Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
An YQ, McDowell JM, Huang S, McKinney EC, Chambliss S, Meagher RB (1996) Strong constitutive expression of the Arabidopsis ACT2/ACT8 actin subclass in vegetative tissues. Plant J 10:107–121
Cai CQ, Doyon Y, Ainley WM, Miller JC, DeKelver RC, Moehle EA, Rock JM, Lee YL, Garrison R, Schulenberg L, Blue R, Worden A, Baker L, Faraji F, Zhang L, Holmes MC, Rebar E, Collingwood TN, Rubin-Wilson B, Gregory PD, Urnov FD, Petolino JF (2009) Targeted transgene integration in plant cells using designed zinc finger nucleases. Plant Mol Biol 69:699–709
Callis J, Raasch JA, Vierstra RD (1990) Ubiquitin extension proteins of Arabidopsis thaliana. Structure, localization and expression of their promoters in transgenic tobacco. J Biol Chem 265:12486–12493
Callis J, Carpenter T, Sun CW, Vierstra RD (1995) Structure and evolution of genes encoding polyubiquitin and ubiquitin-like proteins Arabidopsis thaliana ecotype Columbia. Genetics 139:921–939
Dale EC, Ow DW (1991) Gene transfer with subsequent removal of the selection gene from the host genome. Proc Natl Acad Sci USA 88:10558–10562
de Pater S, Neuteboom LW, Pinas JE, Hooykaas PJJ, van der Zaal BJ (2009) ZFN-induced mutagenesis and gene-targeting in Arabidopsis through Agrobacterium-mediated floral dip transformation. Plant Biotechnol J 7:821–835
DeArruda M (2002) Invader technology for DNA and RNA analysis: principles and applications. Expert Rev Mol Diagn 2:487–496
Evdokimov AG, Pokross ME, Egorov NS, Zaraisky AG, Yampolsky IV, Merzlyak EM, Shkoporov AN, Sander I, Lukyanov KA, Chudakov DM (2006) Structural basis for the fast maturation of Arthropoda green fluorescent protein. EMBO Rep 7:1006–1012
Fang J, Qian JJ, Yi S, Harding TC, Tu GH, VanRoey M, Jooss K (2005) Stable antibody expression at therapeutic levels using the 2A peptide. Nat Biotechnol 23:584–590
Gelvin SG (1987) TR-based sub-Ti plasmids. EP Patent 222493
Hare PD, Chua NH (2002) Excision of selectable marker genes from transgenic plants. Nature Biotechnol 20:575–580
Hoa TTC, Bong BB, Huq E, Hodges TK (2002) Cre/lox site-specific recombination controls the excision of a transgene from the rice genome. Theor Appl Genet 104:518–525
Hoess RH, Wierzbicki A, Abremski K (1986) The role of the loxP spacer region in P1 site-specific recombination. Nucleic Acids Res 14:2287–2300
Hohn B, Levy AA, Puchta H (2001) Elimination of selection markers from transgenic plants. Curr Opin Biotechnol 12:139–143
Huang ML, Cangelosi GA, Halperin W, Nester EW (1990) A chromosomal Agrobacterium gene required for effective plant signal transduction. J Bacteriol 172:1814–1822
Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907
Kim YG, Cha J, Chandrasegaran S (1996) Hybrid restriction enzymes: zinc finger fusions to FokI cleavage domain. Proc Natl Acad Sci USA 93:1156–1160
Komari T, Hiei Y, Saito Y, Murai N, Kumashiro T (1996) Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J 10:165–174
Kopertekh L, Juetner G, Schiemann J (2004) PVX-Cre-mediated marker gene elimination from transgenic plants. Plant Mol Biol 55:491–500
Lee HJ, Kim E, Kim JS (2010) Targeted chromosomal deletions in human cells using zinc finger nucleases. Genome Res 20:81–89
Lie YS, Petropoulos CJ (1998) Advances in quantitative PCR technology. Curr Opin Biotechnol 9:43–48
Lloyd A, Plaisier CL, Carroll D, Drews GN (2005) Targeted mutagenesis using zinc finger nucleases in Arabidopsis. Proc Natl Acad Sci USA 102:2232–2237
Looney MC, Moran LS, Jack WE, Feehery RG, Benner JS, Slatko BE, Wilson GG (1989) Nucleotide sequence of the Fok1 restriction-modification system: separate strand-specificity domains in the methyltransferase. Gene 80:193–208
Luo K, Duan H, Zhao D, Zheng X, Deng W, Chen Y, Stewart CN, McAvoy R, Jiang X, Wu Y, He A, Pei Y, Li Y (2007) ‘GM-gene-deleter’: fused loxP-FRT recognition sequences dramatically improve the efficiency of FLP or CRE recombinase on transgene excision from pollen and seed of tobacco plants. Plant Biotechnol J 5:263–274
Maddaloni M, di Fonzo N, Hartings H, Lazzaroni N, Salamini F, Thompson R, Motto M (1989) The sequence of the zein regulatory gene opaque-2 (O2) of Zea mays. Nucleic Acids Res 17:7532
Maeder ML, Thibodeau-Beganny S, Osiak A, Wright DA, Anthony RM, Eichtinger M, Jiang T, Loley JE, Winfrey RJ, Townsend JA, Unger-Wallace E, Sander JD, Muller-Lerch F, Fu F, Pearlberg J, Gobel C, Dassie JP, Pruett-Miller SM, Porteus MH, Sgroi DC, Iafrate AJ, Dobbs D, McCray PB, Cathomen T, Voytas DF, Joung JK (2008) Rapid “open-source” engineering of customized zinc-finger nucleaes for highly efficient gene modification. Mol Cell 31:294–301
Miki B, McHugh S (2004) Selectable marker genes in transgenic plants: applications, alternatives, biosafety. J Biotechnol 107:193–232
Odell J, Caimi P, Sauer B, Russell S (1990) Site-directed recombination in the genome of transgenic tobacco. Mol Gen Genet 223:369–378
Perez EE, Wang J, Miller JC, Jouvenot Y, Kim KA, Liu O, Wang N, Lee G, Bartsevich VV, Lee YL, Guschin DY, Rupniewski I, Waite AJ, Carpenito C, Carroll RG, Orange JS, Urnov FD, Rebar EJ, Andlo D, Gregory PD, Riley JL, Holmes MC, June CH (2008) Establishment of HIV-1 resistance in CD4 + T cells by genome editing using zinc finger nucleases. Nature Biotechnol 26:808–816
Russell SH, Hoopes JL, Odell JT (1992) Directed excision of a transgene from the plant genome. Mol Gen Genet 234:49–59
Shukla V, Doyon Y, Miller J, DeKelver RC, Moehle EA, Worden SE, Mitchell JC, Arnold NL, Gopalan S, Meng X, Choi VM, Rock JM, Wu YY, Katibah GE, Zhifang G, McCaskill D, Simpson MA, Blakeslee B, Greenwalt SA, Butler HJ, Hinkley SJ, Zhang L, Rebar EJ, Gregory PD, Urnov FD (2009) Precise genome modification in the crop species Zea mays using zinc-finger nucleases. Nature 459:437–441
Siebert R, Puchta H (2002) Efficient repair of genomic double-strand breaks by homologous recombination between directly repeated sequences in the plant genome. Plant Cell 14:1121–1131
Song HS, Lai FM, Roche CE, Brown JA (2008) Method of excising a nucleic acid sequence from a plant genome. WO 2008/145731
Thompson W, Hall G, Spiker S, Allen G (1997) A nuclear scaffold attachment region wihich increases gene expression. WO 1997/27207
Tovkach A, Zeevi V, Tzfira T (2008) A toolbox and procedural notes for characterizing novel zinc finger nucleases for genome editing in plant cells. Plant J 57:747–757
Townsend JA, Wright DA, Winfrey RJ, Fu F, Maeder ML, Joung JK, Voytas DF (2009) High-frequency modification of plant genes using engineered zinc-finger nucleases. Nature 459:442–445
Verdaguer B, de Kochko A, Beachy RN, Fauquet C (1996) Isolation and expression in transgenic tobacco and rice plants of the cassava vein mosaic virus (CVMV) promoter. Plant Mol Biol 31:1129–1139
Waldron C, Murphy E, Roberts J, Gustafson G, Armour S, Malcolm S (1985) Resistance to hygromycin B: a new marker for plant transformation studies. Plant Mol Biol 5:103–108
Wohlleben W, Arnold W, Broer I, Hillemann D, Strauch E, Puehler A (1988) Nucleotide sequence of the phosphinothricin N-acetyl-transferase gene from Streptomyces viridochromogenes Tu494 and its expression in Nicotiana tobacum. Gene 70:25–38
Wright DA, Townsend JA, Winfrey RJ Jr, Irwin PA, Rajagopal J, Lonosky PM, Hall BD, Jondle MD, Voytas DF (2005) High frequency homologous recombination in plants mediated by zinc-finger nucleases. Plant J 44:693–705
Zhang W, Subbarao S, Addae P, Shen A, Armstrong C, Peschke V, Gilbertson L (2003) Cre/lox mediated marker gene excision in transgenic maize (Zea mays L.) plants. Theor Appl Genet 107:1157–1168
Zuo J, Niu QW, Moller SG, Chua NH (2001) Chemical-regulated site-specific DNA excision in transgenic plants. Nat Biotechnol 19:157–161
Acknowledgments
Thanks to Fyodor Urnov and the staff at Sangamo BioSciences for providing the CCR5 binding sites and corresponsing ZFN and to Rebekah Bark, Sushma Ram and Greg Schulenberg for assistance with progeny GUS analysis, qRT-PCR and Southern blots/PCR, respectively.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Petolino, J.F., Worden, A., Curlee, K. et al. Zinc finger nuclease-mediated transgene deletion. Plant Mol Biol 73, 617–628 (2010). https://doi.org/10.1007/s11103-010-9641-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11103-010-9641-4