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1.
Methods Mol Biol ; 1864: 117-130, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30415333

RESUMEN

Biolistic transformation of wheat is one of the most commonly used methods for gene function study and trait discovery. It has been widely adapted as a fundamental platform to generate wheat plants with new traits and has become a powerful tool for facilitating the crop improvement. In this chapter, we present a complete and straightforward protocol for wheat transformation via biolistic bombardment system. Although wheat is still one of the hardest plant species to transform, this protocol offers an optimized and efficient system to produce transgenic plants. To demonstrate the application of this protocol, in this chapter we describe an example of obtaining transgenic wheat by the co-bombardment of two plasmids, containing a green fluorescent protein gene and a glufosinate herbicide selection gene, respectively. In addition, procedures for the screening and testing of putative transgenic plants are described. This protocol has been successfully applied to generate stable transgenic bread wheat (Triticum aestivum) in both spring and winter varieties.


Asunto(s)
Biolística/métodos , Plantas Modificadas Genéticamente/genética , Transformación Genética , Triticum/genética , Biolística/instrumentación , ADN de Plantas/administración & dosificación , ADN de Plantas/genética
2.
PLoS One ; 6(11): e27177, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-22132091

RESUMEN

We assessed the effect of short-term feeding of genetically modified (GM: Bt MON810) maize on immune responses and growth in weanling pigs and determined the fate of the transgenic DNA and protein in-vivo. Pigs were fed a diet containing 38.9% GM or non-GM isogenic parent line maize for 31 days. We observed that IL-12 and IFNγ production from mitogenic stimulated peripheral blood mononuclear cells decreased (P<0.10) following 31 days of GM maize exposure. While Cry1Ab-specific IgG and IgA were not detected in the plasma of GM maize-fed pigs, the detection of the cry1Ab gene and protein was limited to the gastrointestinal digesta and was not found in the kidneys, liver, spleen, muscle, heart or blood. Feeding GM maize to weanling pigs had no effect on growth performance or body weight. IL-6 and IL-4 production from isolated splenocytes were increased (P<0.05) in response to feeding GM maize while the proportion of CD4(+) T cells in the spleen decreased. In the ileum, the proportion of B cells and macrophages decreased while the proportion of CD4(+) T cells increased in GM maize-fed pigs. IL-8 and IL-4 production from isolated intraepithelial and lamina propria lymphocytes were also increased (P<0.05) in response to feeding GM maize. In conclusion, there was no evidence of cry1Ab gene or protein translocation to the organs and blood of weaning pigs. The growth of pigs was not affected by feeding GM maize. Alterations in immune responses were detected; however, their biologic relevance is questionable.


Asunto(s)
Bacillus thuringiensis/fisiología , ADN de Plantas/administración & dosificación , Porcinos/crecimiento & desarrollo , Porcinos/inmunología , Transgenes/genética , Zea mays/genética , Zea mays/microbiología , Aclimatación , Administración Oral , Animales , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/sangre , Proteínas Bacterianas/metabolismo , Peso Corporal , ADN de Plantas/genética , Dieta , Endotoxinas/sangre , Endotoxinas/metabolismo , Conducta Alimentaria , Alimentos Modificados Genéticamente , Genes de Plantas/genética , Proteínas Hemolisinas/sangre , Proteínas Hemolisinas/metabolismo , Inmunidad/inmunología , Leucocitos/metabolismo , Micotoxinas/análisis , Especificidad de Órganos , Residuos de Plaguicidas/análisis , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa , Factores de Tiempo
4.
Nat Biotechnol ; 22(2): 204-9, 2004 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-14730317

RESUMEN

The inclusion of genetically modified (GM) plants in the human diet has raised concerns about the possible transfer of transgenes from GM plants to intestinal microflora and enterocytes. The persistence in the human gut of DNA from dietary GM plants is unknown. Here we study the survival of the transgene epsps from GM soya in the small intestine of human ileostomists (i.e., individuals in which the terminal ileum is resected and digesta are diverted from the body via a stoma to a colostomy bag). The amount of transgene that survived passage through the small bowel varied among individuals, with a maximum of 3.7% recovered at the stoma of one individual. The transgene did not survive passage through the intact gastrointestinal tract of human subjects fed GM soya. Three of seven ileostomists showed evidence of low-frequency gene transfer from GM soya to the microflora of the small bowel before their involvement in these experiments. As this low level of epsps in the intestinal microflora did not increase after consumption of the meal containing GM soya, we conclude that gene transfer did not occur during the feeding experiment.


Asunto(s)
Daño del ADN , ADN de Plantas/química , ADN de Plantas/metabolismo , Alimentos Modificados Genéticamente , Intestino Delgado/metabolismo , Plantas Modificadas Genéticamente/química , Secuencia de Bases , ADN de Plantas/administración & dosificación , ADN de Plantas/análisis , Humanos , Intestino Delgado/cirugía , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente/microbiología , Análisis de Secuencia de ADN , Transgenes
5.
Vaccine ; 20(21-22): 2764-71, 2002 Jun 21.
Artículo en Inglés | MEDLINE | ID: mdl-12034103

RESUMEN

Genomic DNA sequences (bacteria, insect, nematodes and molluscs) or synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs (CpG-DNA/ODN) are regarded as promising candidates for new medical adjuvants for their ability to stimulate the mammalian immune system and enhance immune responses to specific antigens. Here, we first report the immunostimulatory activity of total genomic DNA from two plants, Brassica chinensis L. and Zea may, the CpG methylation status of which is incomplete compared with E. coli DNA. These plant DNA can activate B cells to proliferate. Plant DNA promotes secretion of IL-12, and increases expression of MHC and costimulatory molecules by bone marrow-derived dendritic cells (BMDC). Plant DNA can also enhance antigen presentation capacity of BMDC and macrophages. When administrated in vivo, plant DNA can inhibit tumor growth in situ or metastasis in tumor-bearing mice. The immunostimulatory activity of plant DNA could be abolished by methylation. Our data showed that plant DNA can activate antigen-presenting cells (APC) including DC, macrophages and B cells, indicating that plant DNA is a new kind of potential adjuvant. Therefore, we conclude that plant DNA is another natural source of CpG-DNA, and that green plants may provide abundant resources for this potential medical adjuvant.


Asunto(s)
Adyuvantes Inmunológicos , Células Presentadoras de Antígenos/inmunología , Brassica/genética , ADN de Plantas/inmunología , Animales , Linfocitos B/inmunología , Vacunas contra el Cáncer/inmunología , Metilación de ADN , ADN de Plantas/administración & dosificación , Femenino , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Oligodesoxirribonucleótidos/inmunología , Oligodesoxirribonucleótidos/farmacología , Linfocitos T Citotóxicos/inmunología , Células Tumorales Cultivadas , Vacunas de ADN/inmunología
7.
Nat Med ; 5(4): 387-91, 1999 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-10202926

RESUMEN

Food allergy is a common and often fatal disease with no effective treatment. We describe here a new immunoprophylactic strategy using oral allergen-gene immunization to modulate peanut antigen-induced murine anaphylactic responses. Oral administration of DNA nanoparticles synthesized by complexing plasmid DNA with chitosan, a natural biocompatible polysaccharide, resulted in transduced gene expression in the intestinal epithelium. Mice receiving nanoparticles containing a dominant peanut allergen gene (pCMVArah2) produced secretory IgA and serum IgG2a. Compared with non-immunized mice or mice treated with 'naked' DNA, mice immunized with nanoparticles showed a substantial reduction in allergen-induced anaphylaxis associated with reduced levels of IgE, plasma histamine and vascular leakage. These results demonstrate that oral allergen-gene immunization with chitosan-DNA nanoparticles is effective in modulating murine anaphylactic responses, and indicate its prophylactic utility in treating food allergy.


Asunto(s)
Anafilaxia/terapia , Arachis/inmunología , Quitina/análogos & derivados , ADN de Plantas/uso terapéutico , Vacunas de ADN/uso terapéutico , Albuminas 2S de Plantas , Administración Oral , Anafilaxia/inmunología , Animales , Anticuerpos/sangre , Antígenos de Plantas , Arachis/genética , Quitina/administración & dosificación , Quitina/uso terapéutico , Quitosano , ADN de Plantas/administración & dosificación , Modelos Animales de Enfermedad , Expresión Génica , Genes de Plantas , Glicoproteínas/genética , Glicoproteínas/inmunología , Histamina/sangre , Inmunoglobulina E/análisis , Ratones , Ratones Endogámicos AKR , Tamaño de la Partícula , Proteínas de Plantas , Transformación Genética
8.
Plant Cell ; 11(3): 323-34, 1999 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-10072394

RESUMEN

The Dof proteins are a large family of plant transcription factors that share a single highly conserved zinc finger. The tobacco Dof protein NtBBF1 was identified by its ability to bind to regulatory domain B in the promoter of the rolB oncogene. In this study, we show that the ACT T TA target sequence of NtBBF1 in domain B is necessary for tissue-specific expression of rolB. beta-Glucuronidase (GUS) activity of tobacco plants containing a rolB promoter-GUS fusion with a mutated NtBBF1 target sequence within domain B is almost completely suppressed in apical meristems and is severely abated in the vascular system. The ACT T TA motif is shown here also to be one of the cis-regulatory elements involved in auxin induction of rolB. The pattern of NtBBF1 expression in plants is remarkably similar to that of rolB, except in mesophyll cells of mature leaves, in which only NtBBF1 expression could be detected. Ectopic expression of rolB in mesophyll cells was achieved by particle gun delivery if the NtBBF1 binding sequence was intact. These data provide evidence that in the plant, a Dof protein DNA binding sequence acts as a transcriptional regulatory motif, and they point to NtBBF1 as the protein involved in mediating tissue-specific and auxin-inducible expression of rolB.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Regulación de la Expresión Génica de las Plantas , Ácidos Indolacéticos/fisiología , Proteínas Oncogénicas/genética , Oncogenes , Proteínas de Plantas , Factores de Transcripción/metabolismo , beta-Glucosidasa/genética , Sitios de Unión , Biolística , ADN de Plantas/administración & dosificación , Mutagénesis Sitio-Dirigida , Plantas Tóxicas , Regiones Promotoras Genéticas , Nicotiana
9.
J Allergy Clin Immunol ; 102(3): 469-75, 1998 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-9768590

RESUMEN

BACKGROUND: DNA vaccines reduce IgE responses to selected allergens, but severe reactions to the expressed antigen may limit the usefulness of the technique in allergen immunotherapy. OBJECTIVE: We sought to determine the extent of spread of an injected DNA vaccine in mice. METHODS: We placed the gene encoding the potent Hevea latex allergen Hev b 5 in a mammalian expression vector and injected this DNA vaccine subcutaneously into BALB/c mice. At several times after injection, the presence of Hev b 5 transcript was determined in multiple tissues by RT-PCR. The identity of the amplification product was confirmed by Southern hybridization and restriction analyses. RESULTS: Hev b 5 RNA appeared at the injection site and in the lymph nodes, spleen, and lungs within 1 day after injection and persisted for at least 14 days. Hev b 5 RNA was also identified in the blood and tongue 14 days after injection. Antibody and cell-mediated responses to Hev b 5 were also noted in the immunized animals at later time points. As expected, animals injected with the identical plasmid containing the Hev b 5 DNA in the antisense orientation mounted no immune response to Hev b 5. CONCLUSIONS: The rapid and widespread appearance of the Hev b 5 transcript in the injected mice confirms that DNA is translocated from the injection site, transcribed, and expressed in immune and nonimmune tissues after injection. Controlling the extent and degree of expression in specific target tissues may allow therapeutic DNA vaccination with plasmids that encode potentially toxic allergens.


Asunto(s)
Alérgenos/biosíntesis , Alérgenos/genética , ADN de Plantas/administración & dosificación , Vacunas de ADN/farmacocinética , Alérgenos/inmunología , Animales , Antígenos de Plantas , Southern Blotting , ADN de Plantas/genética , ADN de Plantas/metabolismo , Inyecciones Subcutáneas , Látex/inmunología , Hipersensibilidad al Látex/inmunología , Ganglios Linfáticos/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteínas de Plantas , Plásmidos , ARN de Planta/análisis , ARN de Planta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/metabolismo , Vacunas de ADN/administración & dosificación
10.
Planta ; 201(3): 245-51, 1997.
Artículo en Inglés | MEDLINE | ID: mdl-9129334

RESUMEN

A cDNA clone encoding a WD-40 repeat protein (BGB1) was characterized in Brassica napus L. The clone contained an open reading frame of 327 amino acid residues almost entirely composed of seven segments of WD-40 repeats. Among the WD-40 repeat proteins, BGB1 showed high similarity (63% identity) to a rat intracellular receptor for protein kinase C (RACK1) that functions in the translocation of activated protein kinase C (PKC) from the cytosolic fraction to the membrane fraction. BGB1 also had two sequence motifs involved in binding of RACK1 to PKC. The cDNA clone, when carried in a Xenopus oocyte expression vector and injected into Xenopus laevis oocytes, inhibited insulin-induced maturation of the oocytes, a PKC-mediated pathway, and this inhibition was accompanied by reduction of PKC in the membrane fraction, as in the case of mammalian RACKs. The data show that BGB1 shares some common functional characteristics with the mammalian RACK1 along with the structural similarity, suggesting that a mammalian RACK1-related cellular process might be operating in plants. Southern blot analyses of the genome of B. napus and Arabidopsis thaliana (L.) Heynh. revealed that BGB1-related genes constitute a small multigene family in both species. An approximately 1.4-kb transcript was constitutively expressed in all organs examined.


Asunto(s)
ADN Complementario/administración & dosificación , ADN de Plantas/administración & dosificación , Insulina/farmacología , Oocitos/fisiología , Péptidos/química , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Animales , Brassica , Secuencia de Consenso , Femenino , Mamíferos , Microinyecciones , Datos de Secuencia Molecular , Oocitos/efectos de los fármacos , Proteínas de Plantas/biosíntesis , Proteína Quinasa C/metabolismo , Ratas , Receptores de Cinasa C Activada , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Transcripción Genética , Xenopus laevis
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