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1.
N Engl J Med ; 380(16): 1525-1534, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30995372

RESUMO

BACKGROUND: Allogeneic hematopoietic stem-cell transplantation for X-linked severe combined immunodeficiency (SCID-X1) often fails to reconstitute immunity associated with T cells, B cells, and natural killer (NK) cells when matched sibling donors are unavailable unless high-dose chemotherapy is given. In previous studies, autologous gene therapy with γ-retroviral vectors failed to reconstitute B-cell and NK-cell immunity and was complicated by vector-related leukemia. METHODS: We performed a dual-center, phase 1-2 safety and efficacy study of a lentiviral vector to transfer IL2RG complementary DNA to bone marrow stem cells after low-exposure, targeted busulfan conditioning in eight infants with newly diagnosed SCID-X1. RESULTS: Eight infants with SCID-X1 were followed for a median of 16.4 months. Bone marrow harvest, busulfan conditioning, and cell infusion had no unexpected side effects. In seven infants, the numbers of CD3+, CD4+, and naive CD4+ T cells and NK cells normalized by 3 to 4 months after infusion and were accompanied by vector marking in T cells, B cells, NK cells, myeloid cells, and bone marrow progenitors. The eighth infant had an insufficient T-cell count initially, but T cells developed in this infant after a boost of gene-corrected cells without busulfan conditioning. Previous infections cleared in all infants, and all continued to grow normally. IgM levels normalized in seven of the eight infants, of whom four discontinued intravenous immune globulin supplementation; three of these four infants had a response to vaccines. Vector insertion-site analysis was performed in seven infants and showed polyclonal patterns without clonal dominance in all seven. CONCLUSIONS: Lentiviral vector gene therapy combined with low-exposure, targeted busulfan conditioning in infants with newly diagnosed SCID-X1 had low-grade acute toxic effects and resulted in multilineage engraftment of transduced cells, reconstitution of functional T cells and B cells, and normalization of NK-cell counts during a median follow-up of 16 months. (Funded by the American Lebanese Syrian Associated Charities and others; LVXSCID-ND ClinicalTrials.gov number, NCT01512888.).


Assuntos
Bussulfano/administração & dosagem , Terapia Genética , Vetores Genéticos , Subunidade gama Comum de Receptores de Interleucina/genética , Lentivirus , Condicionamento Pré-Transplante , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia , Antígenos de Diferenciação de Linfócitos T/sangue , Linfócitos B/fisiologia , Transplante de Células-Tronco Hematopoéticas , Humanos , Imunoglobulina M/sangue , Lactente , Células Matadoras Naturais , Contagem de Linfócitos , Masculino , Linfócitos T , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/imunologia
2.
Gene Ther ; 27(12): 545-556, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32341484

RESUMO

Autologous gene therapy using lentiviral vectors (LVs) holds promise for treating monogenetic blood diseases. However, clinical applications can be limited by suboptimal hematopoietic stem cell (HSC) transduction and insufficient quantities of available vector. We recently reported gene therapy for X-linked severe combined immunodeficiency using a protocol in which patient CD34+ cells were incubated with two successive transductions. Here we describe an improved protocol for LV delivery to CD34+ cells that simplifies product manipulation, reduces vector consumption, and achieves greater vector copy number (VCN) of repopulating HSCs in mouse xenotransplantation assays. Notable findings include the following: (1) the VCN of CD34+ cells measured shortly after transduction did not always correlate with the VCN of repopulating HSCs after xenotransplantation; (2) single-step transduction at higher CD34+ cell concentrations (2-4 × 106/ml) conserved LV without compromising HSC VCN; (3) poloxamer F108 (LentiBOOST) increased HSC VCN by two- to threefold (average from three donors); (4) although LentiBOOST + prostaglandin E2 combination further increased VCN in vitro, the VCN observed in vivo were similar to LentiBOOST alone; (5) cyclosporine H increased the HSC VCN to a similar or greater extent with LentiBOOST in vivo. Our findings delineate an improved protocol to increase the VCN of HSCs after CD34+ cell transduction with clinically relevant LVs.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Lentivirus , Animais , Antígenos CD34 , Terapia Genética , Vetores Genéticos/genética , Células-Tronco Hematopoéticas , Humanos , Lentivirus/genética , Camundongos , Transdução Genética
3.
Mol Genet Metab ; 117(2): 199-209, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26766614

RESUMO

New enzyme delivery technologies are required for treatment of lysosomal storage disorders with significant pathologies associated with the so-called "hard-to-treat" tissues and organs. Genetic deficiencies in the GLB1 gene encoding acid ß-galactosidase lead to GM1-gangliosidosis or Morquio B, lysosomal diseases with predominant disease manifestation associated with the central nervous system or skeletal system, respectively. Current lysosomal ERTs are delivered into cells based on receptor-mediated endocytosis and do not effectively address several hard-to-treat organs including those critical for GM1-gangliosidosis patients. Lectins provide alternative cell-uptake mechanisms based on adsorptive-mediated endocytosis and thus may provide unique biodistribution for lysosomal disease therapeutics. In the current study, genetic fusions of the plant galactose/galactosamine-binding lectin, RTB, and the human acid ß-galactosidase enzyme were produced using a plant-based bioproduction platform. ß-gal:RTB and RTB:ß-gal fusion products retained both lectin activity and ß-galactosidase activity. Purified proteins representing both fusion orientations were efficiently taken up into GM1 patient fibroblasts and mediated the reduction of GM1 ganglioside substrate with activities matching mammalian cell-derived ß-galactosidase. In contrast, plant-derived ß-gal alone was enzymatically active but did not mediate uptake or correction indicating the need for either lectin-based (plant product) or mannose-6-phosphate-based (mammalian product) delivery. Native ß-galactosidase undergoes catalytic activation (cleavage within the C-terminal region) in lysosomes and is stabilized by association with protective protein/cathepsin A. Enzymatic activity and lysosomal protein processing of the RTB fusions were assessed following internalization into GM1 fibroblasts. Within 1-4h, both ß-gal:RTB and RTB:ß-gal were processed to the ~64kDa "activated" ß-gal form; the RTB lectin was cleaved and rapidly degraded. The activated ß-gal was still detected at 48h suggesting interactions with protective protein/cathepsin A. Uptake-saturation analyses indicated that the RTB adsorptive-mediated mechanisms of ß-gal:RTB supported significantly greater accumulation of ß-galactose activity in fibroblasts compared to the receptor-mediated mechanisms of the mammalian cell-derived ß-gal. These data demonstrate that plant-made ß-gal:RTB functions as an effective replacement enzyme for GM1-gangliosidosis - delivering enzyme into cells, enabling essential lysosomal processing, and mediating disease substrate clearance at the cellular level. RTB provides novel uptake behaviors and thus may provide new receptor-independent strategies that could broadly impact lysosomal disease treatments.


Assuntos
Gangliosidose GM1/tratamento farmacológico , Proteínas Recombinantes de Fusão/metabolismo , beta-Galactosidase/metabolismo , Células Cultivadas , Terapia de Reposição de Enzimas , Fibroblastos/enzimologia , Humanos , Cinética , Lisossomos/metabolismo , Lectinas de Plantas/química , Lectinas de Plantas/genética , Lectinas de Plantas/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Nicotiana , beta-Galactosidase/química , beta-Galactosidase/genética
4.
Sci Adv ; 9(40): eadg9959, 2023 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-37801507

RESUMO

Lentiviral vector (LV)-based gene therapy holds promise for a broad range of diseases. Analyzing more than 280,000 vector integration sites (VISs) in 273 samples from 10 patients with X-linked severe combined immunodeficiency (SCID-X1), we discovered shared LV integrome signatures in 9 of 10 patients in relation to the genomics, epigenomics, and 3D structure of the human genome. VISs were enriched in the nuclear subcompartment A1 and integrated into super-enhancers close to nuclear pore complexes. These signatures were validated in T cells transduced with an LV encoding a CD19-specific chimeric antigen receptor. Intriguingly, the one patient whose VISs deviated from the identified integrome signatures had a distinct clinical course. Comparison of LV and gamma retrovirus integromes regarding their 3D genome signatures identified differences that might explain the lower risk of insertional mutagenesis in LV-based gene therapy. Our findings suggest that LV integrome signatures, shaped by common features such as genome organization, may affect the efficacy of LV-based cellular therapies.


Assuntos
Vetores Genéticos , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X , Humanos , Vetores Genéticos/genética , Terapia Genética , Retroviridae/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia , Linfócitos T
5.
Mol Ther Methods Clin Dev ; 21: 693-701, 2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34141824

RESUMO

Vector-mediated mutagenesis remains a major safety concern for many gene therapy clinical protocols. Indeed, lentiviral-based gene therapy treatments of hematologic disease can result in oligoclonal blood reconstitution in the transduced cell graft. Specifically, clonal expansion of hematopoietic stem cells (HSCs) highly expressing HMGA2, a chromatin architectural factor found in many human cancers, is reported in patients undergoing gene therapy for hematologic diseases, raising concerns about the safety of these integrations. Here, we show for the first time in vivo multilineage and multiclonal expansion of non-human primate HSCs expressing a 3' UTR-truncated version of HMGA2 without evidence of any hematologic malignancy >7 years post-transplantation, which is significantly longer than most non-human gene therapy pre-clinical studies. This expansion is accompanied by an increase in HSC survival, cell cycle activation of downstream progenitors, and changes in gene expression led by the upregulation of IGF2BP2, a mRNA binding regulator of survival and proliferation. Thus, we conclude that prolonged ectopic expression of HMGA2 in hematopoietic progenitors is not sufficient to drive hematologic malignancy and is not an acute safety concern in lentiviral-based gene therapy clinical protocols.

6.
Cancers (Basel) ; 12(6)2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-32604839

RESUMO

T cells engineered with chimeric antigen receptors (CARs) show great promise in the treatment of some cancers. Modifying T cells to express CARs generally relies on T-cell transduction using viral vectors carrying a transgene, resulting in semi-random DNA integration within the T-cell genome. While this approach has proven successful and is used in generating the Food and Drug Administration (FDA, USA) approved B-lymphocyte antigen CD19-specific CAR T cells, it is possible the transgene could integrate into a locus that would lead to malignant transformation of the engineered T cells. In addition, manufacturing viral vectors is time-consuming and expensive. One way to overcome these challenges is site-specific gene integration, which can be achieved through clustered regularly interspaced short palindromic repeat (CRISPR) mediated editing and non-viral DNA, which serves as a template for homology-directed repair (HDR). This non-viral gene editing approach provides a rapid, highly specific, and inexpensive way to engineer T cells. Here, we describe an optimized protocol for the site-specific knock-in of a large transgene in primary human T cells using non-viral double stranded DNA as a repair template. As proof-of-principle, we targeted the T-cell receptor alpha constant (TRAC) locus for insertion of a large transgene containing green fluorescence protein (GFP) and interleukin-15 (IL-15). To optimize the knock-in conditions we tested template DNA concentration, homology arm length, cell number, and knock-in efficiency over time. We then applied these established guidelines to target the TRAC or interleukin-13 (IL-13) locus for the knock-in of synthetic molecules, such as a CAR, bispecific T-cell engager (BiTE), and other transgenes. While integration efficiency depends on the targeted gene locus and selected transgene, this optimized protocol reliably generates the desired insertion at rates upwards of 20%. Thus, it should serve as a good starting point for investigators who are interested in knocking in transgenes into specific loci.

7.
Plant Cell Rep ; 28(4): 589-99, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19116720

RESUMO

The expression and functionality of a resveratrol synthase (RS) gene from peanut (Arachis hypogaea) was studied using an Agrobacterium tumefaciens-mediated transient expression system in Nicotiana benthamiana leaves. Functional analysis of RS was demonstrated by tracking its expression during 96 h. To measure the transcripts levels of RS transgene, real-time qRT-PCR was used and revealed that the highest level of transcripts was at 48 h post-transfection. Western blot analyses showed that RS protein was accumulated to the highest levels at 72 h post-transfection. Finally, HPLC and mass spectrometry analyses revealed the production of trans-piceid (resveratrol glucoside) as the major stilbenoid compound confirming the functional activity of the RS enzyme in planta. No activity of RS transgene was detected in negative controls. This strategy showed advantages over conventional systems because it does not require establishment of cell cultures, feeding with appropriate substrates or generation of stable transgenic plants. This transient system proved to be a rapid and direct approach to perform functional analysis of stilbene synthases, such as resveratrol synthase. Furthermore, this approach can be useful to study the metabolic effects of over-expressing or silencing specific genes within a short period of time.


Assuntos
Aciltransferases/metabolismo , Arachis/enzimologia , Nicotiana/enzimologia , Proteínas de Plantas/metabolismo , Aciltransferases/genética , Arachis/genética , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Glucosídeos/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Estilbenos/metabolismo , Nicotiana/genética
8.
Phytochemistry ; 68(14): 1992-2003, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17574636

RESUMO

Resveratrol and its derivatives are natural stilbenes associated with many health benefits that include those conferred by their antioxidant and anticancer properties. While stilbenes can be recovered as an extract from a selected number of plants, these products are not suitable for many applications in the food/pharmaceutical sectors due to high levels of impurities as well as the overall low concentration of resveratrol and its derivatives in the extract. To deliver a highly defined and enriched resveratrol product, hairy root cultures of peanut (Arachis hypogaea) were established and tested as a bioproduction system for resveratrol and associated derivatives. Analyses by HPTLC and GC-MS of ethyl acetate extracts showed that a single 24 h sodium acetate elicitation resulted in a 60-fold induction and secretion of trans-resveratrol into the medium of peanut hairy root cultures. trans-Resveratrol accumulated to levels of 98 microg/mg of the dried extract from the medium representing 99% of the total resveratrol produced. Other stilbenes, including trans-pterostilbene, were also detected in the medium. Our results demonstrate the capacity of hairy root cultures as an effective bioprocessing system for valued nutraceuticals like resveratrol and resveratrol derivatives. In being able to effectively induce and recover high levels of resveratrol and associated derivatives from the media fraction, hairy roots may offer a scalable and continuous product recovery platform for naturally-derived, high quality, enriched nutraceuticals.


Assuntos
Arachis/metabolismo , Raízes de Plantas/metabolismo , Estilbenos/metabolismo , Arachis/química , Arachis/crescimento & desenvolvimento , Arachis/microbiologia , Cromatografia Gasosa-Espectrometria de Massas , Isomerismo , Estrutura Molecular , Raízes de Plantas/química , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Resveratrol , Rhizobium/fisiologia , Estilbenos/química
9.
J Agric Food Chem ; 63(15): 3942-50, 2015 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-25837778

RESUMO

Peanut (Arachis hypogaea) produces stilbenoids upon exposure to abiotic and biotic stresses. Among these compounds, the prenylated stilbenoids arachidin-1 and arachidin-3 have shown diverse biological activities with potential applications in human health. These compounds exhibit higher or novel biological activities in vitro when compared to their nonprenylated analogues piceatannol and resveratrol, respectively. However, assessment of these bioactivities in vivo has been challenging because of their limited availability. In this study, hairy root cultures of peanut were induced to produce stilbenoids upon treatment with elicitors. Co-treatment with 100 µM methyl jasmonate (MeJA) and 9 g/L methyl-ß-cyclodextrin (CD) led to sustained high levels of resveratrol, piceatannol, arachidin-1, and arachidin-3 in the culture medium when compared to other elicitor treatments. The average yields of arachidin-1 and arachidin-3 were 56 and 148 mg/L, respectively, after co-treatment with MeJA and CD. Furthermore, MeJA and CD had a synergistic effect on resveratrol synthase gene expression, which could explain the higher yield of resveratrol when compared to treatment with either MeJA or CD alone. Peanut hairy root cultures were shown to be a controlled and sustainable axenic system for the production of the diverse types of biologically active stilbenoids.


Assuntos
Acetatos/farmacologia , Arachis/efeitos dos fármacos , Ciclopentanos/farmacologia , Hemiterpenos/metabolismo , Oxilipinas/farmacologia , Reguladores de Crescimento de Plantas/farmacologia , Raízes de Plantas/efeitos dos fármacos , Estilbenos/metabolismo , Arachis/química , Arachis/crescimento & desenvolvimento , Arachis/metabolismo , Técnicas de Cultura de Células , Meios de Cultura/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Hemiterpenos/análise , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Resveratrol , Estilbenos/análise , beta-Ciclodextrinas/farmacologia
10.
Plant Physiol Biochem ; 74: 50-69, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24269870

RESUMO

Stilbenoids are polyphenolic phytoalexins that exhibit potential health applications in humans. Hairy root cultures of muscadine grape (Vitis rotundifolia Michx.) were used to study the biochemical and molecular regulation of stilbenoid biosynthesis upon treatment with 100 µM methyl jasmonate (MeJA) or 10 mM hydrogen peroxide (H2O2) over a 96-h period. Resveratrol, piceid, and ε-viniferin were identified in higher concentrations in the tissue whereas resveratrol was the most abundant stilbenoid in the medium under either treatment. An earlier increase in resveratrol accumulation was observed for the MeJA-treated group showing a maximum at 12 h in the tissue and 18 h in the medium. Furthermore, the antioxidant capacity of extracts from the tissue and medium was determined by the 2,2'-azinobis[3-ethylbenzthiazoline sulfonic acid] (ABTS) and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays showing correlation with the stilbenoid content. Fourteen candidate reference genes for qPCR were tested under the described experimental conditions and resulted in the selection of 5 reference genes. Quantitative analyses of transcripts for phenylalanine ammonia-lyase (PAL), resveratrol synthase (RS), and two stilbene synthases (STS and STS2) showed the highest RNA level induction at 3 h for both treatments with a higher induction for the MeJA treatment. In contrast, the flavonoid-related chalcone synthase (CHS) transcripts showed induction and a decrease in expression for MeJA and H2O2 treatments, respectively. The observed responses could be related to an oxidative burst triggered by the exposure to abiotic stressor compounds with signaling function such as MeJA and H2O2 which have been previously related to the synthesis of secondary metabolites.


Assuntos
Acetatos/farmacologia , Antioxidantes/metabolismo , Ciclopentanos/farmacologia , Peróxido de Hidrogênio/farmacologia , Oxilipinas/farmacologia , Raízes de Plantas/efeitos dos fármacos , Estilbenos/metabolismo , Vitis/metabolismo , Benzotiazóis/química , Compostos de Bifenilo/química , Meios de Cultura , Genes de Plantas , Picratos/química , Raízes de Plantas/metabolismo , RNA de Plantas/isolamento & purificação , Ácidos Sulfônicos/química , Vitis/genética
11.
Methods Mol Biol ; 824: 535-64, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22160919

RESUMO

Plant-based expression technologies for recombinant proteins have begun to receive acceptance for pharmaceuticals and other commercial markets. Protein products derived from plants offer safer, more cost-effective, and less capital-intensive alternatives to traditional manufacturing systems using microbial fermentation or animal cell culture bioreactors. Moreover, plants are now known to be capable of expressing bioactive proteins from a diverse array of species including animals and humans. Methods development to assess the quality and performance of proteins manufactured in plants are essential to support the QA/QC demands as plant-produced protein products transition to the commercial marketplace. Within the pharmaceutical arena, process validation and acceptance criteria for biological products must comply with the Food and Drug Administration (FDA) and ICH Q6B guidelines in order to initiate the regulatory approval process. Detailed product specifications will also need to be developed and validated for plant-made proteins for the bioenergy, food, chemical synthesis, or research reagent markets.We have, therefore, developed assessment methods for important qualitative and quantitative parameters of the products and the manufacturing methods utilized in plant-based production systems. In this chapter, we describe a number of procedures to validate product identity and characteristics including mass analyses, antibody cross-reactivity, N-terminal sequencing, and bioactivity. We also address methods for routine assessment of yield, recovery, and purity. The methods presented are those developed for the synthesis and recovery of the avian cytokine, chicken interleukin-12 (ChIL-12), produced in the leaves of Nicotiana benthamiana. The ChIL-12 protein used as a model for this chapter includes a C-terminal histidine epitope (HIS-tag) and, thus, these methods may be directly applicable to other HIS-tagged proteins produced in plants. However, the overall strategy presented using the ChIL-12(HIS) example should provide the basis of standard procedures for assessing the quality of other plant-based protein products and manufacturing systems.


Assuntos
Reatores Biológicos , Biotecnologia/normas , Interleucina-12/biossíntese , Nicotiana/metabolismo , Folhas de Planta/metabolismo , Proteínas Recombinantes/biossíntese , Animais , Biotecnologia/métodos , Western Blotting , Galinhas , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Interleucina-12/metabolismo , Controle de Qualidade , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
12.
BMC Res Notes ; 4: 392, 2011 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-21985172

RESUMO

BACKGROUND: Hairy root cultures produced via Agrobacterium rhizogenes-mediated transformation have emerged as practical biological models to elucidate the biosynthesis of specialized metabolites. To effectively understand the expression patterns of the genes involved in the metabolic pathways of these compounds, reference genes need to be systematically validated under specific experimental conditions as established by the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines. In the present report we describe the first validation of reference genes for RT-qPCR in hairy root cultures of peanut which produce stilbenoids upon elicitor treatments. RESULTS: A total of 21 candidate reference genes were evaluated. Nineteen genes were selected based on previous qPCR studies in plants and two were from the T-DNAs transferred from A. rhizogenes. Nucleotide sequences of peanut candidate genes were obtained using their homologous sequences in Arabidopsis. To identify the suitable primers, calibration curves were obtained for each candidate reference gene. After data analysis, 12 candidate genes meeting standard efficiency criteria were selected. The expression stability of these genes was analyzed using geNorm and NormFinder algorithms and a ranking was established based on expression stability of the genes. Candidate reference gene expression was shown to have less variation in methyl jasmonate (MeJA) treated root cultures than those treated with sodium acetate (NaOAc). CONCLUSIONS: This work constitutes the first effort to validate reference genes for RT-qPCR in hairy roots. While these genes were selected under conditions of NaOAc and MeJA treatment, we anticipate these genes to provide good targets for reference genes for hairy roots under a variety of stress conditions. The lead reference genes were a gene encoding for a TATA box binding protein (TBP2) and a gene encoding a ribosomal protein (RPL8C). A commonly used reference gene GAPDH showed low stability of expression suggesting that its use may lead to inaccurate gene expression profiles when used for data normalization in stress-stimulated hairy roots. Likewise the A. rhizogenes transgene rolC showed less expression stability than GAPDH. This study proposes that a minimum of two reference genes should be used for a normalization procedure in gene expression profiling using elicited hairy roots.

13.
Plant Physiol Biochem ; 48(5): 310-8, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20138774

RESUMO

Previously, we have shown that hairy root cultures of peanut provide a controlled, sustainable and scalable production system that can be induced to produce stilbenoids. However to leverage peanut hairy roots to study the biosynthesis of this polyphenolic biosynthetic pathway, growing conditions and elicitation kinetics of these tissue cultures must be defined and understood. To this end, a new peanut cv. Hull hairy root (line 3) that produces resveratrol and its prenylated analogues arachidin-1 and arachidin-3 upon sodium acetate-mediated elicitation was established. Two culture media were compared for impact on root growth and stilbenoid biosynthesis/secretion. The levels of ammonium, nitrate, phosphate and residual sugars were monitored along growth and elicitation period. A modified MS (MSV) medium resulted in higher root biomass when compared to B5 medium. The stilbenoid profile after elicitation varied depending on the age of the culture (6, 9, 12, and 15-day old). After elicitation at day 9 (exponential growth in MSV medium), over 90% of the total resveratrol, arachidin-1 and arachidin-3 accumulated in the medium. Our studies demonstrate the benefits of the hairy root culture system to study the biosynthesis of stilbenoids including valuable prenylated polyphenolic compounds.


Assuntos
Arachis/metabolismo , Hemiterpenos/metabolismo , Extratos Vegetais/metabolismo , Raízes de Plantas/metabolismo , Estilbenos/metabolismo , Técnicas de Cultura de Tecidos , Arachis/crescimento & desenvolvimento , Células Cultivadas , Meios de Cultura , Raízes de Plantas/crescimento & desenvolvimento , Acetato de Sódio/farmacologia
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