - genetics; Sequence Homology, Nucleic Acid; site-directed mutagenesis; Transgene deletion; Transgenes - genetics; Zinc Fingers
• ISSN: 0167-4412; 1573-5028
• DOI: 10.1007/s11103-010-9641-4

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 T₀ 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 ~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 F₂ 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.