这项工作是由摆围赠款＃TR-1250，电阻RT1-01108，CL1-00502。

注：所有动物的程序必须由IACUC或相当于批准。 所有手术设备，手术前必须经过消毒。必须使用无菌手套，窗帘和纱布。 1。手术前准备<ol><li>获得6周大的小家鼠CbySmn.CB17-Prkdc SCID鼠/ J的雄性小鼠或其他免疫功能低下小鼠品系。 </li><li>消毒所有的手术器械，手套和纱布。 </li><li>游离hPSCs被与accutase注射。 </li><li>计数细胞和悬浮1,000,000每20-30μL细胞基底膜稀释DMEM/F-12 1:1。细胞置于冰上，直到完成。 </li><li>请注意，往往通过注射台盼蓝实验运行是有益的事情。这将有助于识别任何潜在的问题，在任何细胞都浪费掉了。 </li><li>在饲养箱，麻醉你的鼠标，根据你的机构接受的程序。在我们的实验中，我们使用了麻醉ESIA机与异氟醚。小鼠最初被放置在一个感应室1L/min氧和3-4％的异氟醚。一旦麻醉，鼻锥与1L/min的氧气和2-3％的异氟醚。 </li><li>剃光与快船队的腹部，并清理鼠标腹部前壁，从腹部的中心开始向外顺时针方向和工作。首次使用聚维酮碘溶液，然后用70％乙醇。重复3次，每次改变拭子。动物转移到一个加热垫，保持温暖的动物组织培养或解剖罩内。 </li><li>使1厘米的纵向切口，通过皮肤与腹膜无菌手术剪刀，略低于髋关节水平。 </li><li>腹膜，用镊子走向右臀达到另一个无菌forcept和白色脂肪组织连同附睾丸拉出。 </li><li>睾丸放在无菌纱布。 </li><li>补充结核菌素或哈密尔顿（1CC）注射器无线TH的hPSCs被注入。请注意，这是一个好主意，包括一个控制的HPSC行，你知道是WA09（也称为H9亚型），如多能干。这样，你有一个积极的控制，你可以用它来确定是否是有缺陷的注​​射或手术。 </li><li>慢慢的hPSCs（20-30μL）注入睾丸胶囊中心远离任何大血管，停止，如果睾丸胶囊开始膨胀。 </li><li>慢慢取下针，以避免回流的细胞。 </li><li>使用镊子，转移到其原来的位置在腹部的睾丸和脂肪组织回。 </li><li>关闭了2个或3个re​​absorbable缝合腹膜和皮肤与autoclips关闭。 </li><li>鼠标应保持温暖，直到它恢复和手术后的一些镇痛药的形式（见什么是你的饲养箱中接受）一天两次1-2天。 请注意，既不是麻醉剂，也不镇痛干扰与肿瘤的发展。 </li><li>监测日é动物为6-12周的肿瘤生长。很少，肿瘤可能增长至6个星期前，注射后5毫米大小，因此它是重要的监测动物。如果发生这种情况，动物应安乐死和肿瘤处理和分析，像往常一样。 </li><li>一旦肿瘤扪及达到约5毫米大小，麻醉鼠标，根据公认的动物协议的程序和牺牲。 </li><li>取出肿瘤和文件 - 照片，测量尺寸，重量。 </li><li>切成小块的肿瘤，并固定在4％多聚甲醛溶液。商店在4％多聚甲醛，直到样品被送到病理学家切片，染色和分析。 </li></ol> 2。代表性成果： 当此协议的描述和注射的细胞是多能干，一个明显的，直观明显，肿瘤形成大多数在12个星期内。对于像WA09建立HPSC线，我们通常看到的6个星期内肿瘤。 IPSC的线，它是不寻常地看到8-10个星期内肿瘤。这是非常重要的，给老鼠注入了线，被称为是能干，作为阳性对照，以确保该程序是正确执行。肿瘤通常看起来非常庞杂，有许多附加囊肿（图1）。由病理学家的肿瘤样本分析显示分化组织所有三个胚层（图2）。 <img alt="图1" src="/files/ftp_upload/3177/3177fig1.jpg" /> 图1。典型HPSC派生畸胎瘤在睾丸胶囊。 所描述的协议之后，一百万WA09细胞注入免疫受损小鼠的睾丸胶囊。六个星期后畸胎瘤进行了观察。一）的畸胎瘤拉鼠标。 b）关闭了畸胎瘤的图片。注意结构的异质性和囊肿。 <img alt="图2" srC =“/ files/ftp_upload/3177/3177fig2.jpg的”/&gt; 图2。苏木精和曙红染色切片从畸胎瘤显示每个胚层组织。 固定之后，畸胎瘤切片和苏木精伊红染色。由病理学家的分析显示，从每3个胚层细胞的存在。

<ol>
	<li>Bradley, A., Evans, M., Kaufman, M. H., Robertson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+germ-line+chimaeras+from+embryo-derived+teratocarcinoma+cell+lines.">Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines.</a> Nature. 309, 255-255 (1984).</li><li>Evans, M. J., Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+in+culture+of+pluripotential+cells+from+mouse+embryos.">Establishment in culture of pluripotential cells from mouse embryos.</a> Nature. 292, 154 (1981).</li><li>Martin, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+pluripotent+cell+line+from+early+mouse+embryos+cultured+in+medium+conditioned+by+teratocarcinoma+stem+cells.">Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.</a> Proc. Natl. Acad. Sci. U. S. A. 78, 7634-7634 (1981).</li><li>Downing, G. J., Battey, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+assessment+of+the+first+20+years+of+research+using+mouse+embryonic+stem+cell+lines.">Technical assessment of the first 20 years of research using mouse embryonic stem cell lines.</a> Stem Cells. 22, 1168-1168 (2004).</li><li>Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1145 (1998).</li><li>Reubinoff, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+from+human+blastocysts:+somatic+differentiation+in+vitro.">Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro.</a> Nat Biotechnol. 18, (2000).</li><li>Muller, F. J., Goldmann, J., Loser, P., Loring, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+call+to+standardize+teratoma+assays+used+to+define+human+pluripotent+cell+lines.">A call to standardize teratoma assays used to define human pluripotent cell lines.</a> Cell Stem Cell. 6, 412-412 (2010).</li><li>Brivanlou, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells.+Setting+standards+for+human+embryonic+stem+cells.">Stem cells. Setting standards for human embryonic stem cells.</a> Science. 300, 913-913 (2003).</li><li>Lensch, M. W., Schlaeger, T. M., Zon, L. I., Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Teratoma+formation+assays+with+human+embryonic+stem+cells:+a+rationale+for+one+type+of+human-animal+chimera.">Teratoma formation assays with human embryonic stem cells: a rationale for one type of human-animal chimera.</a> Cell Stem Cell. 1, 253-25 (2007).</li><li>Gertow, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organized+development+from+human+embryonic+stem+cells+after+injection+into+immunodeficient+mice.">Organized development from human embryonic stem cells after injection into immunodeficient mice.</a> Stem. Cells. Dev. 13, 421-421 (2004).</li><li>Gertow, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+embryonic+stem+cell-derived+teratomas+for+the+assessment+of+pluripotency.">Isolation of human embryonic stem cell-derived teratomas for the assessment of pluripotency.</a> Curr Protoc Stem Cell Biol. Chapter 1, Unit1B 4-Unit1B 4 (2007).</li></ol>

没有利益冲突的声明。

这里介绍的方法，提供一个高度可靠的，从在睾丸胶囊hPSCs产生畸胎瘤直截了当的方法。这项技术有几个关键参数。特别是，它是重要的注入HPSC细胞线，被称为是多能干细胞作为对照。其他重要参数包括注射液和肿瘤的观察之间的时间间隔。对于像WA09细胞株，畸胎瘤应观察6-8周。为IPSC的新线，我们发现，往往需要10个星期。另一个值得关注的是注入细胞的数量。我们注入一百万个细胞，但可以很容易地用较少的细胞进行检测。此外，注入介质是非常重要的。我们发现，我们得到最好的结果，当细胞被注入在基底膜和DMEM/F-12的1:1混合，PBS或DMEM/F-12反对。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> accutase </td><td> Invitrogen公司</td><td> A1110501 </td></tr><tr><td> DMEM/F-12 </td><td> Invitrogen公司</td><td> 113300-032 </td></tr><tr><td>基底膜</td><td> BD公司</td><td> 354277 </td></tr></tbody></table>

teratoma generation in the testis capsule

多能干细胞（PSCS）有独特的特点，他们可以分化成所有三个胚层的细胞。这使得他们为许多不同疾病的治疗具有潜在价值的工具。 （胚胎干细胞）与诱导多能干细胞（iPS细胞），并继续与人类胚胎干细胞研究的到来需要有一个实验可以证明，一个特定的细胞是多能干。生殖传输已经证明小鼠胚胎干细胞（MESC）线1,2,3 pluripotence的黄金标准。使用这个实验中，研究人员可以表明，卓制线可以使所有类型的细胞在胚胎生殖细胞4。随着一代人类ESC线5,6，相应的检测来证明这些细胞的pluripotence不清楚，因为人类胚胎干细胞不能为生殖传输测试。作为替代，畸胎瘤法是目前用来展示pluripotency人类多能干细胞（hPSCs）7,8,9。虽然此法最近受到审查，并正在积极探索新技术，畸胎瘤法是目前的黄金标准7。在这个实验中，细胞免疫受损小鼠注入。如果是多能干细胞，最终将发展畸胎瘤和肿瘤的部分，将显示所有3个胚层组织。在畸胎瘤检测，hPSCs可以被注入老鼠的不同领域。最常见的注射部位，包括睾丸胶囊，补肾胶囊，肝，或到腿部皮下或肌肉注射11。在这里，我们描述了一个强大的协议畸胎瘤的肿瘤生长的部位使用睾丸胶囊hPSCs代。

Watch this Scientific Journal Video about Teratoma Generation in the Testis Capsule at JoVE.com

Teratoma Generation in the Testis Capsule

人类多能干细胞（hPSCs）有无数不同的疾病的潜在治疗。这些细胞的效用在于事实，即他们可以分化成人体任何细胞类型。在这里，我们描述畸胎瘤实验，这用于演示的hPSCs pluripotence的。

畸胎瘤代睾丸胶囊

Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers.  This makes them a ...

teratoma-generation-in-the-testis-capsule

Research

JoVE Journal

Medicine

20.6K 次观看.  Scripps Research Institute. 人类多能干细胞（hPSCs）有无数不同的疾病的潜在治疗。这些细胞的效用在于事实，即他们可以分化成人体任何细胞类型。在这里，我们描述畸胎瘤实验，这用于演示的hPSCs pluripotence的。

视频: Teratoma Generation in the Testis Capsule

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

听力：IMAP测试电池的决策意识

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

科研

医学

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

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, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

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, ...

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

在的鼠标Aortocaval瘘的技术方面

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.

We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth.

肾小囊和异种移植皮下颗粒植入前列腺癌发生和良性前列腺增生症的评价

New therapies for two common prostate diseases,  prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend  critically on experiments ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

生成和动脉病变的小鼠的三维定量使用光学投影层析成像

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

收集和唾液DNA提取用于新一代测序

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs&#160;that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d&#160;with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Here we report a rapid and efficient gene editing method based on RMCE in the AAVS1 locus of human Pluripotent Stem Cells (hPSCs) that improves upon previously described systems. Using this technique, isogenic lines can be rapidly and reliably generated for proper comparative studies, facilitating transgenesis-mediated research with hPSCs.

通过重组酶介导的盒式交换在重组人多能干细胞的快速高效生成<em&gt; AAVS1</em&gt;轨迹

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is ...

Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The goal of computational methods for drug repurposing is to enable solutions for safer, cheaper and faster drug discovery. Towards this end, we developed a novel method that integrates genetic and clinical phenotype data from large-scale GWAS and PheWAS studies with detailed drug information on the concept of transitive Drug-Gene-Disease triads. We created "RE:fine Drugs," a freely available, interactive dashboard that automates gene, disease and drug-based searches to identify drug repurposing candidates. This web-based tool supports a user-friendly interface that includes an array of advanced search and export options. Results can be prioritized in a variety of ways, including but not limited to, biomedical literature support, strength and statistical significance of GWAS and/or PheWAS associations, disease indications and molecular drug targets. Here we provide a protocol that illustrates the functionalities available in the "RE:fine Drugs" system and explores the different advanced options through a case study.

Drug Repurposing Hypothesis Generation Using the &quot;RE:fine Drugs&quot; System

Here we describe a protocol using the web-based drug repurposing hypothesis generation tool: "RE:fine Drugs." This protocol can be modified to a user's preferences at the level of the query type (gene, drug or disease) and/or the range of available advanced options.

药品再利用产生假设使用“RE：精药品”系统

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

小鼠闭合性股骨骨折的形成: 骨愈合模型的研究

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

Generation of Porcine Testicular Organoids with Testis Specific Architecture using Microwell Culture

Organoids are three dimensional structures composed of multiple cell types that are capable of recapitulating tissue architecture and functions of organs in vivo. Formation of organoids has opened up different avenues of basic and translational research. In recent years, testicular organoids have garnered interest in the field of male reproductive biology. Testicular organoids allow for the study of cell-cell interactions, tissue development, and the germ cell niche microenvironment and facilitate high throughput drug and toxicity screening. A method is needed to reliably and reproducibly generate testicular organoids with testis specific tissue architecture. The microwell culture system contains a dense array of pyramid-shaped microwells. Testicular cells derived from pre-pubertal testes are centrifuged into these microwells and cultured to generate testicular organoids with testis-specific tissue architecture and cell associations. Thousands of homogeneous organoids can be generated via this process. The protocol reported here will be of broad interest to researchers studying male reproduction.

Here, we present a protocol for the reproducible generation of porcine testicular organoids with testis specific tissue architecture using the commercially available microwell culture system.

使用微孔培养法生成具有睾丸特定结构的猪睾丸组织

Organoids are three dimensional structures composed of multiple cell types that are capable of recapitulating tissue architecture and functions of organs ...