资金来源：这项工作是由一个斯图尔特总部及维多利亚泉Predoctoral奖学金（RMW），美国国立卫生研究院先锋奖（RAS）的支持

一般健康检查一个更全面的治疗，一般健康检查可以发现杰奎琳克劳利的优良行为的表型资源，什么是错的， 我的鼠标？1。我们首先使用修改后的观察测试电池，以评估主体的一般健康。电池包括塞缪尔欧文的全面评估，协议2项，快速识别这些科目将不适合进一步的测试。简单地说，修改后的电池标识，身体异常的动物（如严重不足或过重，缺乏培养，呼吸困难），表现出异常的姿势，运动或行为。这些都可能是疾病的症状。任何不健康的科目，同样标识的初步评价是适当的。 同样重要的是要注意动物是用于人道都在一般性的健康检查，以及在随后的测试。没有麻醉或镇痛用于这些测试不包括任何潜在的痛苦过程。 嗅觉灵敏测试这个测试是用来检测在两组小鼠（如野生型和突变的动物组）之间的嗅觉灵敏的区别。在这项测试中，香味多个稀释。一个天真的动物，然后调查了这种香味和总探索的时间（3分钟内）单一的稀释被记录下来。多个稀释度的气味不同，天真的科目测试和探索的总时间是用来产生各组小鼠的剂量反应曲线。两个研究人员也应该参与测试，盲目受基因型和/或条件。 主研究员<ol><li>获得4个干净笼子（8（高）x 10宽x 18 L英寸，），所有没有被褥。排队在生物安全柜中的四个笼。 </li><li>设置使用不透明的滤纸舞台之间的每个分区。横跨最终分区一个黑色的海报板等，黑色的侧面线条笼侧面和底部。一个明确的有机玻璃盖的舞台上方，让光线照亮的舞台。 （一） <img src="/files/ftp_upload/old/949_2009_1_14_8/figure1new.png" alt="图1" /> （二） <img src="/files/ftp_upload/old/949_2009_1_14_9/image15.png" alt="图1.5" /> 图1。竞技场设置 （一）适当地搭着，空舞台（二）主体和香味的滤纸竞技场。 </li> <li>开始habituating科目。将在第一的习惯笼的第一个主题。 15分钟后，移动到第二个笼子的第一个主题。清洁Quatricide结核病的第一个笼子里。下一步，放置在第一，清洁的笼子里的第二个问题。这一进程继续进行后续的科目。最终，每个主题应习惯于在所有三个笼习惯测试前15分钟之前被放置在舞台上（第四个也是最后一个笼子）。 </li></ol>盲人研究员<ol><li>适当数量的2英寸2英寸正方形滤纸气味样本。 </li><li>使用预先选定的气味，适当稀释。对于疏水样品，如花生酱，用作溶剂油，为人人，人，使用蒸馏水。涡街流量计。 </li><li>在生物安全柜等，整个笼结束的重点是的尽头，集数字录像设备。采取了初步的视频，以确保有足够的参数。 </li><li>记录在其中的稀释，照顾随机试验的顺序将被测试的顺序。 </li><li>记录主体的枯枝落叶和身份证号码。 </li><li>一旦这个问题已经习惯于在舞台上15分钟，开始编写一审。移液器的稀释和锥形管到滤纸广场前吹打。滤纸表面应优化〜85％覆盖（约0.50 MLS解决方案）。使用kimwipe，以确保不是过于饱和的滤纸。 </li><li>开始视频录制。 </li><li>在笼子里的动物的当前位置的另一端放在滤纸广场。 </li><li>开始计时。 </li><li> 3分钟后，停止录制，然后取出稀释后的气味。 </li><li>如果只有一个气味（如肉桂）进行测试，从舞台上删除的第一次测试科目。重复步骤6-11，每个动物适当稀释为每个主题的气味。如果要测试的是一个以上的香味，但是，没有从舞台中删除的测试题目。相反，开始计时。 </li><li>适当稀释未来的香味，用同样的方法，如上面列出。 </li><li> 1分钟后，开始录制视频。放置在笼子里的下一个气味稀释的动物的电流positio的另一端上的滤纸广场N. </li><li>重复步骤6-13，每个气味注意到每次试验的视频文件。 </li><li>重复步骤6-14为每个主题。 <img src="/files/ftp_upload/old/949_2009_1_14_10/image2new.png" alt="图2" /> 图2。每个代表一个嗅觉灵敏的测试的会议结果。对照组（A组）或实验组（B组）鼠标暴露肉桂五种不同的稀释液在滤纸上提取一，三分钟的会议之一。花费的总时间探索的滤纸进行了评估。组小鼠有一个更大的嗅觉灵敏度比B组</li></ol>嗅觉偏好测试这个测试的目的是确定具体的检测的不足之处，即的能力感吸引力或厌恶的气味。下列程序借用从优从小早川协议，K.，al.3等。再次，两位研究人员应参与测试，有一个盲人受基因型和/或条件。总的探索时间再次记录（视频观看后）各种气味。总探索大于水的气味被指定为“有吸引力”，而与时并进小于水的人被称为“厌恶”。然后研究人员认为，相同的气味是否“有吸引力”或“厌恶”，针对不同的考试科目。 嗅觉灵敏的主要研究人员的测试组按照步骤1-3。 盲人研究员嗅觉偏好测试的过程是相同的，稍作修改步骤2和4。请遵循以下变化嗅觉灵敏盲目研究员测试组中的步骤1-15，对于第2步使用预先选定的气味（吸引力和厌恶的气味的组合），使每个适当稀释的解决方案锥形管。对于疏水样品，如花生酱，用作溶剂油，为人人，人，使用蒸馏水。涡街流量计。步骤4，记录要测试的稀释，而不是记录的，将被测试的不同气味的顺序，随机试验的顺序。 这个测试的目的是确定具体的检测的不足之处，即的能力感吸引力或厌恶的气味。下列程序借用宽松的小早川，K 等协议3。再次，两位研究人员应参与测试，有一个盲人受基因型和/或条件。总的探索时间再次记录（视频观看后）各种气味。总探索大于水的气味被指定为“有吸引力”，而与时并进小于水的人被称为“厌恶”。然后研究人员认为，相同的气味是否“有吸引力”或“厌恶”，针对不同的考试科目。 嗅觉灵敏的主要研究人员的测试组按照步骤1-3。 盲人研究员嗅觉偏好测试的过程是相同的，与步骤2，4和13稍作修改。请遵循以下变化嗅觉灵敏盲目研究员测试组中的步骤1-15，对于第2步使用预先选定的气味（吸引力和厌恶的气味的组合），使每个适当稀释的解决方案锥形管。对于疏水样品，如花生酱，用作溶剂油，为人人，人，使用蒸馏水。涡街流量计。步骤4，记录要测试的稀释，而不是记录的，将被测试的不同气味的顺序，随机的顺序，在每个审判。第13步，在笼子里的动物的当前位置的另一端上的下一个气味过滤纸平方米。 <img src="/files/ftp_upload/old/949_2009_1_14_11/image3new.png" alt="图3" /> 图3。为代表的嗅觉偏好测试的会议结果 。每只小鼠暴露在滤纸上的每一个不同的气味，一，三分钟的会议。花费的总时间探索的滤纸进行了评估。花生酱和香草有吸引力的气味（探索时间的香味&gt;探索时间水）和2 -甲基丁酸是厌恶（探索时间的香味&lt;探索时间水） 。 计分的嗅觉测试获取所有的嗅觉灵敏和偏好的视频后，有必要分析花费的总时间探索每个稀释度/香味。有失明的研究员分析一致性的所有影片。 <ol><li>预览的VIDEO，指出以下几点：一）滤纸放置在舞台上时，b）当鼠标接近滤纸，C）当鼠标调查滤纸和D）时，三分钟过去了，，（从当过滤器文件被放置在舞台上）。知道什么时候把重点放在鼠标的行为，使得它更容易准确地得分探索经审查花费的总时间。 </li><li>查看视频。注意当鼠标首次调查的滤纸（延迟）的时间。 </li><li>用秒表，时间三分钟，在舞台上放置滤纸内的所有调查。调查的时间是指任何时候鼠标的鼻孔&lt;1毫米的滤纸。重要的是不包括任何的时间，当鼠标在接近滤纸，但不积极调查。不包括咀嚼和舔的文件或纸张上的总的探索时间坐在较量较量。这是必要的，始终坚持密切这些规则，精确地分析嗅觉行为。 </li><li>记录的延迟时间，花费的总时间调查滤纸和视频文件数量。 </li></ol>

<ol>
	<li>Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What&amp;rsquo;s+Wrong+with+my+Mouse?+Behavioral+Phenotyping+of+Transgenic+and+Knockout+Mice.">What&amp;rsquo;s Wrong with my Mouse? Behavioral Phenotyping of Transgenic and Knockout Mice.</a> , 42-60 (2007).</li><li>Irwin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+observational+assessment:+Ia.+A+systematic,+quantitative+procedure+for+assessing+the+behavioral+and+physiologic+state+of+the+mouse.">Comprehensive observational assessment: Ia. A systematic, quantitative procedure for assessing the behavioral and physiologic state of the mouse.</a> Psychopharmacologia. 13, 222-257 (1968).</li><li>Kobayakawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+versus+learned+odour+processing+in+the+mouse+olfactory+bulb.">Innate versus learned odour processing in the mouse olfactory bulb.</a> Nature. 450, 503-508 (2007).</li></ol>

上述测试的基本嗅觉功能允许快速和可靠的评估。它不是被忽视，然而，这种可靠性要求的正确选择和测试科目的准备。动物患病或其他无行为能力的往往是不一致的考试科目。此外，考试科目的习惯是关键。我们已经发现多个笼的习惯过程中的使用，使动物成为实验者处理和不断变化的环境舒适。我们还发现，对于习惯上所标明的短不足以减轻焦虑。焦虑动物也往往是不可预知的考试科目，并?...

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olfactory behavioral testing in the adult mouse

啮齿动物的嗅觉系统是越来越大的兴趣，科学家，研究，在系统生物学中的一部分，由于其千篇一律，但访问的电路。此外，这方面的独特能力，一个生物体的整个生命周期内产生新的神经元，使得它有吸引力的系统发育和再生生物学家的一致好评。这种利益必须的嗅觉功能的快速而可靠的评估手段。许多嗅觉能力测试，是复杂的，变量或没有专门对小鼠。另外，一些试验是敏感的记忆障碍以及嗅觉能力，得到的结果混杂的缺陷。 在这里，我们描述了一个简单的电池测试的目的是找出在嗅觉灵敏和偏好的缺陷。首先，最初的一般健康状况评估，可以对动物进行进一步的测试适合的鉴定。其次，老鼠接触到不同的气味稀释，以确定是否有是一个阈值差异。第三，小鼠的各种气味，吸引力和厌恶，允许嗅觉偏好评估。这些简单的研究，应使不同的资源和专门知识的实验室访问的嗅觉行为的初步鉴定。

Watch this Scientific Journal Video about Olfactory Behavioral Testing in the Adult Mouse at JoVE.com

Olfactory Behavioral Testing in the Adult Mouse

的啮齿类动物嗅觉系统的基础，但独特的属性，导致其日益增加的研究，生物学家。然后还需要一个相对简单的评估，其功能是。在这里，我们描述了小鼠嗅觉灵敏的特性和喜好的敏感测试。

在成年小鼠的嗅觉行为测试

The rodent olfactory system is of increasing interest to scientists, studied, in part, in systems biology because of its stereotyped, yet accessible ...

olfactory-behavioral-testing-in-the-adult-mouse

Research

JoVE Journal

Biology

19.4K 次观看.  Dana Farber Cancer Institute. 的啮齿类动物嗅觉系统的基础，但独特的属性，导致其日益增加的研究，生物学家。然后还需要一个相对简单的评估，其功能是。在这里，我们描述了小鼠嗅觉灵敏的特性和喜好的敏感测试。

视频: Olfactory Behavioral Testing in the Adult Mouse

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

高分辨率视频跟踪移动的，在成年果蝇。

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

科研

生物学

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

活体成像和治疗卵巢癌的原位小鼠模型

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

腹腔注射到成年斑马鱼

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavaging成年斑马鱼

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

从成年小鼠切牙牙源性上皮干细胞的分离和培养

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

鼠标离体肾技术

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

胚胎干细胞的跨内细胞质量注射导致嵌合体率增高

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina propria. EGCs play important roles in gut homeostasis through the release of various trophic factors and contribute to the integrity of the epithelial barrier. Most studies of primary enteric glial cultures use cells isolated from the myenteric plexus after enzymatic dissociation. Here, a non-enzymatic method to isolate and culture EGCs from the intestinal submucosa and lamina propria is described. After manual removal of the longitudinal muscle layer, EGCs were liberated from the lamina propria and submucosa using sequential HEPES-buffered EDTA incubations followed by incubation in commercially available non-enzymatic cell recovery solution. The EDTA incubations were sufficient to strip most of the epithelial mucosa from the lamina propria, allowing the cell recovery solution to liberate the submucosal EGCs. Any residual lamina propria and smooth muscle was discarded along with the myenteric glia. EGCs were easily identified by their ability to express glial fibrillary acidic protein (GFAP). Only about 50% of the cell suspension contained GFAP+ cells after completing tissue incubations and prior to plating on the poly-D-lysine/laminin substrate. However, after 3 days of culturing the cells in glial cell-derived neurotrophic factor (GDNF)-containing culture media, the cell population adhering to the substrate-coated plates comprised of &#62;95% enteric glia. We created a hybrid mouse line by breeding a hGFAP-Cre mouse to the ROSA-tdTomato reporter line to track the percentage of GFAP+ cells using endogenous cell fluorescence. Thus, non-myenteric enteric glia can be isolated by non-enzymatic methods and cultured for at least 5 days.

Here, we describe the isolation of enteric-glial cells from the intestinal-submucosa using sequential EDTA incubations to chelate divalent cations and then incubation in non-enzymatic cell recovery solution. Plating the resultant cell suspension on poly-D-lysine and laminin results in a highly enriched culture of submucosal glial cells for functional analysis.

成年小鼠黏膜下层和固有层肠道胶质细胞的分离

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

成人小鼠心脏干细胞的分离、鉴定及分化

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...