Endosperm-Specific Co-Expression of Recombinant Soybean Ferritin and Aspergillus Phytase in Maize Results in Significant Increases in the Levels of Bioavailable Iron

Jeon

et al. 2009

314

254

The most widespread dietary problem in the world is mineral deficiency. We used the nicotianamine synthase (NAS) gene to increase mineral contents in rice grains. Nicotianamine (NA) is a chelator of metals and a key component of metal homeostasis. We isolated activation-tagged mutant lines in which expression of a rice NAS gene, OsNAS3, was increased by introducing 35S enhancer elements. Shoots and roots of the OsNAS3 activationtagged plants (OsNAS3-D1) accumulated more Fe and Zn. Seeds from our OsNAS3-D1 plants grown on a paddy field contained elevated amounts of Fe (2.9-fold), Zn (2.2-fold), and Cu (1.7-fold). The NA level was increased 9.6-fold in OsNAS3-D1 seeds. Analysis by size exclusion chromatography coupled with inductively coupled plasma mass spectroscopy showed that WT and Os-NAS3-D1 seeds contained equal amounts of Fe bound to IP6, whereas OsNAS3-D1 had 7-fold more Fe bound to a low molecular mass, which was likely NA. Furthermore, this activation led to increased tolerance to Fe and Zn deficiencies and to excess metal (Zn, Cu, and Ni) toxicities. In contrast, disruption of OsNAS3 caused an opposite phenotype. To test the bioavailability of Fe, we fed anemic mice with either engineered or WT seeds for 4 weeks and measured their concentrations of hemoglobin and hematocrit. Mice fed with engineered seeds recovered to normal levels of hemoglobin and hematocrit within 2 weeks, whereas those that ate WT seeds remained anemic. Our results suggest that an increase in bioavailable mineral content in rice grains can be achieved by enhancing NAS expression. activation tagging ͉ bioavailability ͉ hemoglobin ͉ metal homeostasis

Section: Discussionmentioning

confidence: 99%

Iron fortification of rice seeds through activation of the nicotianamine synthase gene

Jeon

et al. 2009

314

254

“…Thirteen-dayold immature zygotic embryos were bombarded with 10 mg of gold particles coated with the carotenogenic constructs and the selectable marker bar (41) at a molar ratio of 3:1 as reported (42) and returned to osmoticum medium for 12 h before selection. Bombarded callus was selected on phosphinothricinsupplemented medium as described (43,44), and transgenic plantlets were regenerated and hardened off in soil. Seventy independent events were selected for further analysis and either pollinated with nontransformed white maize (M37W) pollen or self-pollinated to produce T1 seeds.…”

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

Self Cite

328

263

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

“…Immature zygotic embryos of the South African elite white maize genotype M37W were transformed by particle bombardment at 10 -14 days after pollination as described in ref. 26. Bombarded embryos were transferred in the dark every 2 weeks to fresh N6-based medium containing 3 mg/liter phosphinothricin (PPT).…”

Section: Methodsmentioning

confidence: 99%

“…The plants were either selfed or crossed with wild-type M37W, and Southern blot analysis was used to confirm transgene integration and stability. Most of the transgenic plants contained all three input transgenes linked within a single genetic locus, as anticipated (25,26). Single genetic loci are beneficial, because they favor high-level and stable transgene expression over future generations (27)(28)(29)(30)(31).…”

mentioning

confidence: 99%

Cost-effective production of a vaginal protein microbicide to prevent HIV transmission

Ramessar

Rademacher

Sack

et al. 2008

Self Cite

148

124

A series of small-molecule microbicides has been developed for vaginal delivery to prevent heterosexual HIV transmission, but results from human clinical trials have been disappointing. Proteinbased microbicides, such as HIV-specific monoclonal antibodies, have been considered as an alternative approach. Despite their promising safety profile and efficacy, the major drawback of such molecules is the economy of large-scale production in mammalian cells, the current system of choice. Here, we show that an alternative biomanufacturing platform is now available for one of the most promising anti-HIV antibodies (2G12). Our data show that the HIV-neutralization capability of the antibody is equal to or superior to that of the same antibody produced in CHO cells. We conclude that this protein production system may provide a means to achieve microbicide ingredient manufacture at costs that would allow product introduction and manufacture in the developing world.