在这项研究中所描述的这项研究是由一个国家科学基金会的资助（DEB - 0805818）WAF的支持，AWS和ID这项研究也支持补助的IOS - 0845847 AWS，IOS - 0919855 AWS和ID和IOS - 0920720 WAF

<html> 1。饲养和操纵的饮食生产在车身尺寸和机翼大小的变化 理性和概述。缩放关系最终取决于的发展，产生最后的器官和身体大小的变化的监管过程中的表达。这些进程是最好的工作， 如 D.完全变质（即holometabolous）昆虫果蝇 ，在器官内生长为未分化的"成虫光盘"，喜欢GRUB 幼虫 11,15-17 。在果蝇中，最后的器官和身体大小是受经济增长在幼虫阶段，通过第一和第二的幼虫龄幼虫成长，直到他们达到最小的羽化可行的重量（MVW五）对第三龄幼虫18开始。实现MVW 发送前的饥饿防止成人羽化。然而，MVW 发送饿死的幼虫产生可行的成年人，尽管在一个非常小的的尺寸。这是因为在开发过程中的营养减少，降低最终的身体和器官的大小，不只是在果蝇，但在几乎所有的动物19-21。通过操纵MVW 发送后的粮食供应达到，但化蛹发生之前，有可能诱发极端在车身尺寸的表型可塑性，大大超过了其他来源的变化（例如，个人的遗传变异）。 <ol><li>鸡蛋收集phenotyped人口，分为很多50放置在含瓶标飞食品10毫升。由于产卵不断发生，卵被收集三天每24小时，生产3个年龄组。 </li><li>当最古老的人群达到徘徊阶段，或刚刚开始化蛹，饮食操纵应用。在这一点上，最古老的人群，远远超出了 MVW发送，最年轻的世代是非常接近MVW发送，其余的队列是介于这两个极端之间。因此，在这一点上去除食物会产生非常大的成年人，从最古老的队列，从剩余的队列从最年轻的队列和中等大小的成人非常小大人。要删除从小瓶的幼虫，大约40％的蔗糖溶液5毫升的被添加到所有小瓶。为了自由的幼虫，食物是激动摇床表上15-20分钟设置一个中间速度。 </li><li>罚款（00）画笔的​​蔗糖溶液，放入含有湿棉塞的小瓶，并允许化蛹的幼虫从浮动。另外，幼 ​​虫可能被放置在一个不易消化的食品添加剂，如甲基纤维素 22 。 </li></ol> 2。车身尺寸的估计 理性和概述 。以往的研究表明，胸部的长度是较理想的整体机身尺寸为 22代理。相反，我们使用蛹的大小作为衡量的车身尺寸。最大的车身尺寸是固定在化蛹之前停止喂养。因此，有一个蛹的大小和成年体大小23之间的紧密相关。 <ol><li>蛹腹侧被安排在显微镜幻灯片和影像使用数码相机连接在解剖显微镜。每幅图像被捕获，分配一个唯一的识别代码，并转移到一台计算机，通过现场的数码相机连接到计算机的饲料。 </li><li>成像蛹被转移到个人2毫升epitubes含飞食品1毫升，标有个别的独特代码，并刺破气孔，使气体交换。 </li><li>蛹的规模估计为蛹的轮廓的像素数量。也可以用于其他规格指标。 </li></ol> 3。性状（翼）规模估计 理性和概述 。图像的翅膀，我们使用了"右翼采集"24日 ，拉，并拥有现场，麻醉飞垂直于它的身体翼。两片玻璃之间的压力，这使机翼作为一个二维对象的影像。 <ol><li>苍蝇麻醉轻轻CO 2和放置在机翼上装有一个连接到电脑上的数码相机，在解剖显微镜采集的地位。 </li><li>翼下，个人的独特代码成像和保存。 </li><li>翼的大小，可以使用最小的凸多边形，circumscribes像素数为翼的剪影边际固定标志或估计。 </li></ol> 4。比例关系估计<ol><li>跨世代的数据汇集，但按性别分开数转换。 </li><li>比例关系是适合使用II型（即减少主要轴）回归（见12检讨），并为每个组估计参数的大小数据。 </li></ol> 5。代表性的成果: 帐篷"我们的饮食操纵将产生广泛的身体尺寸（图1）和特征尺寸（图2）。绘图针对对方的日志转化特征，揭示了在一个给定的生物组的协变格局，并允许符合比例关系车身尺寸的形状尺度组（图3）提取的比例关系参数描述如何允许跨组的大小形状covaries比较。 <img alt="图1" src="/files/ftp_upload/3162/3162fig1.jpg" /> 图1。蛹后通过饮食操纵产生的粒度分布的极端个人实现MVW发送。实际蛹的图像显示在左蛹的大小在右边的测量及其相应的剪影。比例尺为1mm。 <img alt="图2" src="/files/ftp_upload/3162/3162fig2.jpg" /> 从图2。翅膀后通过饮食操纵产生的车身尺寸分布的极端个人实现 MVW发送。实际翼图像显示在左侧显示在右侧的机翼尺寸测量其相应的剪影。比例尺为1mm。 <img alt="图3" src="/files/ftp_upload/3162/3162fig3.jpg" /> 图3。翼身果蝇的性别大小缩放。数转换为每个性别完全美联储（黑眼圈），饿死（灰色圆圈）或两天（空心圆）作为幼虫的个人数据。 II型回归线显示每个性别。每个年龄组之间，平均机翼面积下降〜0.1毫米（〜7％）和〜0.2毫米（〜8％）时，增加观察到表型的总范围。回归分析显示，女性稍hypoallometric这个比例关系（坡度为0.96），而男性稍hyperallometric（斜率1.06）。

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+regulation+of+the+insulin-like+growth+factors.">Nutritional regulation of the insulin-like growth factors.</a> Endocr. Rev. 15, 80-101 (1994).</li><li>Boots, M., Begon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resource+limitation+and+the+lethal+and+sublethal+effects+of+a+viral+pathogen+in+the+Indian+meal+moth,+Plodia+interpunctella.">Resource limitation and the lethal and sublethal effects of a viral pathogen in the Indian meal moth, Plodia interpunctella.</a> Ecol. Entomol. 19, 319-326 (1994).</li><li>Shingleton, A. W., Mirth, C. K., Bates, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+model+of+static+allometry+in+holometabolous+insects.">Developmental model of static allometry in holometabolous insects.</a> Proc. R. Soc. B. 275, 1875-1885 (2008).</li><li>Stern, D. L., Emlen, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+basis+of+allometry+in+insects.">The developmental basis of allometry in insects.</a> Development. 126, 1091-1101 (1999).</li><li>Emlen, D. J., Allen, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotype+to+phenotype:+Physiological+control+of+trait+size+and+scaling+in+insects.">Genotype to phenotype: Physiological control of trait size and scaling in insects.</a> Integr. Comp. 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没有利益冲突的声明。

形态缩放已经近一个世纪以来，在激烈的进化生物学研究的主题。最近，切近的基础上的增长和缩放调控已成为越来越重要的发展，生理学和遗传学（例如，看到在12,15,23-25 ​​评论）。我们的方法允许估计整个形态特征和身体大小的整个范围缩放关系。这可能是重要的，为特征的大小可能不与车身尺寸（如13,14）线性规模。然而，这可能不很明显，如果动物只在理想的实验室条件下饲养。 虽然我们的协议是明确允许翼的车身尺寸在果蝇中的比例关系估计，它可以用来与特质，具体的修改，估计任何苍蝇的形态特征的比例关系。同样，饮食操纵也可能被用来生产其他完全变质昆虫的特征和身体大小的全方位，这些应用仅仅需要知道什么时候该MVW E是在这一点上取得和运用在个体发育的操纵。

experimental manipulation of body size to estimate morphological scaling relationships in drosophila

身体各部位的比例是动物形态 1-7的核心特征。内，物种形态特征需要​​正确的比例，对身体功能的有机体;较大的人通常有较大的身体部位和个体较小，一般都较小的身体部位，这样，整个体形是保持整个成年体尺寸范围。正确比例的规定，是指物种内的个体通常表现在低相对性状的大小变化。相反，相对性状的大小显着不同物种之间和形态多样，是生产的一个主要机制。超过一个世纪的比较工作，建立这些区域内和种间模式3,4。 也许使用最广泛的方法来描述这种变化是计算使用异速生长方程的两个形态特征的大小之间的比例关系为Y =bxα，其中X和Y是两个特征，如器官和身体的大小为8，大小9。该方程描述了两个性状之间的组内（例如，物种，种群）的比例关系，无论在规模各不相同。此方程的日志转化生成一个简单的线性方程组，日志（Y）= LOG（B）+αlog（x）和日志记录图，同一物种的个体之间的不同性状的大小通常揭示一个拦截线性缩放日志（B）和α斜坡，被称为"异速系数"9,10 。群体间的形态变异是描述一个给定的特征对不同比例关系截获或斜坡。因此，异速生长方程的参数（b和α）的变化，优雅的描述的形状变化，器官和身体大小之间的关系内部和相互之间的生物群体（见11,12）抓获。 并非所有的特征相互或与车身尺寸（ 如 13,14），因此线性规模，形态比例关系最翔实数据时是采取全方位的特征尺寸。在这里，我们描述了如何简单的饮食实验操作可用于在昆虫产生全方位的车身尺寸。这允许任何性状对全比例关系的估计，从而使一个如何塑造covaries规模和强大的生物群体之间的比例关系参数的比较完整的描述。虽然我们对果蝇的重点，我们的方法应该适用于几乎任何完全变质的昆虫。

Watch this Scientific Journal Video about Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila at JoVE.com

Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila

形态缩放关系的捕获和描述有机体的形状。我们提出了一个方法来衡量整个完全变质的昆虫体型自然形态缩放关系。使用一个简单的饮食操控，我们增加的特征尺寸的分布，允许的形状和大小如何变化的准确描述。

实验操作的车身尺寸估计​​形态缩放关系果蝇</em

The scaling of body parts is a central feature of animal morphology1-7. Within species, morphological traits need to be correctly proportioned to the body ...

experimental-manipulation-body-size-to-estimate-morphological-scaling

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila

13.7K 次观看.  University of Houston. 形态缩放关系的捕获和描述有机体的形状。我们提出了一个方法来衡量整个完全变质的昆虫体型自然形态缩放关系。使用一个简单的饮食操控，我们增加的特征尺寸的分布，允许的形状和大小如何变化的准确描述。

视频: Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila

Experimental Approaches to Tissue Engineering

组织工程的实验方法

科研

生物学

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

在芽殖酵母中的蛋白浓度的功能形态学表型的发展分析

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

影像生活细胞的形状变化果蝇胚胎

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

使用屏幕在车身尺寸规例中的线虫基因介导的RNA干扰取食策略<em&gt; C.线虫</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

对于外来体定量和尺寸测量新方法研究

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ecologically-important endpoints, though they are rarely considered in toxicological studies. D. melanogaster is emerging as a toxicology model for the behavioral, neurological, and genetic impacts of toxicants, to name a few. More importantly, species in the genus Drosophila can be utilized as a model system for an integrative framework approach to incorporate emergent properties and answer ecologically-relevant questions in&#160;toxicology research. The aim of this paper is to provide a protocol for exposing species in the genus Drosophila to pollutants to be used as a model system for a range of phenotypic outputs and ecologically-relevant questions. More specifically, this protocol can be used to 1) link multiple biological levels of organization and understand the impact of toxicants on both individual- and population-level fitness; 2) test the impact of toxicants at different stages of developmental exposure; 3) test multigenerational and evolutionary implications of pollutants; and 4) test multiple contaminants and stressors simultaneously.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.

利用果蝇作为无脊椎动物毒性试验模型系统的实验协议

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ...

Noninvasive Monitoring of Lesion Size in a Heterologous Mouse Model of Endometriosis

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time through noninvasive monitoring of fluorescence emitted by implanted ectopic human endometrial tissue. For this purpose, biopsies of human endometrium are obtained from donor women ongoing oocyte donation. Human endometrial fragments are cultured in the presence of adenoviruses engineered to express cDNA for the reporter fluorescent protein mCherry. Upon visualization, labeled tissues with an optimal rate of fluorescence after infection are subsequently chosen for the implantation in recipient mice. One week prior to the implantation surgery, recipient mice are oophorectomized, and estradiol pellets are placed subcutaneously to sustain the survival and growth of lesions. On the day of surgery mice are anesthetized, and peritoneal cavity accessed through a small (1.5 cm) incision by the linea-alba. Fluorescently labeled implants are tweezed, briefly soaked in glue and attached to the peritoneal layer. Incisions are sutured, and animals left to recover for a couple of days. Fluorescence emitted by endometriotic implants is usually non-invasively monitored every 3 days for 4 weeks with an in vivo imaging system. Variations in the size of endometriotic implants can be estimated in real time by quantification of the mCherry signal and normalization against the initial time-point showing maximal fluorescence intensity.
Traditional preclinical rodents of models of endometriosis do not allow non-invasive monitoring of lesion in real time but rather allow evaluation of the effects of drugs assayed at the end point. This protocol allows one to track lesions in real time and is more useful to explore the therapeutic potential of drugs in preclinical models of endometriosis. The main limitation of the model thus generated is that non-invasive monitoring is not possible over long periods of time due to the episomal expression of Ad-virus.

Here, we present a protocol for live-imaging of fluorescently labeled human endometrial fragments grafted in mice. The method allows studying the effects of drugs of choice on endometriotic lesion size through monitoring and quantification of fluorescence emitted by the fluorescent reporter on real time

子宫内膜异位症异种小鼠病变大小的无创监测

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time ...

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of assisted reproduction techniques such as oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and cloning of domestic animals by nuclear transfer from somatic cell. IVM is the method particularly of significance. It is the platform technology for the supply of mature, good quality oocytes for applications such as reduction of the generation interval in commercially important or endangered species, research concerning in vitro human reproduction, and production of transgenic animals for cell therapies. The term oocyte quality includes its competence to complete maturation, be fertilized, thereby resulting in healthy offspring. This means that oocytes of good quality are paramount for successful fertilization including IVF procedures. This poses many difficulties to develop a reliable culture method that would support growth not only of human oocytes but also of other large mammalian species. The first step in IVM is the in vitro culture of oocytes. This work describes two protocols for the 3D culture of porcine oocytes. In the first, 3D model cumulus-oocyte complexes (COCs) are encapsulated in a fibrin-alginate bead interpenetrating network, in which a mixture of fibrin and alginate are gelled simultaneously. In the second one, COCs are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles to form microbioreactors defined as Liquid Marbles (LM). Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, thereby preserving the functional relationship between the oocyte, and surrounding follicular cells.

Presented here are two protocols for the encapsulation of porcine oocytes in 3D culture conditions. In the first, cumulus-oocyte complexes (COCs) are encapsulated in fibrin-alginate beads. In the second, they are enclosed with fluorinated ethylene propylene powder particles (microbioreactors). Both systems ensure optimal conditions to maintain their 3D organization.

蛋白酶降解藻酸盐水凝胶和疏水性微生物反应器，用于猪卵母细胞封装

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of ...

Establishment and Genetic Manipulation of Murine Hepatocyte Organoids

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the executor for most of the liver functions, primary hepatocytes have limited proliferating capacity. This requires the establishment of ex vivo hepatocyte expansion models for liver physiological and pathological research. Here, we isolated murine hepatocytes by two steps of collagenase perfusion and established a 3D organoid culture as the 'mini-liver' to recapitulate cell-cell interactions and physical functions. The organoids consist of heterogeneous cell populations including progenitors and mature hepatocytes. We introduce the process in detailed to isolate and culture the murine hepatocytes or fetal hepatocyte to form organoids within 2-3 weeks and show how to passage them by mechanically pipetting up and down. In addition, we will also introduce how to digest the organoids into single cells for lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering. The organoids can be used for drug screens, disease modelling, and basic liver research by modeling liver biology and pathobiology.

A long-term ex vitro 3D organoid culture system was established from mouse hepatocytes. These organoids can be passaged and genetically manipulated by lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering.

实验操作的车身尺寸估计形态缩放关系果蝇

摘要

探索更多视频

此视频中的章节

组织工程的实验方法

在芽殖酵母中的蛋白浓度的功能形态学表型的发展分析

影像生活细胞的形状变化果蝇胚胎

使用屏幕在车身尺寸规例中的线虫基因介导的RNA干扰取食策略

凋亡细胞清除过程中实时成像

对于外来体定量和尺寸测量新方法研究

利用果蝇作为无脊椎动物毒性试验模型系统的实验协议

子宫内膜异位症异种小鼠病变大小的无创监测

蛋白酶降解藻酸盐水凝胶和疏水性微生物反应器，用于猪卵母细胞封装

小鼠肝细胞类器官的建立与遗传操作

实验操作的车身尺寸估计​​形态缩放关系果蝇

摘要

探索更多视频

此视频中的章节

组织工程的实验方法

在芽殖酵母中的蛋白浓度的功能形态学表型的发展分析

影像生活细胞的形状变化果蝇胚胎

使用屏幕在车身尺寸规例中的线虫基因介导的RNA干扰取食策略

凋亡细胞清除过程中实时成像

对于外来体定量和尺寸测量新方法研究

利用果蝇作为无脊椎动物毒性试验模型系统的实验协议

子宫内膜异位症异种小鼠病变大小的无创监测

蛋白酶降解藻酸盐水凝胶和疏水性微生物反应器，用于猪卵母细胞封装

小鼠肝细胞类器官的建立与遗传操作

实验操作的车身尺寸估计形态缩放关系果蝇