RESUMO
Targeted transgene integration in plants remains a significant technical challenge for both basic and applied research. Here it is reported that designed zinc finger nucleases (ZFNs) can drive site-directed DNA integration into transgenic and native gene loci. A dimer of designed 4-finger ZFNs enabled intra-chromosomal reconstitution of a disabled gfp reporter gene and site-specific transgene integration into chromosomal reporter loci following co-transformation of tobacco cell cultures with a donor construct comprised of sequences necessary to complement a non-functional pat herbicide resistance gene. In addition, a yeast-based assay was used to identify ZFNs capable of cleaving a native endochitinase gene. Agrobacterium delivery of a Ti plasmid harboring both the ZFNs and a donor DNA construct comprising a pat herbicide resistance gene cassette flanked by short stretches of homology to the endochitinase locus yielded up to 10% targeted, homology-directed transgene integration precisely into the ZFN cleavage site. Given that ZFNs can be designed to recognize a wide range of target sequences, these data point toward a novel approach for targeted gene addition, replacement and trait stacking in plants.
Assuntos
Endonucleases/metabolismo , Transgenes/genética , Dedos de Zinco/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Quitinases/genética , Endonucleases/genética , Glucuronidase/genética , Glucuronidase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Recombinação Genética , Nicotiana/citologia , Nicotiana/genética , Nicotiana/metabolismo , Transfecção/métodosRESUMO
Agricultural biotechnology companies have been asked to provide intact transgenic seed to regulatory agencies as reference materials for evaluating transgene and protein detection methods (PCR and immunoassay). Due to intellectual-property and product-stewardship considerations, submission of devitalized seed prior to regulatory approval is preferable in any given country. Commonly used devitalization procedures, such as heating or autoclaving, degrade the protein and/or DNA rendering the seed unfit as a reference material for these tests. A novel method for devitalizing seed was developed that involves hydration, freezing in liquid nitrogen, and lyophilization. The devitalization method described here was found to preserve the transgenic DNA and protein in cotton (Gossypium hirsutum) and maize (Zea mays) seed allowing its use as a reference material for evaluating detection methods.
Assuntos
DNA de Plantas/química , DNA de Plantas/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Sementes/química , Sementes/genética , Biotecnologia , Southern Blotting , Western Blotting , Criopreservação , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Liofilização , Engenharia Genética/legislação & jurisprudência , Engenharia Genética/normas , Germinação , Gossypium/química , Gossypium/genética , Preservação Biológica/métodos , Proteínas Recombinantes/genética , Padrões de Referência , Sementes/crescimento & desenvolvimento , Zea mays/química , Zea mays/genéticaRESUMO
The underlying redox-sensitive mechanisms that regulate hepatocyte expression of inducible nitric-oxide synthase (iNOS) and its antioxidant functions are largely unknown. We have demonstrated previously that oxidative stress induced by benzenetriol-mediated superoxide production increases interleukin-1beta-induced iNOS protein synthesis, steady state iNOS mRNA expression, NO production, iNOS gene transcription, and trans-activation of the iNOS promoter in primary cultures of rat hepatocytes. In this study, we extend these studies by establishing the sequence specificity and binding of nuclear protein to the previously described 15-base cis-regulatory element of the rat hepatocyte iNOS promoter, isolating and identifying the cis-regulatory element transcription factor as hepatocyte nuclear factor-4alpha (HNF-4alpha), and confirming the functional role of HNF-4alpha in mediating redox-sensitive iNOS promoter trans-activation. In addition, we demonstrate that binding of HNF-4alpha to the transcriptional coactivator, PC4, in the presence of oxidative stress and interleukin-1beta stimulation is essential for increased iNOS promoter activity in this setting. Our results indicate that HNF-4alpha is the transcription factor that mediates redox regulation of hepatocyte iNOS gene transcription.
Assuntos
Proteínas de Ligação a DNA , Regulação Enzimológica da Expressão Gênica , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Oxirredução , Fosfoproteínas/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Sítios de Ligação , Western Blotting , Células Cultivadas , Fator 4 Nuclear de Hepatócito , Hepatócitos/metabolismo , Proteínas Imediatamente Precoces , Masculino , Proteínas de Membrana , Mutagênese Sítio-Dirigida , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II , Estresse Oxidativo , Testes de Precipitina , Regiões Promotoras Genéticas , Ligação Proteica , Ratos , Ratos Endogâmicos Lew , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Ativação Transcricional , Transfecção , Raios UltravioletaRESUMO
The production of nitric oxide (NO) in response to endotoxin (LPS)-stimulation is associated with a myriad of NO-dependent regulatory functions. The study of NO-dependent genetic programs in the setting of endotoxin stimulation can be aided by determination of genes whose transcription is upregulated in the presence of NO and LPS. Using subtractive suppression hybridization analysis, we demonstrate that ANA-1 murine macrophages produce the CC class chemokines, monocyte chemoattractant protein-1 (JE/MCP-1) and macrophage inflammatory protein-related protein-1 (C10/MRP-1), in response to LPS-mediated nitric oxide (NO) production. MCP-1 and MRP-1 gene transcription and protein synthesis are upregulated in the setting of LPS-induced NO synthesis. NO alone is necessary but insufficient for induction of chemokine protein expression; an additional LPS-dependent signaling pathway is also required. This study suggests a novel mechanism by which NO induces chemokine expression and regulates the host inflammatory response to endotoxin.