我们感谢我们的资金来源加拿大基因组和基因组魁北克和蒙特利尔大学以及资金从加拿大创新基金会资助我们的“突触病”（S2D）项目。

1。可能从头突变引起的疾病选择符合下列条件的一种疾病，可配合新生突变假说： <ol><li>生殖健身是减少。 </li><li>尽管各种不同的环境，疾病的频​​率是比较高的和不断的。 </li><li>具有较高的父亲年龄相关的疾病。 </li><li>经典的连锁和关联研究无法解释的疾病遗传的重要部分。 </li><li>双胞胎的一致性数据支持一种新模式。 </li></ol>分析的可能性，一种常见的疾病，诺突变可能部分解释的遗传基础，是关键的第一步。 2。选择的情况下，DNA样本选择适当的样本是新突变的鉴定成功的关键。为了最大限度地寻找新突变的机会，我们提出以下建议： <ol><li>从小发病，严重的表型选择的情况下，未受影响的父母，和老父亲没有关于该疾病的大家庭历史。 </li><li>选择患者的足够可用的DNA进行研究。尤其重要的是从一个没有受到培养的原代细胞源（如血液DNA或唾液中的DNA）的DNA可用性， </li><li>父母双方的DNA的可用性是关键，以确定突变传输状态继承与从头。额外的影响同伙的可用性和正常对照组进行基因验证研究是必要的，一旦发现候选基因。 </li><li>估计样本大小的基础上的突变率，量的基因进行筛选， 并从新生突变的情况下，可能会导致分数的估计的。 </li></ol> 3。基因测序;两个主要途径<ol><li>高品质低通量测序 这种方法是基于对候选基因的方法。 <ol><li>候选基因的选择（S） 选择的最佳人选，根据计分制，这是对6个主要的标准建造的基因。然后计算出对应的总的归结点使用表1中列出的六个标准的总和。看到从我们的项目选择和未选择的基因分布在图1的例子。 <img src="/files/ftp_upload/2534/2534table1.jpg" alt="表1"/> 用于候选基因的选择标准见表1 。 <img src="/files/ftp_upload/2534/2534fig1.jpg" alt="图1"/> 图1显示在我们的项目排序的选择，而不是选定的基因分布图。我们获得了候选人属性，根据自己的分数值排名基因排序的基因分布。例如，SHANK3 NRXN1基因，两个基因，我们发现新生突变，产生了7 ​​分和6（最大为12）。 </li><li>设计引物，通过Exonprimer使用Primers3软件。只有编码区和剪接交界处应涵盖包括一个额外的50外显子两侧的碱基对。 </li><li>优化PCR条件的Taq的选择，反应体积等。 </li><li>优化PCR片段</li><li>根据标准程序，从血液样本中提取的基因组DNA扩增5纳克</li><li>在测序发送之前，做您的PCR产物的质量控制，加载2％琼脂糖凝胶。选定的随机样本。 </li><li>在一个链上的DNA分析仪的序列的PCR产物。如果超过90％的痕迹分析是可能的一个片段被认为是成功测序。这是适用于大规模筛查。 </li><li>变异检测<ol><li>如PolyPhred，Polyscan和突变测量师的基因组变异的基因分型检测和使用工具。 2个以上的检测工具的组合是理想的。例如，PolyPhred V.5和PolyPhred V.6使用默认设置不检测的相同变化。 Polyscan V.3有INDELS（93％）的SNPs（96％）和较高的假阳性突变率。 PolyPhred V.6没有发现真正INDELS的多数，但有假阳性突变率（INDELS）低于Polyscan V.3整体（90％）。我们应该删除Polyscan V.3的单核苷酸多态性检测的选项，都保持PolyPhred V.5和PolyPhred V.6变异检测。突变测量师和Polyscan是为更好地检测INDELS。注：在使用Polyscan时，不应适用于SNP的检测选项。只有“INDEL”选项应。 Polyscan产生许多虚假的SNP的检测阳性。 </li><li>对于每一个独特的小说外显子变异检测，确认手动reamplifying片段和resequencing先证者和父母双方使用反向和正向引物，以消除任何技术神器。 </li></ol></li></ol></li><li>全外显子组测序 这种方法是针对大部分地区的人类基因组编码的高通量测序。我们现在在我们的实验室目前正在使用这种新方法，加速潜在候选基因的检测。 <ol><li>订购“SureSelect人类所有外显子”，针对超过1.6万个基因的编码区（50 MB基因组），罗氏等其他由安捷伦或任何其他同类产品的设计。为了捕捉套件之前，测序平台应确定（Illumina公司对固体）。 </li><li>根据安捷伦的协议，不要捕捉</li><li>提供各自的下一代平台上的产品序列</li><li>从整个外显子组测序的Variant检测：新一代测序平台的基因变异的检测和基因分型的几个生物信息学工具，如宽带无线接入，Bfast，放映机将执行对齐。之后，额外的免费提供的工具（例如SAM工具，Varscan，Annovar）下游将需要它来调用和注释的变种。采用序列比对和变体的调用和注释的商业软件，也可用，如下一代（Softgenetics），中图分类号生物和其他</li></ol></li></ol> 4。优先级基因变种优先跟进根据他们在诺和有害的蛋白或mRNA的功能和/或结构的概率确定的变种。跟进的变种新变种的检测重点应如下： <ol><li> （在一个单一的情况下观察到一次）独特的变化</li><li>变化不存在的父母</li><li>截断蛋白的变化：废话，INDELS导致移码和剪接突变。 </li><li>错义和沉默变化的预测功能破坏性的（例如，影响mRNA剪接）。 Polyphen，上海对外贸易学院和Panther对蛋白质功能的预测效果。 </li></ol>如果使用全外显子组测序，选择候选基因可以被用来作为进一步研究的优先次序变种战略。 5。遗传验证<ol><li>重新排序在其他病例（找出其他致病突变）和控制的整个基因。对照样品将被用来评估优先次序的变异的等位基因频率。包含至少两个不同的从头有害突变（废话，拼接，frameshifts，并预言损害错义）在不同的病人（但不是在控制样品或公共数据库）中发现的任何基因，应高度优先为进一步验证研究。这包括：1，从病人的淋巴母细胞系中的一个潜在的剪接缺陷）测试。根据我们的经验，大多数基因产生淋巴细胞系的RT - PCR产物; 2）调查改变蛋白的表达水平，蛋白提取物的定量免疫印迹分析淋巴细胞系和3）进一步的测试/功能水平的突变基因动物（C线虫，斑马鱼）和细胞株作为先前由本集团（如：5,6）模型。 </li></ol> 6。代表性的成果： 根据这一协议，我们能够识别新的精神分裂症和自闭症的基因。一个例子是我们最近SHANK3基因的发现（图2）。两种不同的从头SHANK3基因突变，无义突变在三个受灾的兄弟和一个错义突变，在一个受影响的女性。 <img src="/files/ftp_upload/2534/2534fig2.jpg" alt="图2"/> 图2。 （一）分隔R1117X家庭PED的419中的影响在三个兄弟的无义突变。先证者的箭头表示（二）R536W错义突变的先证者的分离，但不是在PED的56她不影响弟弟。

<ol>
	<li>Bassett, A. S., Bury, A., Hodgkinson, K. A., Honer, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproductive+fitness+in+familial+schizophrenia.">Reproductive fitness in familial schizophrenia.</a> Schizophr Res. 21, 151-160 (1996).</li><li>Jablensky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schizophrenia:+manifestations,+incidence+and+course+in+different+cultures.+A+World+Health+Organization+ten-country+study.">Schizophrenia: manifestations, incidence and course in different cultures. A World Health Organization ten-country study.</a> Psychol Med Monogr. , 1-97 (1992).</li><li>Malaspina, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schizophrenia+risk+and+paternal+age:+a+potential+role+for+de+novo+mutations+in+schizophrenia+vulnerability+genes.">Schizophrenia risk and paternal age: a potential role for de novo mutations in schizophrenia vulnerability genes.</a> CNS Spectr. 7, 26-29 (2002).</li><li>DeLisi, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+scan+for+linkage+to+chromosomal+regions+in+382+sibling+pairs+with+schizophrenia+or+schizoaffective+disorder.">A genome-wide scan for linkage to chromosomal regions in 382 sibling pairs with schizophrenia or schizoaffective disorder.</a> Am J Psychiatry. 159, 803-812 (2002).</li><li>Gauthier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+Novo+SHANK3+Mutations+in+Patients+Ascertained+for+Schizophrenia.">De Novo SHANK3 Mutations in Patients Ascertained for Schizophrenia.</a> Proc Natl Acad Sci U S A. , (2010).</li><li>Piton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+the+calcium-related+gene+IL1RAPL1+are+associated+with+autism.">Mutations in the calcium-related gene IL1RAPL1 are associated with autism.</a> Hum Mol Genet. 17, 3965-3974 (2008).</li><li>Krawczak, M., Ball, E. V., Cooper, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neighboring-nucleotide+effects+on+the+rates+of+germ-line+single-base-pair+substitution+in+human+genes.">Neighboring-nucleotide effects on the rates of germ-line single-base-pair substitution in human genes.</a> Am J Hum Genet. 63, 474-488 (1998).</li></ol>

这里列出的程序的目的是确定具体的常见疾病，可能导致部分，从诺突变，并证明这一假说。从头突变的一些疾病，例如遗传性癌症综合征发展为建立良好的机制，但一直不佳，常见疾病的探讨。这从头突变，这就需要大量的DNA，它只有测序鉴定所涉及的技术挑战的一部分结果最近成为新一代测序的到来有效的成本。此外， 重新在人类的突变率，直到最近，只是一个估计。只是最近，有报道直接确定在人类的突变率。这些测量前，这是很难预测这类研究所需的样本大小，以确定所观察到的新生突变率大于基准率。全基因组测序与候选基因？由于大多数疾病突变错义/无义突变和剪接位点突变（根据HGMD网站）我们的筛选策略将确定已知的突变超过68％。也有一个氨基酸替换的严重性和可能性的临床表型之间的明确关系。一个保守的氨基酸替换相比，废话变化是9.0倍，可能目前临床上7。因此，在这个时候候选基因测序是最具成本效益的策略。 概述过程的成功取决于几个关键步骤，这是中详细概述，并举例说明了使用了两个例子，自闭症和精神分裂症。有很多陷阱需要避免，如选择何种疾病，患者到屏幕上，源DNA，和如何有效地识别诺突变的详细信息，。我们提供最有效的方法，确定任何疾病，这种自发突变的结果的情况下的一小部分。

a strategy to identify de novo mutations in common disorders such as autism and schizophrenia

有证据支持机制，为常见的疾病，如自闭症和精神分裂症，从头突变作用的几行。首先， 重新在人类的突变率是比较高的的，所以在人口中的高频率产生新的基因突变。然而， 从头突变没有被报道的最常见的疾病。导致严重的疾病，在胚胎阶段或减少生殖健身有一个强烈反对负选择表型，如杀伤力的基因突变，不会被发送到多个家庭成员，并因此不会被检测联动基因图谱或协会研究。同卵双胞胎中的一致性非常高的观察和非常低的异卵双胞胎的一致性也强烈支持这一假设，相当一部分的情况下，可能会导致从新的基因突变。疾病，如自闭症和精神分裂症的情况下是这样。其次，尽管降低生殖健身1和一个变数极大的环境因素，对一些疾病的发病率是全世界保持在一个相对较高的恒定速率。这是孤独症和精神分裂症的情况下，与全世界的发病率约为1％。突变负荷可以被认为是一个新生突变的有害突变和其生产或反对之间选择平衡。繁殖率较低，构成了一个消极的选择，应减少在人群中的突变​​的等位基因的数量，最终导致疾病的患病率下降的因素。这些选择性的压力往往是在不同的环境不同强度。然而，这些严重的精神疾患，一直保持在一个恒定在跨文化和国家的广泛的全球人口的患病率相对较高，尽管一个强烈反对他们2负选择。这是不是什么人会预测在降低生殖健身疾病，除非有新的突变率。最后，父亲年龄的影响：有是一个疾病的风险显着增加，随着父亲年龄的增加，这可能导致从年龄增加从头在父亲的基因突变有关。这是自闭症和精神分裂症3。突变率的男性与女性的比例估计约为4-6:1，大概是由于岁男性的生殖细胞分裂。因此，人会预测， 从头突变会更频繁的男性，尤其是老年男性4。一个新的基因突变率很高，可能部分解释遗传研究为什么迄今未能确定诱发复合物疾病的基因，如自闭症和精神分裂症有关的许多基因，以及为什么疾病已经确定人类基因组中只有3％的基因。鉴定从头突变引起的一种疾病，需要有针对性的分子的方法，其中包括学习的父母和受影响的学科。确定某种疾病的遗传基础，可能会导致部分从头突变和分子的方法来建立这个链接将作为例子说明，使用自闭症和精神分裂症的过程。

Watch this Scientific Journal Video about A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia at JoVE.com

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

寻找从头突变导致自闭症和精神分裂症等常见疾病的分子遗传学战略。

一项战略，以确定在常见的疾病，如自闭症和精神分裂症的新突变

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

a-strategy-to-identify-de-novo-mutations-common-disorders-such-as

Research

JoVE Journal

Medicine

25.6K 次观看.  Universite de Montreal. 寻找从头突变导致自闭症和精神分裂症等常见疾病的分子遗传学战略。

视频: A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

双侧颈总动脉闭塞足够的预处理刺激诱导局灶性脑缺血早期缺血耐受

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

科研

医学

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

该<em&gt;在OVO</em&gt; CAM作为一个肉瘤Xenograftmodel的检测

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e. smooth muscle, mucosa, nerves) in healthy and pathological conditions.
The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.
The in vitro smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.

This manuscript presents a simple, yet powerful, in vitro method for evaluating smooth muscle contractility in response to pharmacological agents or nerve stimulation. Main applications are drug screening and understanding tissue physiology, pharmacology,&#160;and pathology.

膀胱平滑肌收缩带作为一种方法来评估下尿路药理学

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties ...

Fundus Photography as a Convenient Tool to Study Microvascular Responses to Cardiovascular Disease Risk Factors in Epidemiological Studies

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an important role in maintaining cardiovascular health. The retina is a tissue that lines the interior of the eye and it is the only tissue that allows for a non-invasive analysis of the microvasculature. Nowadays, high-quality fundus images can be acquired using digital cameras. Retinal images can be collected in 5 min or less, even without dilatation of the pupils. This unobtrusive and fast procedure for visualizing the microcirculation is attractive to apply in epidemiological studies and to monitor cardiovascular health from early age up to old age.
Systemic diseases that affect the circulation can result in progressive morphological changes in the retinal vasculature. For example, changes in the vessel calibers of retinal arteries and veins have been associated with hypertension, atherosclerosis, and increased risk of stroke and myocardial infarction. The vessel widths are derived using image analysis software and the width of the six largest arteries and veins are summarized in the Central Retinal Arteriolar Equivalent (CRAE) and the Central Retinal Venular Equivalent (CRVE). The latter features have been shown useful to study the impact of modifiable lifestyle and environmental cardiovascular disease risk factors.
The procedures to acquire fundus images and the analysis steps to obtain CRAE and CRVE are described. Coefficients of variation of repeated measures of CRAE and CRVE are less than 2% and within-rater reliability is very high. Using a panel study, the rapid response of the retinal vessel calibers to short-term changes in particulate air pollution, a known risk factor for cardiovascular mortality and morbidity, is reported. In conclusion, retinal imaging is proposed as a convenient and instrumental tool for epidemiological studies to study microvascular responses to cardiovascular disease risk factors.

Retinal image analysis is an unobtrusive procedure for visualizing the microcirculation. The impact of cardiovascular disease risk factors can result in changes of retinal vessel calibers. The procedures to acquire fundus images and the steps for calculating the vessel calibers are described.

眼底摄影作为一种方便的工具，研究微血管回应流行病学研究心血管疾病的风险因素

The microcirculation consists of blood vessels with diameters less than 150 &#181;m. It makes up a large part of the circulatory system and plays an ...

Sublingual Immunotherapy as an Alternative to Induce Protection Against Acute Respiratory Infections

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been shown to effectively induce humoral and cellular responses at systemic and mucosal sites, namely the lungs and urogenital tract. Sublingual vaccination can promote protection against infections at the lower and upper respiratory tract; it can also promote tolerance to allergens and ameliorate asthma symptoms. Modulation of lung&#8217;s immune response by sublingual immunotherapy (SLIT) is safer than direct administration of formulations by intranasal route because it does not require delivery of potentially harmful molecules directly into the airways. In contrast to intranasal delivery, side effects involving brain toxicity or facial paralysis are not promoted by SLIT. The immune mechanisms underlying SLIT remain elusive and its use for the treatment of acute lung infections has not yet been explored. Thus, development of appropriate animal models of SLIT is needed to further explore its potential advantages. 
This work shows how to perform sublingual administration of therapeutic agents in mice to evaluate their ability to protect against acute pneumococcal pneumonia. Technical aspects of mouse handling during sublingual inoculation, precise identification of sublingual mucosa, draining lymph nodes and isolation of tissues, bronchoalveolar lavage and lungs are illustrated. Protocols for single cell suspension preparation for FACS analysis are described in detail. Other downstream applications for the analysis of the immune response are discussed. Technical aspects of the preparation of Streptococcus pneumoniae inoculum and intranasal challenge of mice are also explained.
SLIT is a simple technique that allows screening of candidate molecules to modulate lungs&#8217; immune response. Parameters affecting the success of SLIT are related to molecular size, susceptibility to degradation and stability of highly concentrated formulations.

The present work illustrates the convenience of using sublingual immunotherapy to boost the innate immune response in the lungs and confer protection against acute pneumococcal pneumonia in mouse.

舌下免疫治疗作为一种替代起到保护作用对急性呼吸道感染

Sublingual route has been widely used to deliver small molecules into the bloodstream and to modulate the immune response at different sites. It has been ...

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

使用腺相关病毒作为一种工具来研究疾病中的视网膜障碍

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Surgical Injury to the Mouse Pancreas through Ligation of the Pancreatic Duct as a Model for Endocrine and Exocrine Reprogramming and Proliferation

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide new expandable sources of beta cells to alleviate the donor scarcity in human islet transplantation as therapy for diabetes. Although recent advances have been made towards this aim, mechanisms that regulate beta cell expansion and differentiation from a stem/progenitor cell remain to be characterized. Here, we describe a protocol for an injury model in the adult mouse pancreas that can function as a tool to study mechanisms of tissue remodeling and beta cell proliferation and differentiation. Partial duct ligation (PDL) is an experimentally induced injury of the rodent pancreas involving surgical ligation of the main pancreatic duct resulting in an obstruction of drainage of exocrine products out of the tail region of the pancreas. The inflicted damage induces acinar atrophy, immune cell infiltration and severe tissue remodeling. We have previously reported the activation of Neurogenin (Ngn) 3 expressing endogenous progenitor-like cells and an increase in beta cell proliferation after PDL. Therefore, PDL provides a basis to study signals involved in beta cell dynamics and the properties of an endocrine progenitor in adult pancreas. Since, it still remains largely unclear, which factors and pathways contribute to beta cell neogenesis and proliferation in PDL, a standardized protocol for PDL will allow for comparison across laboratories.

This protocol describes an injury model involving the surgical ligation of the pancreatic duct in the adult mouse pancreas, resulting in severe injury that establishes an environment that allows beta cell neogenesis and proliferation. This model can be used as a tool to study mechanisms involved in beta cell formation.

通过胰管结扎手术损伤的小鼠胰腺作为模型内分泌和外重新规划和扩散

Expansion of pancreatic beta cells in vivo or ex vivo, or generation of beta cells by differentiation from an embryonic or adult stem cell, can provide ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

体内兔颈总动脉内皮的基因转移

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...

An Experimental Model of Diet-Induced Metabolic Syndrome in Rabbit: Methodological Considerations, Development, and Assessment

In recent years, obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased prevalence due to the rise of sedentary lifestyles and unhealthy eating habits. Thanks to animal models, basic research can investigate the mechanisms underlying pathological processes such as MetS. Here, we describe the methods used to develop an experimental rabbit model of diet-induced MetS and its assessment. After a period of acclimation, animals are fed a high-fat (10% hydrogenated coconut oil and 5% lard), high-sucrose (15% sucrose dissolved in water) diet for 28 weeks. During this period, several experimental procedures were performed to evaluate the different components of MetS: morphological and blood pressure measurements, glucose tolerance determination, and the analysis of several plasma markers. At the end of the experimental period, animals developed central obesity, mild hypertension, pre-diabetes, and dyslipidemia with low HDL, high LDL, and an increase of triglyceride (TG) levels, thus reproducing the main components of human MetS. This chronic model allows new perspectives for understanding the underlying mechanisms in the progression of the disease, the detection of preclinical and clinical markers that allow the identification of patients at risk, or even the testing of new therapeutic approaches for the treatment of this complex pathology.

We describe methods to develop an experimental model of diet-induced metabolic syndrome (MetS) in rabbits using a high-fat, high-sucrose diet. Animals developed central obesity, mild hypertension, pre-diabetes, and dyslipidemia, thus reproducing the main components of human MetS. This chronic model will allow acquisition of knowledge underlying mechanisms of disease progression.

家兔膳食诱导代谢综合征的实验模型: 方法论考虑、发展和评价

In recent years, obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased ...

A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, intimal hyperplasia, and atherosclerosis. The goal of this article is to demonstrate a method for placing bilateral venous interposition grafts in a rabbit, then transducing the grafts with a gene transfer vector that achieves durable transgene expression. The method allows the investigation of the biological roles of genes and their protein products in normal vein graft homeostasis. It also allows the testing of transgenes for the activities that could prevent vein graft failure, e.g., whether the expression of a transgene prevents the neointimal growth, reduces the vascular inflammation, or reduces atherosclerosis in rabbits fed with a high-fat diet. During an initial survival surgery, the segments of right and left external jugular vein are excised and placed bilaterally as reversed end-to-side common carotid artery interposition grafts. During a second survival surgery, performed 28 days later, each of the grafts is isolated from the circulation with vascular clips and the lumens are filled (via an arteriotomy) with a solution containing a helper-dependent adenoviral (HDAd) vector. After a 20-min incubation, the vector solution is aspirated, the arteriotomy is repaired, and flow is restored. The veins are harvested at time points dictated by individual experimental protocols. The 28-day delay between the graft placement and the transduction is necessary to ensure the adaptation of the vein graft to the arterial circulation. This adaptation avoids rapid loss of transgene expression that occurs in vein grafts transduced before or immediately after grafting. The method is unique in its ability to achieve durable, stable transgene expression in grafted veins. Compared to other large animal vein graft models, rabbits have advantages of low cost and easy handling. Compared to rodent vein graft models, rabbits have larger and easier-to-manipulate blood vessels that provide abundant tissue for analysis.

This method describes the placement of interposition vein grafts in rabbits, the transduction of the grafts, and the achievement of durable transgene expression. This allows the investigation of physiological and pathological roles of transgenes and their protein products in grafted veins, and testing of gene therapies for vein graft disease.

颈动脉介入移植中颈静脉持久性转基因表达的家兔模型

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, ...