Biochemistry and Molecular Biology Core

生物化学和分子生物学核心

• 批准号: 7596518
• 负责人: ASISH K. SAHA
• 金额: $ 20.12万
• 依托单位: BOSTON MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-15 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7596518
• 关键词: 1,2-diacylglycerol; Ablation; Academic Medical Centers; Acetylation; Adenine Nucleotides; Adenoviruses; Adopted; Adult; Age; Aging; Animals; Antibodies; Arteries; Arts; Backcrossings; Bacteria; Basal metabolic rate; Behavior; Biochemistry; Biological; Biological Assay; Blood Vessels; Blood capillaries; Body Composition; Bone Marrow Cells; Boston; Breeding; Budgets; Cataloging; Catalogs; Cattle; Cell Culture Techniques; Cell Lineage; Cells; Ceramides; Characteristics; Chemicals; Collection; Computer software; Computers; Cultured Cells; Custom; Data; Deletion Mutation; Development; Devices; Diabetes Mellitus; Diet; Diglycerides; Disease; Dominant-Negative Mutation; DsRed; Embryo; Endothelial Cells; Endothelium; Ensure; Excision; Exercise; Facility Designs; Female; Fiber; Fluorescence; Frequencies; Functional disorder; Funding; Gene Expression Regulation; Genetic; Genetic Recombination; Genotype; Health; Heating; Hematopoietic; Housing; Human; Human Resources; Image; Image Analysis; Immunofluorescence Immunologic; Immunoprecipitation; In Vitro; Individual; Insecta; Institutes; Insulin; Intervention; Laboratories; Laboratory Animals; LacZ Genes; Length; Link; Lipids; Literature; Liver; Longitudinal Studies; Lung; Lysine; Magnetic Resonance; Maintenance; Malonyl Coenzyme A; Measurement; Measures; Mechanics; Mediating; Medical; Medicine; Metabolic; Metabolic syndrome; Metabolism; Methods; Microscope; Microscopy; Minor; Modeling; Molecular Biology; Monitor; Mouse Strains; Mus; Muscle; NADH; NF-kappa B; Operative Surgical Procedures; Oxidation-Reduction; Penetrance; Performance; Personal Communication; Phase; Phenotype; Phosphotransferases; Photography; Physical activity; Plasma; Production; Protein Isoforms; Protein Kinase C; Protocols documentation; Pyruvate; Pyruvates; Rattus; Reagent; Records; Regulation; Relative (related person); Reporter; Reporting; Research Personnel; Resolution; Rest; Rodent; Rodent Model; STK11 gene; Scheme; Services; Signal Transduction; Source; Specificity; Spottings; Staging; Staining method; Stains; Subfamily lentivirinae; Supervision; System; Tamoxifen; Technology; Testing; Thermogenesis; Time; Tissues; Training; Transgenes; Transgenic Mice; Transgenic Organisms; Translational Research; Triglycerides; Update; Variant; Vertebral column; Viral; Weight; Western Blotting; Work; atherogenesis; base; cadherin 5; capillary; case control; cost; cryostat; density; experience; feeding; fluorescence microscope; glucose tolerance; grasp; in vivo; instrument; malformation; metabolomics; meter; minimally invasive; mouse model; muscle strength; mutant; pathogen; postnatal; pregnant; professor; programs; promoter; recombinase; research facility; research study; respiratory; retinal rods; small hairpin RNA; transgene expression

Mouse breeding and characterization: An important function of Core B that will be utilized by all components involves the breeding and characterization of conditional LKB1 and SIRT1 mice. The "mouse" component of Aim B will implement a cost-effective facility for the production and maintenance of new mouse strains. The core will perform initial experiments and continually update methods associated with Cre-mediated transgene expression. The core will optimize the production of the mouse models and will provide training to investigators in the Ruderman, Cohen and Walsh laboratory regarding the optimal methods associated with the excision of SIRT1 and LKB1 transgenes. The core will provide investigators with mice to be amplified for their individual experiments. The core will construct conditional SIRT1- and LKB1-deficient mice predominantly using VEcad-Cre- ERT2 (1), but will also use Tie2-Cre if necessary (2). This core will utilize at least 2 transgenic strains of Cre recombinase to circumvent problems that could arise from potential embryonic lethality as well as from the potential lack of endothelial cell specificity of the Cre transgene. The major rationale for the Tie2-Cre transgenic line is that these mice can be obtained commercially as certifiably pathogen-free (The Jackson Laboratory), and work with this strain does not involve an induction protocol. Thus, we are able to initiate our experiments sooner and there is much prior experience with this line. However, we recognize that the Tie-2 line expresses Cre in approximately 90% of cells of the hematopoietic lineage (M. Luisa Iruela-Arispe, personal communication), despite some claims to the opposite in the literature. Thus, the core will undertake a second approach using the tamoxifen-inducible Cre-recombinase under the control of the VE cadherin promoter, and we have obtained these mice from Dr. Iruela-Arispe. This approach is potentially more powerful because the transgene can be induced at different stages of embryonic and postnatal development, it can give rise to a high frequency of endothelial-specific recombination in adult mice, and under some induction conditions can result in a minor (0.3%) subpopulation of bone marrow cells that are positive for the transgene (1). Construction ofSIRTI- and LKB1-deficient mice- The core will utilize the conditional Cre-loxP gene regulation system where the transgene expression is disrupted in a tissue-specific manner. LKB1-floxed mice (3) in the FVB/N background have been provided by Reuben Shaw (The Salk Institute). The SIRT1-floxed mice in a C57/BI6 background were provided by Drs. Sinclair and Alt. The breeding scheme is shown in the Figure. Genotyping will be performed by the core using PCR methods. The core will also backcross mice into the C57/BI6 background when required. The core will also provide investigators with control mice. In most cases control mice will be transgenic (Tg)-Cre-LKB1/SIRT1(+/+)mice. The core will characterize the degree of transgene excision. Using the tamoxifen-inducible Cre in adult mice, endothelial-specific recombination is reported to range from 71-92% (1). For adult experiments, tamoxifen (2 mg) can be delivered IP for 5 consecutive days. To delete LKB or SIRT1 in embryos, tamoxifen is injected IP into pregnant females at specific embryonic stages, and it is reported to achieve 95% excision frequencies (1). Of relevance for endothelial vs. hematopoietic excision, constitutive VE-cadherin-Cre displays a 50-60% penetrance in hematopoietic lineage cells (4). However, the VEcad-Cre-ERT2 can be induced with tamoxifen in adult mice and show a minimal (-0.3%) fraction of bone marrow cells that are positive for recombination (1). Thus, the VEcad-Cre-ERT2 model can potentially be very useful for studies proposed in the PPG because it can be used in a manner to promote excision of LKB1 or SIRT1 in the endothelium vs. hematopoietic lineage and it potentially circumvents problems associated with embryonic vascular malformations and lethality. It should be noted that the Walsh lab is familiar with the manipulation of strains of inducible transgenic mice (5-7), and Walsh lab personnel will materially participate in the operation of Core B which will provide my lab and the other PPG investigators with the "floxed" LKB1 and SIRT1 mice. >4na/yses of LKB1/SIRT1 excision- A number of analyses will be performed to assess the ablation of transgene in the endothelium. First, immunohistochemical analyses with LKB1 or SIRT1 staining will be performed on artery sections from wild-type and floxed mice. Second, LKB1 expression can be assessed in cultured endothelial cells isolated from wild-type, heterozygous- or homozygous-deficient mice by Western blot or real-time PCR analysis. Third, the core will estimate the degree of LKB1/SIRT1 excision by performing parallel experiments on mice where the Tg-Cre strain is crossed with ROSAR26R reporter mice that have a floxed 'stop' cassette upstream of LacZ. Thus the degree of excision can be assessed under different experimental conditions by analyzing histological sections from embryonic and adult mice. These experiments may be warranted because the degree of excision can be dependent on background strain, age, weight and pathological situations (1), and analysis with the ROSAR26R reporter mice would provide information about the behavior of the conditional Cre-loxP gene regulation system under the conditions of our assays. Biochemistry Component (A.K. Saha. PI) (Y. Ido CO-PI): This core will carry out selected analyses that will be utilized by 2 or more projects in the program. In addition, it will assist investigators in setting up certain assays that they will use intensively in their own laboratories. We anticipate that the measurements could include the following a) Tissue and endothelial cell concentrations of adenine nucleotides, (all projects), lactate, pyruvate, malonyl CoA, diacylglycerol, ceramides and triglycerides {S. Itani Diabetes 2003; Saha, AJP 1997} and NAD and NADH and plasma FFA levels. All of these measurements have been carried out by Dr. Saha in connection with studies of malonyl CoA and AMPK regulation in muscle and liver (DK19514), and/or by Dr. Ido in vascular cells (see Project 1 Literature Cited section for full references). It is anticipated that these measurements will be of especial use in determining the basis for AMPK activation (energy state vs redox), and in characterizing the tissue lipid abnormalities induced by the high fed diet when it stimulates atherogenesis. b) Assays related to SIRT1/LKB1 signaling. Dr. Lan working with Dr. Ido has set up assays for LKB Kinase (LKB tide) and many other parameters related to the SIRT1/LKB1 signaling mechanism (see project 1, Aim 1). They will be made available to other program investigators. c) Protein Kinase C: western blots and in some instances activity measurements of specific PKC isoforms (after immunoprecipitation) will be performed {Itani 2003}. Dr. Ido has set up most of these methods for endothelial cells and Dr. Saha has a long experience in studying PKC in other tissues {Itani, S., et. al., Diabetes 2003; Saha, A.K., et. al. Am. J. Physiol. 1997}. Based on recent experience, an increasing use of these methods by all projects is anticipated. 3. Molecular Biology and Cell Culture Components (Y. Ido. PI: K. Walsh. Consultant). A. Viral Constructs and Cell Tagging: Dr. Lan in Dr. Ruderman's lab has been marking and cataloging a number of adenoviruses (listed later) including those for LKB1, LKB1 various mutants, SIRT1 and SIRT1 dominant negative mutants, and dominant negative and constitutively active AMPK. In addition to these adenoviruses, she has made lentivirus expressing shRNA for human and mouse SIRT1. Dr. Ido's lab has been adopting a system which upon request, allows him to produce many different tagged (including flag, his, GST, GFP, DsRed) and backbone (adenovirus, lentivirus, AAV, bacteria and insect expression) constructs using the Gateway system (Invitrogen). His lab has also produced NF-kappaB reporter adenoviruses that detect NF-kB activation status in endothelial cells. The core will provide services to produce these adenoviruses by amplification and purification, as well as lentivirus and various tagged constructs upon request. B. Antibodies: The second task of this core will be to provide information about commercially available and custom made antibodies. Dr. Lan has accumulated information concerning the antibodies relevant for all three projects including those for AMPK, p-AMPK and LKB1. The latter include antibodies for pS428 LKB1 and autophosphorylated (pT336) LKB1, and acetyl-lysine antibodies. The core will provide such information to individual laboratories. Our studies indicate that SIRT1 induced acetylation of LKB1 occurs at K48. Therefore, we will produce a specific antibody to detect LKB1 K48 acetylation. If the antibody is successfully made, the core will distribute it to the individual labs. Dr. Lan will carry out these activities under Dr. Ido's supervision. C. Maintenance of BAECs and HAECs: The third task of this core will be to culture bovine and human aortic endothelial cells (BAEC and HAEC) and distribute them to individual labs. The purpose is to avoid variations that might result from culturing cells from different sources in the individual labs. . In addition, to avoid variability amongst different human donors, the core will buy HAECs from several donors, pool them and provide the individual labs with "standardized" HAECs. Since these cells may change in character with higher passage number, the core will also periodically add younger passage cells to the pool and then re-characterize them based on various characteristics to assure relative biological constancy. D. To assist the Animal Core in the production of cultured endothelial cells from mice with endothelial cell-specific SIRT1 and LKB1 deletion mutations. Dr. Walsh's lab is experienced in culturing mouse lung endothelial cells, and these cells from from flox'd SIRT and LKB1 will be cultured and provided by the core so that individual projects may use them as a knockdown strategy

小鼠育种和表征：Core B 的一个重要功能，将被所有组件利用 涉及条件 LKB1 和 SIRT1 小鼠的繁殖和表征。 “鼠标”组件 Aim B 将建立一个经济高效的设施来生产和维护新的小鼠品系。这 core 将进行初始实验并不断更新与 Cre 介导的转基因相关的方法 表达。该核心将优化小鼠模型的生产，并为研究人员提供培训 在 Ruderman、Cohen 和 Walsh 实验室中，研究与切除相关的最佳方法 SIRT1 和 LKB1 转基因。该核心将为研究人员提供可供个体扩增的小鼠 实验。 核心将主要使用 VEcad-Cre- 构建有条件的 SIRT1 和 LKB1 缺陷小鼠 ERT2 (1)，但如有必要也会使用 Tie2-Cre (2)。该核心将利用至少 2 个 Cre 转基因菌株 重组酶以避免潜在的胚胎致死性以及 Cre 转基因可能缺乏内皮细胞特异性。 Tie2-Cre 的主要理由 转基因品系的一个特点是这些小鼠可以在商业上获得，因为可证明无病原体（杰克逊 实验室），并且使用该菌株的工作不涉及诱导方案。因此，我们能够启动我们的 更快地进行实验，并且在这条线上有很多先前的经验。然而，我们认识到，Tie-2 细胞系在大约 90% 的造血细胞系中表达 Cre（M. Luisa Iruela-Arispe，个人 沟通），尽管文献中有一些相反的说法。因此，核心将进行第二次 使用受 VE 钙粘蛋白启动子控制的他莫昔芬诱导型 Cre 重组酶的方法，以及 我们从 Iruela-Arispe 博士那里获得了这些小鼠。这种方法可能更强大，因为 转基因可以在胚胎和出生后发育的不同阶段诱导，它可以产生高 成年小鼠内皮特异性重组的频率，在某些诱导条件下可能会导致 在转基因呈阳性的一小部分 (0.3%) 骨髓细胞亚群中 (1)。 SIRTI-和LKB1缺陷型小鼠的构建-核心将利用条件Cre-loxP基因调控系统，其中转基因表达以组织特异性方式被破坏。 FVB/N 中的 LKB1-floxed 小鼠 (3) 背景由 Reuben Shaw（索尔克研究所）提供。 C57/BI6 背景中的 SIRT1-floxed 小鼠由 Drs 提供。辛克莱和阿尔特。育种方案如图所示。基因分型将由核心使用 PCR 方法进行。需要时，核心还会将小鼠回交到 C57/BI6 背景中。该核心还将为研究人员提供对照小鼠。在大多数情况下，对照小鼠是转基因 (Tg)-Cre-LKB1/SIRT1(+/+) 小鼠。 核心将表征转基因切除的程度。在成人中使用他莫昔芬诱导型 Cre 据报道，在小鼠中，内皮特异性重组的范围为 71-92% (1)。对于成人实验， 他莫昔芬（2 mg）可以连续 5 天腹膜内注射。要删除胚胎中的 LKB 或 SIRT1，他莫昔芬是 在特定胚胎阶段对怀孕雌性进行IP注射，据报道可以实现95%的切除 频率 (1)。与内皮细胞与造血细胞切除相关，组成型 VE-钙粘蛋白-Cre 显示 造血谱系细胞的外显率为 50-60% (4)。然而，VEcad-Cre-ERT2 可以通过以下方式诱导： 他莫昔芬在成年小鼠中显示出极小部分（-0.3%）的骨髓细胞呈阳性 重组（1）。因此，VEcad-Cre-ERT2 模型对于 中提出的研究可能非常有用。 PPG 因为它可以以某种方式促进内皮细胞中 LKB1 或 SIRT1 的切除。 造血谱系，它有可能规避与胚胎血管相关的问题 畸形和致死率。应该指出的是，沃尔什实验室熟悉菌株的操作 诱导转基因小鼠（5-7只），Walsh实验室人员将实质性参与Core B的运营 这将为我的实验室和其他 PPG 研究人员提供“floxed”LKB1 和 SIRT1 小鼠。 >4na/yses LKB1/SIRT1 切除-将进行大量分析来评估 LKB1/SIRT1 切除的情况 内皮中的转基因。首先，使用 LKB1 或 SIRT1 染色进行免疫组织化学分析 对野生型和 flox 小鼠的动脉切片进行。其次，可以评估 LKB1 的表达 通过蛋白质印迹从野生型、杂合子或纯合子缺陷小鼠中分离培养的内皮细胞 或实时 PCR 分析。第三，核心将通过执行来估计 LKB1/SIRT1 切除的程度 在小鼠上进行平行实验，其中 Tg-Cre 品系与 ROSAR26R 报告小鼠杂交，该报告小鼠具有 LacZ 上游的 floxed“停止”盒。因此，可以在不同的情况下评估切除程度 通过分析胚胎和成年小鼠的组织切片来确定实验条件。这些实验 可能是必要的，因为切除程度可能取决于背景应变、年龄、体重和 病理情况 (1)，使用 ROSAR26R 报告小鼠进行分析将提供有关以下方面的信息： 条件 Cre-loxP 基因调控系统在我们的测定条件下的行为。 生物化学组件 (A.K. Saha. PI) (Y. Ido CO-PI)：该核心将进行选定的分析 将由该计划中的 2 个或更多项目使用。此外，它将协助调查人员设置 他们将在自己的实验室中大量使用某些测定方法。我们预计 测量可包括以下内容 a) 组织和内皮细胞的腺嘌呤核苷酸（所有项目）、乳酸、丙酮酸、 丙二酰辅酶A、二酰甘油、神经酰胺和甘油三酯{S. Itani 糖尿病 2003；萨哈，AJP 1997}以及NAD和NADH以及血浆FFA水平。所有这些测量均已进行 由 Saha 博士在肌肉和肌肉中的丙二酰辅酶 A 和 AMPK 调节研究中提出 肝脏 (DK19514)，和/或 Ido 博士在血管细胞中（参见项目 1 文献引用部分了解完整信息） 参考）。预计这些测量将特别用于确定 AMPK 激活（能量状态与氧化还原）和表征组织脂质的基础 高饲饮食刺激动脉粥样硬化而引起的异常。 b) 与 SIRT1/LKB1 信号传导相关的测定。 Lan 博士与 Ido 博士合作建立了 LKB 检测方法 激酶（LKB潮汐）等许多与SIRT1/LKB1信号机制相关的参数 （参见项目 1，目标 1）。它们将提供给其他项目调查人员。 c) 蛋白激酶 C：蛋白质印迹以及某些情况下特定 PKC 的活性测量 将进行亚型（免疫沉淀后）{Itani 2003}。 Ido 博士已经设置了大部分 这些方法适用于内皮细胞，Saha 博士在研究其他领域的 PKC 方面拥有丰富的经验 组织{Itani，S.，et。等人，《糖尿病》2003 年；萨哈，A.K.等。等人。是。 J.生理学。 1997}。 根据最近的经验，预计所有项目都会越来越多地使用这些方法。 3. 分子生物学和细胞培养成分（Y. Ido. PI：K. Walsh. 顾问）。 A、病毒 构建体和细胞标记：Ruderman 博士实验室的 Lan 博士一直在标记和编目 腺病毒数量（稍后列出），包括 LKB1、LKB1 各种突变体、SIRT1 和 SIRT1 显性失活突变体，以及显性失活和组成型活性 AMPK。此外 针对这些腺病毒，她制作了表达人类和小鼠 SIRT1 shRNA 的慢病毒。博士。 Ido 的实验室一直在采用一种系统，根据要求，他可以生产许多不同的标记 （包括flag、his、GST、GFP、DsRed）和骨干（腺病毒、慢病毒、AAV、细菌和昆虫） 表达）使用 Gateway 系统（Invitrogen）构建。他的实验室还生产了 NF-kappaB 检测内皮细胞中 NF-kB 激活状态的报告腺病毒。核心将提供 通过扩增和纯化生产这些腺病毒以及慢病毒和 根据要求提供各种标记结构。 B. 抗体：该核心的第二个任务是提供 有关市售和定制抗体的信息。兰博士已积累 有关与所有三个项目相关的抗体的信息，包括 AMPK、p-AMPK 的抗体 和LKB1。后者包括 pS428 LKB1 和自磷酸化 (pT336) LKB1 的抗体，以及 乙酰赖氨酸抗体。核心将向各个实验室提供此类信息。我们的研究 表明 SIRT1 诱导的 LKB1 乙酰化发生在 K48。因此，我们将制作一个特定的 抗体检测 LKB1 K48 乙酰化。如果抗体制作成功，核心将进行分发 到各个实验室。 Lan 博士将在 Ido 博士的监督下开展这些活动。三、维护保养 BAEC 和 HAEC：该核心的第三个任务是培养牛和人的主动脉 内皮细胞（BAEC 和 HAEC）并将其分发到各个实验室。目的是为了避免 在各个实验室中培养不同来源的细胞可能会导致差异。 。此外， 为了避免不同人类捐赠者之间的差异，核心将从多个捐赠者那里购买 HAEC，池 并为各个实验室提供“标准化”HAEC。由于这些细胞可能会发生变化 具有较高传代数的字符，核心也会定期向其添加较年轻的传代细胞 池，然后根据各种特征重新表征它们，以确保相对生物学 恒心。 D. 协助 Animal Core 从小鼠体内生产培养的内皮细胞 内皮细胞特异性 SIRT1 和 LKB1 缺失突变。 Walsh博士的实验室在培养方面经验丰富 小鼠肺内皮细胞，这些来自 flox'd SIRT 和 LKB1 的细胞将被培养并 由核心提供，以便各个项目可以将它们用作击倒策略