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：核心B的重要功能，所有组件都将使用 涉及条件LKB1和SIRT1小鼠的繁殖和表征。的“鼠标”组件 AIM B将实施一种具有成本效益的设施，以生产和维护新的老鼠菌株。这 核心将执行初始实验，并连续更新与CRE介导的转基因相关的方法 表达。核心将优化鼠标模型的生产，并将为调查人员提供培训 在鲁德曼（Ruderman），科恩（Cohen）和沃尔什（Walsh）实验室有关与切除相关的最佳方法 SIRT1和LKB1转基因。核心将为调查人员提供小鼠的个人 实验。 核心将构建有条件的SIRT1-和LKB1缺陷小鼠，主要使用vecad-cre- ERT2（1），但在必要时也将使用TIE2-CRE（2）。该核心将使用至少2个转基因菌株 重组酶以规避潜在的胚胎致死性以及从 可能缺乏CRE转基因的内皮细胞特异性。 TIE2-CRE的主要理由 转基因线是这些小鼠可以在商业上以证实无病原体的身份获得（杰克逊 实验室），与这种菌株一起工作不涉及归纳方案。因此，我们能够发起我们的 实验更快，这一行有很多先前的经验。但是，我们认识到Tie-2 线在大约90％的造血谱系细胞中表达CRE（M. Luisa Iruela-arispe，个人 沟通），尽管文献中有一些相反的声称。因此，核心将进行第二次 在VE钙粘蛋白启动子的控制下使用他莫昔芬诱导的CRE成年酶的方法， 我们已经从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产后发育的不同阶段诱导，它可以引起高 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（1）阳性的骨髓细胞的小（0.3％）中。 构建sirti-和LKB1缺陷型小鼠 - 核心将利用条件CRE-loxP基因调节系统，其中转基因表达以组织特异性方式破坏。 FVB/N 背景由鲁本·肖（Reuben Shaw）（Salk Institute）提供。 DRS提供了C57/BI6背景中的SIRT1-串联小鼠。 Sinclair和Alt。繁殖方案显示在图中。基因分型将使用PCR方法通过核心进行。当需要时，核心还将将小鼠回到C57/BI6背景中。核心还将为研究人员提供控制小鼠。在大多数情况下，控制小鼠将是转基因（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的CRE 据报道，小鼠，内皮特异性重组范围为71-92％（1）。对于成人实验， 他莫昔芬（2 mg）可以连续5天传递IP。要在胚胎中删除LKB或SIRT1，他莫昔芬是 在特定的胚胎阶段注射IP进入怀孕的女性，据报道可实现95％的切除 频率（1）。与内皮与造血切除，本构VE-Cadherin-CRE显示的相关性 造血谱系细胞中的50-60％渗透率（4）。但是，可以通过 成年小鼠的他莫昔芬，显示最小（-0.3％）的骨髓细胞阳性 重组（1）。因此，Vecad-Cre-ert2模型对于在该研究中提出的研究可能非常有用 PPG是因为它可以以某种方式促进内皮与内皮中的LKB1或SIRT1切除。 造血谱系，它可能阐明与胚胎血管相关的问题 畸形和致死性。应该注意的是，沃尔什实验室熟悉对菌株的操纵 诱导的转基因小鼠（5-7）和沃尔什实验室人员将实质上参与核心B的操作 这将为我的实验室和其他PPG调查人员提供“ Floxed” LKB1和SIRT1小鼠。 > LKB1/SIRT1切除的4NA/YSE-将进行许多分析以评估消融 内皮中的转基因。首先，用LKB1或SIRT1染色进行免疫组织化学分析将是 在野生型和floxed小鼠的动脉切片上进行。其次，可以评估LKB1表达 培养的内皮细胞从野生型，杂合或纯合的小鼠通过蛋白质印迹分离 或实时PCR分析。第三，核心将通过执行LKB1/SIRT1切除的程度 在TG-CRE菌株与Rosar26R报告菌相交的小鼠上进行平行实验 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 实验条件通过分析胚胎和成年小鼠的组织学切片。这些实验 可能有必要，因为切除程度可能取决于背景压力，年龄，体重和 病理状况（1）和对Rosar26R记者小鼠的分析将提供有关有关的信息 条件Cre-loxp基因调节系统在我们的测定条件下的行为。 生物化学组件（又称saha。pi）（Y. Ido Co-Pi）：此核心将进行选定的分析 该计划中的两个或更多项目将使用。此外，它将帮助调查人员设置 提出某些测定法，他们将在自己的实验室中大量使用。我们预计 测量可能包括以下 a）腺嘌呤核苷酸的组织和内皮细胞浓度（所有项目），乳酸，丙酮酸， Malonyl COA，二酰基甘油，神经酰胺和甘油三酸酯{S. Itani Diabetes 2003; Saha，AJP 1997}，NAD，NADH和血浆FFA水平。所有这些测量已携带 Saha博士与Malonyl COA的研究和AMPK调节有关 肝脏（DK19514）和/或由IDO博士在血管细胞中（请参阅第1个文献，请参见完整的部分 参考）。预计这些测量结果将在确定 AMPK激活的基础（能量状态与氧化还原），并在表征组织脂质的方面 高喂养饮食刺激动脉粥样硬化时引起的异常。 b）与SIRT1/LKB1信号传导有关的测定。 Lan博士与Ido博士合作为LKB设置了测定法 激酶（LKB潮汐）和许多与SIRT1/LKB1信号机制有关的其他参数 （请参阅项目1，AIM 1）。它们将提供给其他计划调查员。 c）蛋白激酶C：蛋白质印迹和在某些情况下特定PKC的活性测量 同工型（免疫沉淀后）将进行{Itani 2003}。伊多博士已经建立了大部分 这些用于内皮细胞和Saha博士的方法在研究其他方面具有很长的经验 组织{Itani，S。，et。 Al。，Diabetes 2003; Saha，又名等。 al。是。 J. Physiol。 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。Chanselant）。 A.病毒 构造和单元格标记：鲁德曼博士实验室的LAN一直在标记和分类 腺病毒的数量（稍后列出），包括LKB1，LKB1各种突变体，SIRT1和 SIRT1显性阴性突变体，以及主动和组成性活性AMPK。此外 对于这些腺病毒，她使慢病毒为人类和小鼠SIRT1表达shrna。博士 伊多（Ido）的实验室一直在采用一个系统，可应要求，允许他生产许多不同的标签 （包括国旗，他的GST，GFP，DSRED）和骨干（腺病毒，慢病毒，AAV，细菌和昆虫 表达式）使用网关系统（Invitrogen）构造。他的实验室也生产了NF-kappab 在内皮细胞中检测NF-KB激活状态的记者腺病毒。核心将提供 通过扩增和纯化以及慢病毒和慢病毒和 根据要求进行各种标记的构造。 B.抗体：该核心的第二任务是提供 有关市售和定制抗体的信息。 Lan博士积累了 与所有三个项目有关的抗体有关的信息，包括AMPK，P-AMPK 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。核心将向单个实验室提供此类信息。我们的研究 表明SIRT1诱导LKB1的乙酰化发生在K48。因此，我们将产生一个特定的 检测LKB1 K48乙酰化的抗体。如果成功制成抗体，则核心将分发 到个人实验室。 LAN博士将在IDO博士的监督下进行这些活动。 C.维护 Baecs和Haecs：该核心的第三个任务是培养牛和人主动脉 内皮细胞（BAEC和HAEC）并将其分配给单个实验室。目的是避免 来自单个实验室中不同来源的细胞可能导致的变化。 。此外， 为了避免不同人类捐助者之间的可变性，核心将从几个捐助者，池中购买HAECS 它们并为各个实验室提供“标准化”的HAEC。由于这些细胞可能会改变 字符具有较高的通过数，核心还将定期将年轻传递单元添加到 池，然后根据各种特征对其进行重新表征以确保相对生物学 恒定。 D.为动物核心提供从小鼠中产生培养的内皮细胞 内皮细胞特异性SIRT1和LKB1缺失突变。沃尔什博士的实验室经验丰富 小鼠肺内皮细胞以及来自Flox'd Sirt和Lkb1的这些细胞将被培养，并且 由核心提供，以便单个项目可以将其用作敲低策略