转基因海带配子体的制备与高效增殖

海带具有很高的营养价值和经济社会价值。自20世纪90年代以来，本实验室在借鉴高等植物基因工程原理和方法的基础上，根据海带自身特点，建立了海带遗传转化体系（海带孢子体表达系统），它的基本原理是利用基因枪法转化海带配子体，经孤雌或受精途径再生幼孢子体后，用氯霉素筛选幼孢子体获得转基因海带，然后进行海上安全栽培和转基因产品的检测与提取。目前该表达系统已成功实现报告基因（β-半乳糖苷酶基因，lacZ）和功能基因（乙肝表面抗原基因，HBsAg）的稳定表达。 由于海带孢子体表达系统需经孢子体再生和海上栽培等阶段，周期较长，而且转基因安全性问题也在一定程度上制约其研究与应用。因此，我们在海带孢子体表达系统的基础上又建立和优化了海带配子体表达系统，并成功实现了报告基因（绿色荧光蛋白基因，GFP）的瞬间表达和功能基因（瑞替普酶基因，rt-PA）的稳定表达。虽然海带配子体表达系统能避免转基因安全性问题，周期较短，但在表达量和生物量积累方面，与孢子体表达系统相比还有较大差距。 本文首先在海带配子体表达系统中成功实现了人酸性成纤维细胞生长因子基因（hafgf）和鲎素基因（tac）的稳定表达，制备了转基因海带配子体，然后将光生物反应器培养技术应用于转基因海带配子体的高效增殖，以期解决阻碍海带配子体表达系统发展的量的问题，并为转基因海带配子体的大规模培养提供试验依据和技术支持。 本文的研究结果为： 1、人酸性成纤维细胞生长因子基因和鲎素基因可以稳定整合到海带配子体基因组中，实现转基因产物的表达。 2、根据转基因海带配子体的生长特点，研制开发了一套培养体积为300 ml的鼓泡式光生物反应器，它具有操作简便、成本低廉、适合海带配子体生长等特点。随后将培养体系扩大到2.5 L，并研究了光对转基因海带配子体生长的影响，试验结果显示，转基因海带配子体在光强为30 μE m-2 s-1时即可达到光饱和生长，最优光周期为14:10 LD，而且蓝光可促进转基因海带配子体的生长。 3、在前期研究工作的基础上，为改善反应器内的传质条件，我们又设计研制了2.5 L气升式光生物反应器用于转基因海带配子体的高效增殖。研究发现，气升式光生物反应器较鼓泡式光生物反应器能明显地改善反应器内的传质状态，实现转基因海带配子体更高密度的培养（生物量可达到1,990 mg L-1），是一套高密度悬浮培养转基因海带配子体的有效装置和设备。

饲料中转基因成分检测及对水产动物安全性评价的初步研究

自转基因作物问世以来，转基因产品的安全性问题一直是人们关注的焦点。本文根据GenBank中登录的转基因大豆完整外源DNA序列设计了几对引物，对转基因大豆进行了巢式PCR检测。结果表明，巢式PCR可以扩增10-10g/μl浓度的DNA溶液，检测灵敏度高达0.01％。该巢式PCR技术具有高度特异性、灵敏度和很好的重复性。用巢式PCR对部分市售的水产饲料和豆制食品进行检测，90.6％的水产饲料和46.5％的食品能检测出外源基因片段，表明转基因大豆广泛存在于水产饲料和我们的日常食品中，为食品安全分析和管理提供了方法和依据。环介导的等温扩增技术（LAMP）依赖于能够识别靶序列上6个特异区域的引物和一种具有链置换特性的DNA聚合酶，在等温条件下可高效、快速、高特异地扩增靶序列。本研究建立了转基因大豆的LAMP扩增技术，针对豆制品以及饲料的转基因LAMP检测技术正在研究和开发中。 利用转基因和非转基因豆粕制作的饲料，喂养吉富罗非鱼，分别于4周、7周取样，对其体重和血液指标进行了检测。实验显示，投喂转基因饲料7周以后，增重率和血清指标，转基因组与非转基因饲料组相比没有显著差异。全血指标中白细胞数目、大血小板比率、平均血小板体积和血小板体积分布宽度4项指标显著高于非转基因饲料组，而且差异达到极显著水平。由以上结果可见，转基因大豆与非转基因大豆相比，对罗非鱼的一些生理过程造成了一定的影响，但是并未对其生长造成可见的影响。分别于投喂1h、4h和8h以后取罗非鱼胃内容物、肠道内容物和粪便，并分别于4周、7周和继续饥饿2周后，取罗非鱼不同组织，提取DNA，用巢式PCR法检测转基因大豆中的外源基因在各种组织中的分布，结果显示在胃内容物、肠内容物、粪便、心脏、肝脏、胃、肠、卵巢、精巢、脑、鳃丝、脾脏、胆囊、肌肉等不同部位的DNA中都能检测到外源基因的存在，说明转基因大豆中的外源DNA并不能被罗非鱼的消化道完全降解，其DNA片段可能通过消化吸收转移到鱼体的各种组织。在投喂转基因饲料7周以后以及停止投喂饥饿2周以后分离水体中的微生物，提取其DNA，进行转基因检测。结果显示在所分离纯化的各种微生物中都没有检测到转基因大豆中外源基因35S-EPSPS的存在。

Análise de sensibilidade do modelo de Caprio para simulação de evolução de resistência de pragas a toxinas BT.

2009

Resultados de pesquisa da Embrapa Soja - 2000: ecofisiologia e biologia molecular.

Maximização do aproveitamento das disponibilidades climáticas pela soja; Bases agronômicas e fisiológicas das respostas da soja à disponibilidade hídrica (04.2000.331-01); Bases agronômicas e fisiológicas das respostas da soja às condições térmicas e fotoperiódicas (04.2000.331-02); Identificação, clonagem e sequenciamento de genes diferencialmente expressos em resposta às variações climáticas em soja (04.2000.331-03); Manejo dos recursos disponíveis do ambiente para produção de soja (04.2000.331-04); Estratégias para amenizar impactos decorrentes das adversidades climáticas (04.2000.331-05); Modelos de simulação do desenvolvimento da cultura da soja em resposta às variáveis do ambiente (04.2000.331-06); Genética aplicada ao melhoramento da soja; Identificação de marcadores moleculares ligados a genes de resistência a doenças (04.2000.322-01); Variabilidade genética de patógenos de soja (04.2000.322-02); Caracterização do germoplasma ativo de soja com marcadores moleculares tipo AFLP e micros-satélites (04.2000.322-03); Genética quantitativa aplicada ao melhoramento da soja: diversidade genética e resistência a doenças (04.2000.322-04); Desenvolvimento de soja transgênica com genes de interesse ao melhoramento (04.2000.322-05); Zoneamento agroclimático das principais culturas de grãos do Brasil; Caracterização da aptidão climática de regiões para o cultivo de soja no Brasil (01.2000.051-03).

BRS 399RR: soja precoce e resistente a nematóides.

BRS 399RR: soja precoce e resistente a nematóides, grupo de maturidade relativa: 6.0 ; Precocidade; Sanidade; Regiões Edafoclimáticas de Adaptação (REC); Época de semeadura; Ciclo, Acamamento e Densidade de plantas; Características gerais; Reação a doenças.

Rodent models for resolving extremes of exercise and health

The extremes of exercise capacity and health are considered a complex interplay between genes and the environment. In general, the study of animal models has proven critical for deep mechanistic exploration that provides guidance for focused and hypothesis driven discovery in humans. Hypotheses underlying molecular mechanisms of disease, and gene/tissue function can be tested in rodents in order to generate sufficient evidence to resolve and progress our understanding of human biology. Here we provide examples of three alternative uses of rodent models that have been applied successfully to advance knowledge that bridges our understanding of the connection between exercise capacity and health status. Firstly we review the strong association between exercise capacity and all-cause morbidity and mortality in humans through artificial selection on low and high exercise performance in the rat and the consequent generation of the "energy transfer hypothesis". Secondly we review specific transgenic and knock-out mouse models that replicate the human disease condition and performance. This includes human glycogen storage diseases (McArdle and Pompe) and α-actinin-3 deficiency. Together these rodent models provide an overview of the advancements of molecular knowledge required for clinical translation. Continued study of these models in conjunction with human association studies will be critical to resolving the complex gene-environment interplay linking exercise capacity, health, and disease.

Fitness of hybrids between rapeseed (Brassica napus) and wild Brassica rapa in natural habitats

J. Allainguillaume, M. Alexander, J. M. Bullock, M. Saunders, C. J. Allender, G. King, C. S. Ford, M. J. Wilkinson. (2006). Fitness of hybrids between rapeseed Brassica napus and wild Brassica rapa in natural habitats. Molecular Ecology, 15 (4) 1175-1184. RAE2008

The role of presenilin 1 and presenilin 2 in IL-1β-, LPS- and TNFα- mediated signalling events

The importance of γ-secretase protease activities in development, neurogenesis and the immune system are highlighted by the diversity of its substrates and phenotypic characterization of Presenilin (PS)-deficient transgenic animals. Since the discovery of Amyloid precursor protein (APP) and it’s cleavage by γ-secretase complexes, over 90 other type I membrane proteins have been identified as γ-secretase substrates. We have identified interleukin-1 (IL-1) receptor type I (IL-1R1), toll-like receptor 4 (TLR4) and tumour necrosis factor-α (TNFα) receptor-1 (TNFR1) as novel substrates for - secretase cleavage, which play an important role in innate immunity. In this study, using PS-deficient cells and PS-knockout animal models we examined the role of PS proteins, PS1 and PS2, in IL-1R1-, TLR4- and TNFR1- mediated inflammatory responses. Data presented show that in response to IL- 1β, lipopolysaccharide (LPS) or TNFα, immortalised fibroblasts from PS2- deficient animals have diminished production of specific cytokines and chemokine, with differential reduction in nuclear factor-κB (NF-κB) and (mitogen activated protein kinase) MAPK activities. In contrast, no defect in the response to IL-1β, LPS or TNFα was observed in PS1-deficient immortalised fibroblasts. These observations were confirmed using bone marrow-derived macrophages from PS2-null mice, which also display impaired responsiveness to IL-1β- and LPS, with decreased production of inflammatory cytokines. Furthermore, in whole animal in vivo responses, we show that PS2-deficient animals display ligand (IL-1β, LPS and TNFα)-dependent alterations in the production of specific pro-inflammatory cytokines or chemokines. Importantly, this reduced responsiveness to IL-1β, LPS or TNFα is independent of γ- secretase protease activity and γ-secretase cleavage of TNFR1, IL-1R1 or TLR4. These observations suggest a novel γ-secretase-independent role of PS2 in the regulation of innate immune responsiveness and challenge current concepts regarding the regulation of IL-1β-, LPS- and TNFα-mediated immune signalling.

Akt signal transduction dysfunction in the 3xTg-AD mouse model of Alzheimer's disease

Alzheimer’s disease (AD) is an incurable neurodegenerative disorder, accounting for over 60% of all cases of dementia. The primary risk factor for AD is age, however several genetic and environmental factors are also involved. The pathological characteristics of AD include extracellular deposition of the beta-amyloid peptide (Aβ) and intraneuronal accumulation of neurofibrillary tangles (NFTs) made of aggregated paired helical filaments (PHFs) of the hyperphosphorylated tau protein, along with synaptic loss and neuronal death. There are numerous biochemical mechanisms involved in AD pathogenesis, however the reigning hypothesis points to toxic oligomeric Aβ species as the primary causative factor in a cascade of events leading to neuronal stress and dyshomeostasis that initiate abnormal regulation of tau. The insulin and IGF-1 receptors (IR, IGF-1R) are the primary activators of PI3- K/Akt through which they regulate cell growth, development, glucose metabolism, and learning and memory. Work in our lab and others shows increased Akt activity and phosphorylation of its downstream targets in AD brain, along with insulin and insulin-like growth factor-1 signalling (IIS) dysfunction. This is supported by studies of AD models in vivo and in vitro. Our group and others hypothesise that Aβ activates Akt through IIS to initiate a negative feedback mechanism that desensitises neurons to insulin/IGF-1, and sustains activation of Akt. In this study the functions of endogenous Akt, IR, and the insulin receptor substrate (IRS-1) were examined in relationship to Aβ and tau pathology in the 3xTg-AD mouse model, which contains three mutant human transgenes associated with familial AD or dementia. The 3xTg-AD mouse develops Aβ and tau pathology in a spatiotemporal manner that best recapitulates the progression of AD in human brain. Western blotting and immunofluorescent microscopy techniques were utilised in vivo and in vitro, to examine the relationship between IIS, Akt, and AD pathology. I first characterised in detail AD pathology in 3xTg-AD mice, where an age-related accumulation of intraneuronal Aβ and tau was observed in the hippocampal formation, amygdala, and entorhinal cortex, and at late stages (18 months), extracellular amyloid plaques and NFTs, primarily in the subiculum and the CA1 layer of the hippocampal formation. Increased activity of Akt, detected with antibody to phosphoSer473-Akt, was increased in 3xTg-AD mice compared to age-matched non-transgenic mice (non-Tg), and in direct correlation to the accumulation of Aβ and tau in neuronal somatodendritic compartments. Akt phosphorylates tau at residue Ser214 within a highly specific consensus sequence for Akt phosphorylation, and phosphoSer214-tau strongly decreases microtubule (MT) stabilisation by preventing tau-MT binding. PhosphoSer214-tau increased concomitantly with this in the same age-related and region-specific fashion. Polarisation of tau phosphorylation was observed, where PHF-1 (tauSer396/404) and phosphoSer214-tau both appeared early in 3xTg-AD mice in distinct neuronal compartments: PHF-1 in axons, and phosphoSer214-tau in neuronal soma and dendrites. At 18 months, phosphoSer214-tau strongly colocalised with NFTs positive for the PHF- 1 and AT8 (tauSer202/Thr205) phosphoepitopes. IR was decreased with age in 3xTg-AD brain and in comparison to age-matched non-Tg, and this was specific for brain regions containing Aβ, tau, and hyperactive Akt. IRS-1 was similarly decreased, and both proteins showed altered subcellular distribution. Phosphorylation of IRS-1Ser312 is a strong indicator of IIS dysfunction and insulin resistance, and was increased in 3xTg-AD mice with age and in relation to pathology. Of particular note was our observation that abberant IIS and Akt signalling in 3xTg-AD brain related to Aβ and tau pathology on a gross anatomical level, and specifically localised to the brain regions and circuitry of the perforant path. Finally, I conducted a preliminary study of the effects of synthetic Aβ oligomers on embryonic rat hippocampus neuronal cultures to support these results and those in the literature. Taken together, these novel findings provide evidence for IIS and Akt signal transduction dysfunction as the missing link between Aβ and tau pathogenesis, and contribute to the overall understanding of the biochemical mechanisms of AD.

Traumatic brain injury exacerbates neurodegenerative pathology: improvement with an apolipoprotein E-based therapeutic.

Cognitive impairment is common following traumatic brain injury (TBI), and neuroinflammatory mechanisms may predispose to the development of neurodegenerative disease. Apolipoprotein E (apoE) polymorphisms modify neuroinflammatory responses, and influence both outcome from acute brain injury and the risk of developing neurodegenerative disease. We demonstrate that TBI accelerates neurodegenerative pathology in double-transgenic animals expressing the common human apoE alleles and mutated amyloid precursor protein, and that pathology is exacerbated in the presence of the apoE4 allele. The administration of an apoE-mimetic peptide markedly reduced the development of neurodegenerative pathology in mice homozygous for apoE3 as well as apoE3/E4 heterozygotes. These results demonstrate that TBI accelerates the cardinal neuropathological features of neurodegenerative disease, and establishes the potential for apoE mimetic therapies in reducing pathology associated with neurodegeneration.

Ras controls melanocyte expansion during zebrafish fin stripe regeneration.

Regenerative medicine for complex tissues like limbs will require the provision or activation of precursors for different cell types, in the correct number, and with the appropriate instructions. These strategies can be guided by what is learned from spectacular events of natural limb or fin regeneration in urodele amphibians and teleost fish. Following zebrafish fin amputation, melanocyte stripes faithfully regenerate in tandem with complex fin structures. Distinct populations of melanocyte precursors emerge and differentiate to pigment regenerating fins, yet the regulation of their proliferation and patterning is incompletely understood. Here, we found that transgenic increases in active Ras dose-dependently hyperpigmented regenerating zebrafish fins. Lineage tracing and marker analysis indicated that increases in active Ras stimulated the in situ amplification of undifferentiated melanocyte precursors expressing mitfa and kita. Active Ras also hyperpigmented early fin regenerates of kita mutants, which are normally devoid of primary regeneration melanocytes, suppressing defects in precursor function and survival. By contrast, this protocol had no noticeable impact on pigmentation by secondary regulatory melanocyte precursors in late-stage kita regenerates. Our results provide evidence that Ras activity levels control the repopulation and expansion of adult melanocyte precursors after tissue loss, enabling the recovery of patterned melanocyte stripes during zebrafish appendage regeneration.

Hepcidin as a therapeutic tool to limit iron overload and improve anemia in β-thalassemic mice.

Excessive iron absorption is one of the main features of β-thalassemia and can lead to severe morbidity and mortality. Serial analyses of β-thalassemic mice indicate that while hemoglobin levels decrease over time, the concentration of iron in the liver, spleen, and kidneys markedly increases. Iron overload is associated with low levels of hepcidin, a peptide that regulates iron metabolism by triggering degradation of ferroportin, an iron-transport protein localized on absorptive enterocytes as well as hepatocytes and macrophages. Patients with β-thalassemia also have low hepcidin levels. These observations led us to hypothesize that more iron is absorbed in β-thalassemia than is required for erythropoiesis and that increasing the concentration of hepcidin in the body of such patients might be therapeutic, limiting iron overload. Here we demonstrate that a moderate increase in expression of hepcidin in β-thalassemic mice limits iron overload, decreases formation of insoluble membrane-bound globins and reactive oxygen species, and improves anemia. Mice with increased hepcidin expression also demonstrated an increase in the lifespan of their red cells, reversal of ineffective erythropoiesis and splenomegaly, and an increase in total hemoglobin levels. These data led us to suggest that therapeutics that could increase hepcidin levels or act as hepcidin agonists might help treat the abnormal iron absorption in individuals with β-thalassemia and related disorders.

Evidence that SOX2 overexpression is oncogenic in the lung.

BACKGROUND: SOX2 (Sry-box 2) is required to maintain a variety of stem cells, is overexpressed in some solid tumors, and is expressed in epithelial cells of the lung. METHODOLOGY/PRINCIPAL FINDINGS: We show that SOX2 is overexpressed in human squamous cell lung tumors and some adenocarcinomas. We have generated mouse models in which Sox2 is upregulated in epithelial cells of the lung during development and in the adult. In both cases, overexpression leads to extensive hyperplasia. In the terminal bronchioles, a trachea-like pseudostratified epithelium develops with p63-positive cells underlying columnar cells. Over 12-34 weeks, about half of the mice expressing the highest levels of Sox2 develop carcinoma. These tumors resemble adenocarcinoma but express the squamous marker, Trp63 (p63). CONCLUSIONS: These findings demonstrate that Sox2 overexpression both induces a proximal phenotype in the distal airways/alveoli and leads to cancer.

Level of beta-adrenergic receptor kinase 1 inhibition determines degree of cardiac dysfunction after chronic pressure overload-induced heart failure.

BACKGROUND: Heart failure is characterized by abnormalities in beta-adrenergic receptor (betaAR) signaling, including increased level of myocardial betaAR kinase 1 (betaARK1). Our previous studies have shown that inhibition of betaARK1 with the use of the Gbetagamma sequestering peptide of betaARK1 (betaARKct) can prevent cardiac dysfunction in models of heart failure. Because inhibition of betaARK activity is pivotal for amelioration of cardiac dysfunction, we investigated whether the level of betaARK1 inhibition correlates with the degree of heart failure. METHODS AND RESULTS: Transgenic (TG) mice with varying degrees of cardiac-specific expression of betaARKct peptide underwent transverse aortic constriction (TAC) for 12 weeks. Cardiac function was assessed by serial echocardiography in conscious mice, and the level of myocardial betaARKct protein was quantified at termination of the study. TG mice showed a positive linear relationship between the level of betaARKct protein expression and fractional shortening at 12 weeks after TAC. TG mice with low betaARKct expression developed severe heart failure, whereas mice with high betaARKct expression showed significantly less cardiac deterioration than wild-type (WT) mice. Importantly, mice with a high level of betaARKct expression had preserved isoproterenol-stimulated adenylyl cyclase activity and normal betaAR densities in the cardiac membranes. In contrast, mice with low expression of the transgene had marked abnormalities in betaAR function, similar to the WT mice. CONCLUSIONS: These data show that the level of betaARK1 inhibition determines the degree to which cardiac function can be preserved in response to pressure overload and has important therapeutic implications when betaARK1 inhibition is considered as a molecular target.

Beta-arrestins regulate atherosclerosis and neointimal hyperplasia by controlling smooth muscle cell proliferation and migration.

Atherosclerosis and arterial injury-induced neointimal hyperplasia involve medial smooth muscle cell (SMC) proliferation and migration into the arterial intima. Because many 7-transmembrane and growth factor receptors promote atherosclerosis, we hypothesized that the multifunctional adaptor proteins beta-arrestin1 and -2 might regulate this pathological process. Deficiency of beta-arrestin2 in ldlr(-/-) mice reduced aortic atherosclerosis by 40% and decreased the prevalence of atheroma SMCs by 35%, suggesting that beta-arrestin2 promotes atherosclerosis through effects on SMCs. To test this potential atherogenic mechanism more specifically, we performed carotid endothelial denudation in congenic wild-type, beta-arrestin1(-/-), and beta-arrestin2(-/-) mice. Neointimal hyperplasia was enhanced in beta-arrestin1(-/-) mice, and diminished in beta-arrestin2(-/-) mice. Neointimal cells expressed SMC markers and did not derive from bone marrow progenitors, as demonstrated by bone marrow transplantation with green fluorescent protein-transgenic cells. Moreover, the reduction in neointimal hyperplasia seen in beta-arrestin2(-/-) mice was not altered by transplantation with either wild-type or beta-arrestin2(-/-) bone marrow cells. After carotid injury, medial SMC extracellular signal-regulated kinase activation and proliferation were increased in beta-arrestin1(-/-) and decreased in beta-arrestin2(-/-) mice. Concordantly, thymidine incorporation and extracellular signal-regulated kinase activation and migration evoked by 7-transmembrane receptors were greater than wild type in beta-arrestin1(-/-) SMCs and less in beta-arrestin2(-/-) SMCs. Proliferation was less than wild type in beta-arrestin2(-/-) SMCs but not in beta-arrestin2(-/-) endothelial cells. We conclude that beta-arrestin2 aggravates atherosclerosis through mechanisms involving SMC proliferation and migration and that these SMC activities are regulated reciprocally by beta-arrestin2 and beta-arrestin1. These findings identify inhibition of beta-arrestin2 as a novel therapeutic strategy for combating atherosclerosis and arterial restenosis after angioplasty.