Delivery of Multiple Transgenes to Plant Cells

Dafny-Yelin, Mery; Tzfira, Tzvi

doi:10.1104/pp.107.106104

Cited by 127 publications

(80 citation statements)

References 73 publications

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“…More specifically, the assembly of multigene arrays by traditional cloning or recombination systems typically leaves repetitive sequences within the DNA molecule (Dafny-Yelin and Tzfira, 2007), which can include recombination sites, traces of multicloning sites, and even reparative promoters and terminator regions: these may hinder the use of HR-mediated recombination methods for their targeting. Our approach may facilitate the engineering of multigene arrays by using pairs of ZFNs, which are designed to flank the target genes and to be used in conjunction with matching donor DNA molecules for NHEJ-mediated replacement.…”

Section: Discussionmentioning

confidence: 99%

Nonhomologous End Joining-Mediated Gene Replacement in Plant Cells

2013

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Stimulation of the homologous recombination DNA-repair pathway via the induction of genomic double-strand breaks (DSBs) by zinc finger nucleases (ZFNs) has been deployed for gene replacement in plant cells. Nonhomologous end joining (NHEJ)-mediated repair of DSBs, on the other hand, has been utilized for the induction of site-specific mutagenesis in plants. Since NHEJ is the dominant DSB repair pathway and can also lead to the capture of foreign DNA molecules, we suggest that it can also be deployed for gene replacement. An acceptor DNA molecule in which a green fluorescent protein (GFP) coding sequence (gfp) was flanked by ZFN recognition sequences was used to produce transgenic target plants. A donor DNA molecule in which a promoterless hygromycin B phosphotransferase-encoding gene (hpt) was flanked by ZFN recognition sequences was constructed. The donor DNA molecule and ZFN expression cassette were delivered into target plants. ZFN-mediated sitespecific mutagenesis and complete removal of the GFP coding sequence resulted in the recovery of hygromycin-resistant plants that no longer expressed GFP and in which the hpt gene was unlinked to the acceptor DNA. More importantly, ZFNmediated digestion of both donor and acceptor DNA molecules resulted in NHEJ-mediated replacement of the gfp with hpt and recovery of hygromycin-resistant plants that no longer expressed GFP and in which the hpt gene was physically linked to the acceptor DNA. Sequence and phenotypical analyses, and transmission of the replacement events to the next generation, confirmed the stability of the NHEJ-induced gene exchange, suggesting its use as a novel method for transgene replacement and gene stacking in plants.

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Section: Discussionmentioning

confidence: 99%

Nonhomologous End Joining-Mediated Gene Replacement in Plant Cells

2013

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“…Direct DNA transfer with separate vectors usually results in transgene integration at a random single locus, in the form of a multigene array (22,23) containing any number of transgenes from 1 to n, with the distribution within the transgenic population tending to describe a normal curve as would be expected from random sampling (24). Input transgenes once integrated remain linked and do not segregate in subsequent generation as is the norm for direct DNA transfer of multiple transgenes irrespective of whether these are on 1 cointegrate vector or independent plasmids (cotransformation) (19)(20)(21)(22)(23)(24). Plants carrying 1-5 transgenes in a roughly normal distribution could be identified from colored seed phenotypes arising from the accumulation of specific carotenoids, and these could be correlated with mRNA and metabolite profiles to identify and quantify the specific carotenoid molecules present in the endosperm.…”

Section: Discussionmentioning

confidence: 99%

“…This in turn requires strategies to introduce multiple transgenes into plants and ensure their coordinated expression over many generations (12). Expressing multiple transgenes stably is one of the most significant hurdles currently limiting progress in plant molecular biology (14,19), because the chances of failure for at least 1 of the transgenes increases with the number of genes introduced, requiring the generation of very large populations to ensure complete pathway reconstruction. Alternative approaches such as individual transformation followed by crossing to ''stack'' transgenes are unworkable for large numbers of transgenes because of the time taken to stack all transgenes in 1 line and the risk of segregation of unlinked genes in later generations.…”

Section: Discussionmentioning

confidence: 99%

Combinatorial genetic transformation generates a library of metabolic phenotypes for the carotenoid pathway in maize

Zhu

Naqvi

Breitenbach

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

332

263

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Combinatorial nuclear transformation is a novel method for the rapid production of multiplex-transgenic plants, which we have used to dissect and modify a complex metabolic pathway. To demonstrate the principle, we transferred 5 carotenogenic genes controlled by different endosperm-specific promoters into a white maize variety deficient for endosperm carotenoid synthesis. We recovered a diverse population of transgenic plants expressing different enzyme combinations and showing distinct metabolic phenotypes that allowed us to identify and complement rate-limiting steps in the pathway and to demonstrate competition between ␤-carotene hydroxylase and bacterial ␤-carotene ketolase for substrates in 4 sequential steps of the extended pathway. Importantly, this process allowed us to generate plants with extraordinary levels of ␤-carotene and other carotenoids, including complex mixtures of hydroxycarotenoids and ketocarotenoids. Combinatorial transformation is a versatile approach that could be used to modify any metabolic pathway and pathways controlling other biochemical, physiological, or developmental processes.induced mutation ͉ metabolic engineering ͉ transgenic plant ͉ provitamin A

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“…Such experiments in turn require strategies to introduce multiple transgenes into plants and ensure their coordinated expression over many generations (6). The stable expression of multiple transgenes is one of the most significant hurdles currently limiting progress in plant molecular biology (11,12), because the chances of failure for at least 1 of the transgenes increases with the number of genes introduced, requiring the generation of very large populations to ensure complete pathway reconstruction. Alternative approaches, such as individual transformation fol-lowed by crossing to ''stack'' transgenes, are unworkable for large numbers of genes because of the time taken to stack all transgenes in 1 line and the likelihood that unlinked genes will segregate in later generations.…”

Section: Resultsmentioning

confidence: 99%

Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways

Naqvi

Zhu

Farré

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

460

318

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Vitamin deficiency affects up to 50% of the world's population, disproportionately impacting on developing countries where populations endure monotonous, cereal-rich diets. Transgenic plants offer an effective way to increase the vitamin content of staple crops, but thus far it has only been possible to enhance individual vitamins. We created elite inbred South African transgenic corn plants in which the levels of 3 vitamins were increased specifically in the endosperm through the simultaneous modification of 3 separate metabolic pathways. The transgenic kernels contained 169-fold the normal amount of ␤-carotene, 6-fold the normal amount of ascorbate, and double the normal amount of folate. Levels of engineered vitamins remained stable at least through to the T3 homozygous generation. This achievement, which vastly exceeds any realized thus far by conventional breeding alone, opens the way for the development of nutritionally complete cereals to benefit the world's poorest people.folic acid ͉ metabolic engineering ͉ transgenic maize ͉ vitamin A fortification ͉ vitamin C

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Delivery of Multiple Transgenes to Plant Cells

Cited by 127 publications

References 73 publications

Nonhomologous End Joining-Mediated Gene Replacement in Plant Cells

Nonhomologous End Joining-Mediated Gene Replacement in Plant Cells

Combinatorial genetic transformation generates a library of metabolic phenotypes for the carotenoid pathway in maize

Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways

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