作者要感谢本杰明 J Weigler, D.V.M., 博士和沃尔特曼德维尔, D.V.M. (动物资源 & 校园参加兽医, 内华达大学, 里诺) 为他们提供了优良的动物服务为老鼠并且他们的忠告在手术程序。

以下议定书已获得内华达大学 (UNR) 动物资源机构动物保育和使用委员会 (IACUC) 的批准, 并符合有关使用研究动物的所有机构伦理准则。
1. 动物。
<ol><li>获得成熟 (4-6 周大) 的 C57BL/6 小鼠体重介于20-30 克之间. 在 UNR 动物资源集中的动物设施中, 把实验室老鼠的菌落放在一个中心。</li>
</ol>2. 部分梗阻性手术
注意: 手术是在一个专门用于外科手术的房间里进行的。手术前所有的手术器械都是蒸压的。手术室所有人员都应佩戴无菌的手术袍和手套。
<ol><li>手术小鼠的制备<ol>
<li>检查麻醉提供系统, 以确保氧气和异氟醚的供应是足够的程序。打开供应氧气。打开气体流量计, 并将其调整为500-1000 毫升/分钟. 将动物放在感应腔内并封上顶部。</li>
		<li>打开异氟醚蒸发器到 5%, 并监测动物, 直到它成为卧。将麻醉传递系统切换到鼻锥。</li>
		<li>将任何残余气体的感应腔冲洗成氧气, 然后关闭感应腔线, 同时保持鼻锥线打开。</li>
		<li>把动物从房间里取出, 小心地将眼膏放在动物的眼睛上。</li>
		<li>当气体继续流动时, 将鼻锥放在预热的暖垫上。</li>
		<li>将氧气流量改为100-200 毫升/分, 2-3% 异氟醚。如果动物开始移动, gentlyrestrain 的动物与鼻子锥, 直到完全麻醉再次。</li>
		<li>监测过程中的呼吸和反应, 并根据需要调整异氟醚 (2-5%) 的百分比。动物麻醉水平的监测是缺乏脚趾捏反射前手术进行。</li>
		<li>注射止痛药 (丁丙诺啡, 体重1µg/克) 腹腔远离切口部位。</li>
		<li>使用干净的棉签在腹部涂抹脱毛液。让乳液在鼠标上坐3-5 分钟, 然后用纱布和棉签去除头发。重复此步骤, 直到所有的头发已经从鼠标的腹部被删除。</li>
		<li>用纱布和棉签清洁皮肤, 用70% 乙醇。应用 swabsticks 或聚维酮碘溶液清洗腹部。</li>
	</ol>
</li>
	<li>部分梗阻性手术<ol>
<li>使用 25 x 50 厘米无菌纸, 在手术区中部为 2.5 x 2.5 厘米开口, 将手术部位悬垂。通过在开口和皮肤的边界放置无菌条来保护动物的悬垂性。</li>
		<li>3.0 厘米腹部切口纵向使用15号刀片手术刀, 确保只有皮肤被切割和避免削减到 musculoperitoneal 层在这个时候。</li>
		<li>使用镊子和手术剪刀, 小心地将皮肤从 musculoperitoneal 层分开, 而不会导致任何切口到 musculoperitoneal 层。在层数被充分地分离了 (大约 1 cm x 4 cm) 之后, 辨认 linea 在 musculoperitoneal 层数和切开 ~ 2 cm 沿 linea, 用微钳和剪刀暴露腹腔腔。</li>
		<li>仔细定位并辨认盲肠。用微钳慢慢地从腹腔腔中取出盲肠, 将盲肠和回肠带到不育的褶皱上。立即用0.9% 条无菌盐水浸泡纱布将肠道组织湿润, 并在腹腔外的任何时候保持暴露的组织湿润。</li>
		<li>定位和辨认回肠和近端结肠之间的肠系膜。使切口 (1 厘米) 平行, 并刚好低于回肠, 在肠系膜上, 避免切割任何血管。</li>
		<li>采取蒸压硅胶环 (长度为6毫米, 4 毫米外径, 3.5 毫米内径)。纵向切开打开油管, 用微钳打开环。</li>
		<li>在肠系膜组织的切口上插入开环的一端。将环带到一个完整的圆环形状上, 使一端与另一个接触, 而回肠被环包围。</li>
		<li>确保硅胶环完全围绕回肠, 闭合环与缝合, 并小心地将肠道放回腹腔腔内。</li>
	</ol>
</li>
	<li>手术关闭<ol>
<li>在 linea 的 musculoperitoneal 层上进行简单的连续缝合, 用可吸收缝线闭合 musculoperitoneal 伤口。缝合完成后, 用0.9% 无菌生理盐水浸泡纱布清洗任何出血。</li>
		<li>要完全闭合伤口, 用单独的尼龙缝合, 对皮肤进行简单的连续缝合。</li>
		<li>两针缝合完成后, 用新的 swabstick 或聚维酮碘清洗伤口。</li>
		<li>腹腔注射抗生素 (庆大霉素, 每只老鼠 150 ul 基于20-30 克体重)。</li>
		<li>完成程序后, 关闭异氟醚蒸发器, 让动物呼吸只有流动的氧气, 直到它开始获得意识。</li>
		<li>一旦动物醒了, 把动物带到一个单独的恢复区, 热支持, 直到完全恢复。</li>
	</ol>
</li>
</ol>3. 术后观察。
<ol><li>手术完成后, 将动物移到恢复室的孵化器中, 温度和湿度是调节的。观察动物术后每15分钟的第一小时, 然后每30分钟的第二小时, 而动物在孵化器。</li>
	<li>一旦规定的观察完成, 移动动物到他们自己的笼子并且每日监视他们为临床征兆痛苦18, 并且确保手术伤口是正确愈合没有任何并发症的迹象 (开裂) 目前。</li>
</ol>

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作者没有什么可透露的。

我们表明, 小鼠接受肠道 PO 手术的持续和重现性发展肠平滑肌肥大, 这是模仿人类肠梗阻。为不同的动物开发了肠梗阻手术, 包括老鼠7, 老鼠10, 豚鼠11, 狗12和猫13。小鼠肠梗阻模型具有时间、成本、大小和表型优势, 优于其他大型动物模型。小鼠或大鼠肥大的发展只需要10-14 天10, 与豚鼠、狗...

肠道阻塞是小肠或大肠的部分或全部堵塞, 可防止消化的食物、液体和气体通过肠道移动1。由于梗阻, 堵塞导致肠道壁变厚, 缩小流明2。由于腹部或盆腔手术导致腹部粘连组织形成或胃肠道疾病 (如克罗恩病)、憩、疝、扭转、狭窄、肠套叠, 便秘, 粪便嵌塞, 伪梗阻, 癌症和肿瘤3,4,5。肠道阻塞在这些情况下经常导致肠道肌的肥大6。
PO 的流明诱发肠道扩张, 并增加平滑肌层厚度周围的梗阻, 以响应需要继续功能蠕动7,8,9,10, 11,12,13。开发了肠道动物模型, 研究小鼠的平滑肌肥厚7, 大鼠10, 豚鼠11, 狗12, 猫13 , 持续发展类似的肥厚在肠道肌肉层。
小肠 PO 的老鼠模型是产生和研究肠道障碍的最经济有效的方法在体内。小鼠小肠梗阻是通过使用硅胶环手术放置在回肠周围。在 po 手术后, po 小鼠的细胞数量 (增生) 和肌肉层厚度 (肥厚) 的增加较早,8,15。SMC 是主要的塑料细胞, 生长在平滑的肌肉层, 以响应肥厚条件14, 但其他细胞, 如 ICC 和 PDGFRα+细胞与 SMC 密切关联, 也填充。我们以前报告说, PO 小鼠在小肠发育肥大, 其中 SMC 被去分化肉瘤成PDGFRα + 细胞, 高度增生的7, 15, 16.相反, 在肠道 obsruction 的发育过程中, ICC 在肥厚平滑肌层中退化并丢失了7。PO 模型的另一个主要好处是它有能力诱发肠道神经系统的改变和传播神经源性运动模式。小鼠小肠的主要传播神经源性运动模式是迁移马达复合体 (MMC), 这是神经源性的, 不需要 ICC 或电慢波17。PO 模型可以清楚地了解复合材料和肠神经是如何通过局部梗阻重塑的。
在这里, 我们提出了一个小鼠的肠道 PO 手术的协议使用硅胶环。接受 PO 手术的小鼠在小肠肌中可靠地产生肥大。在肥厚的肌肉内, SMC, ICC, PDGFRα+, 神经元细胞被显著重塑。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Surgical drape</td>
			<td>Medical and veterinary supplies</td>
			<td>SMS40</td>
			<td>40&#8221;X100 yards</td>
		</tr>
		<tr>
			<td>Underpad, econ, pro plus</td>
			<td>Medical and veterinary supplies</td>
			<td>MSC281224</td>
			<td>17x24&#8221;</td>
		</tr>
		<tr>
			<td>Iris scissors</td>
			<td>Braintree scientific, Inc</td>
			<td>SC-i-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris scissors</td>
			<td>Vantage</td>
			<td>V95-304</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont electronic &#38; jeweler tweezers</td>
			<td>Dumont</td>
			<td>98-180-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Braided absorbable suture</td>
			<td>Covidien polysorb</td>
			<td>SL-5687G</td>
			<td>5-0, polyglactin</td>
		</tr>
		<tr>
			<td>Nylon non-absorbable mono filament</td>
			<td>AD surgical</td>
			<td>S-N618R13</td>
			<td>6-0, nylon</td>
		</tr>
		<tr>
			<td>Surgical blade</td>
			<td>Dynarex</td>
			<td></td>
			<td>No.15</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Jacobson microvascular</td>
			<td>36-1342TC</td>
			<td>8.5 inch</td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>Flinn scientific</td>
			<td>AB1049</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsurgical scissor</td>
			<td>WPI</td>
			<td>503305</td>
			<td></td>
		</tr>
		<tr>
			<td>Petrolatum ophthalmic ointment</td>
			<td>Puralube VET</td>
			<td></td>
			<td>3.5 g</td>
		</tr>
		<tr>
			<td>Fluriso (isoflurane)</td>
			<td>Vetone</td>
			<td>V1 502017</td>
			<td>250 ml</td>
		</tr>
		<tr>
			<td>Steri-strip reinforced skin closure</td>
			<td>3M</td>
			<td>R1547</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical gloves</td>
			<td>Medline</td>
			<td>MSG2270</td>
			<td></td>
		</tr>
		<tr>
			<td>Ear loop face mask</td>
			<td>The safety zone</td>
			<td>RS700</td>
			<td></td>
		</tr>
		<tr>
			<td>Avant gauze non-woven sponges</td>
			<td>Caring</td>
			<td>PRM25444</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical cup</td>
			<td>Admiral&#160; craft OYC-2</td>
			<td>725-A42</td>
			<td>2.5 oz</td>
		</tr>
		<tr>
			<td>Swabstick</td>
			<td>ChloraPrep</td>
			<td>260103</td>
			<td>2% w/v Chlorhexidine&#160; Gluconate (CHG) and 70% v/v Isopropyl Alcohol (IPA)</td>
		</tr>
		<tr>
			<td>Cotton tipped applicator</td>
			<td>Puritan</td>
			<td>806-WC</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Zoo pharm</td>
			<td>BZ8069317</td>
			<td>1 mg/ml</td>
		</tr>
		<tr>
			<td>Gentamycin sulfate</td>
			<td>Vetone</td>
			<td>G-6336-04</td>
			<td>100 mg/ml</td>
		</tr>
		<tr>
			<td>Fast acting gel cream remover</td>
			<td>Veet</td>
			<td>8111002</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>AHS</td>
			<td>AH01T2516</td>
			<td>1 ml with needle</td>
		</tr>
		<tr>
			<td>Silicon ring</td>
			<td>VWR</td>
			<td>60985-720</td>
			<td>6 mm in length, 4 mm exterior diameter, 3.5 mm interior diameter</td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>The Jackson Laboratory</td>
			<td></td>
			<td>4-6 weeks old</td>
		</tr>
	</tbody>
</table>

a mouse model of intestinal partial obstruction

肠道阻塞, 阻碍或阻断蠕动运动, 可能是由腹部粘连和大多数胃肠道 (胃肠) 疾病, 包括肿瘤生长引起的。然而, 由于肠道障碍而引起的细胞重塑机制被理解得很差。一些动物模型的肠道障碍已经开发, 但鼠标模型是最成本/时间的有效。老鼠模型使用的外科植入肠道部分梗阻 (PO), 如果它没有正确的执行率高死亡率。此外, 如果不使用适当的封锁或不适当放置, 接受 PO 手术的小鼠就无法发育肥大。在这里, 我们描述了一个详细的协议, 为 PO 手术, 产生可靠和可重复的肠道障碍, 死亡率非常低。该协议利用手术放置的硅胶环包围回肠, 部分阻断消化运动在小肠。部分堵塞使肠道因消化运动停止而扩张。肠道扩张会导致环的口腔侧平滑肌肥大, 逐渐发育超过2周, 直至死亡。手术 PO 鼠标模型提供了一个体内模型, 用于研究肠道细胞的病理变化, 包括平滑肌细胞 (SMC), Cajal (ICC), PDGFRα+和神经元细胞在肠道梗阻的发育过程中。

部分梗阻 (PO) 手术诱导的一个月大鼠通过放置一个硅胶环周围的回肠附近的回盲括约肌。这枚戒指造成了回肠部分堵塞。假手术 (所以) 也没有一个环上的年龄/性别匹配的老鼠, 这些老鼠没有显示任何类似的症状, 在 PO 老鼠发现。几个小时后, 小鼠很快从 PO 手术中恢复过来。他们在第一个星期内没有表现出明显的行为变化或虚弱, 但在第一周之后, 他们逐渐开始显示出了 PO ?...

Watch this Scientific Journal Video about A Mouse Model of Intestinal Partial Obstruction at JoVE.com

A Mouse Model of Intestinal Partial Obstruction

肠道阻塞是肠道的部分或全部堵塞, 可引起严重的腹痛、恶心、呕吐和防止大便的通过。该方法在小鼠肠道部分 obsructions 的建立是可靠的, 研究的机制, 病理细胞生长和死亡的肠道。

小肠部分梗阻的小鼠模型

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

a-mouse-model-of-intestinal-partial-obstruction

Research

JoVE Journal

Biology

20.9K 次观看.  University of Nevada, Reno School of Medicine. 肠道阻塞是肠道的部分或全部堵塞, 可引起严重的腹痛、恶心、呕吐和防止大便的通过。该方法在小鼠肠道部分 obsructions 的建立是可靠的, 研究的机制, 病理细胞生长和死亡的肠道。

视频: A Mouse Model of Intestinal Partial Obstruction

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 ...

科研

生物学

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease (CD). Both UC and CD, when present in the colon, generate a similar symptom profile which can include diarrhea, rectal bleeding, abdominal pain, and weight loss.1 Although the pathogenesis of IBD remains unknown, it is described as a multifactorial disease that involves both genetic and environmental components.2
There are numerous and variable animal models of colonic inflammation that resemble several features of IBD. Animal models of colitis range from those arising spontaneously in susceptible strains of certain species to those requiring administration of specific concentrations of colitis-inducing chemicals, such as dextran sulphate sodium (DSS). Chemical-induced models of gut inflammation are the most commonly used and best described models of IBD. Administration of DSS in drinking water produces acute or chronic colitis depending on the administration protocol.3 Animals given DSS exhibit weight loss and signs of loose stool or diarrhea, sometimes with evidence of rectal bleeding.4,5 Here, we describe the methods by which colitis development and the resulting inflammatory response can be characterized following administration of DSS. These methods include histological analysis of hematoxylin/eosin stained colon sections, measurement of pro-inflammatory cytokines, and determination of myeloperoxidase (MPO) activity, which can be used as a surrogate marker of inflammation.6
The extent of the inflammatory response in disease state can be assessed by the presence of clinical symptoms or by alteration in histology in mucosal tissue. Colonic histological damage is assessed by using a scoring system that considers loss of crypt architecture, inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess.7 Quantitatively, levels of pro-inflammatory cytokines with acute inflammatory properties, such as interleukin (IL)-1&beta;, IL-6 and tumour necrosis factor (TNF)-&alpha;,can be determined using conventional ELISA methods. In addition, MPO activity can be measured using a colorimetric assay and used as an index of inflammation.8
In experimental colitis, disease severity is often correlated with an increase in MPO activity and higher levels of pro-inflammatory cytokines. Colitis severity and inflammation-associated damage can be assessed by examining stool consistency and bleeding, in addition to assessing the histopathological state of the intestine using hematoxylin/eosin stained colonic tissue sections. Colonic tissue fragments can be used to determine MPO activity and cytokine production. Taken together, these measures can be used to evaluate the intestinal inflammatory response in animal models of experimental colitis.

Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.

调查在DSS诱导的鸡传染性法氏囊病模型的肠道炎症

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

代谢谱的实时分析<em&gt;离体</em&gt;鼠标肠隐窝类器官培养

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

协议的慢病毒转导与肠组织体下游分析

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Partial Bile Duct Ligation in the Mouse: A Controlled Model of Localized Obstructive Cholestasis

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile duct proliferation. However, long-term experiments can lead to increased morbidity and mortality. In select mouse strains with underlying liver disease, meaningful comparisons can be made even with ligation of a single lobe of the liver, which can reduce animal losses and expenses. Here, we describe partial bile duct ligation (pBDL) in the mouse, in which only the left hepatic bile duct is ligated, causing biliary obstruction in the left lobe but not the remaining lobes. With careful microsurgical technique, pBDL experiments can be cost-effective, since the unligated lobe serves as an internal control to the ligated lobes, when subjected to the same conditions in the same animal. Unlike cBDL, a separate sham-operated control group is not necessary. pBDL is highly useful to directly compare localized versus systemic effects of cholestasis and other retained bile components. pBDL can also be repurposed as a novel method to investigate mechanisms related to medications and cell migration.

Here, we present partial bile duct ligation as a surgical model of liver injury and regeneration in rodents.

小鼠部分胆管结扎术: 局部梗阻性胆汁淤积症的对照模型

In rodents, complete bile duct ligation (cBDL) of the common bile duct is an established surgical technique for studying obstructive cholestasis and bile ...

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 ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

质谱分析，用于识别 EYFP 标记 CENP-A （EYFP-CENP-A） 的泛化

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

A Mouse Model of Lumbar Spine Instability

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding the pathological processes and evaluating new drugs. Here, we introduced a surgically induced lumbar spine instability (LSI) mouse model that develops IDD starting from 1 week post operation. In detail, the mouse under anesthesia was operated by low back skin incision, L3&#8211;L5 spinous processes exposure, detachment of paraspinous muscles, resection of processes and ligaments, and skin closure. L4&#8211;L5 IVDs were chosen for the observation. The LSI model develops lumbar IDD by porosity and hypertrophy in endplates at an early stage, decrease in intervertebral disc volume, shrinkage in nucleus pulposus at an intermediate stage, and bone loss in lumbar vertebrae (L5) at a later stage. The LSI mouse model has the advantages of strong operability, no requirement of special equipment, reproducibility, inexpensive, and relatively short period of IDD development. However, LSI operation is still a trauma that causes inflammation within the first week post operation. Thus, this animal model is suitable for study of lumbar IDD.

We developed a lumbar intervertebral disc degeneration mouse model by resection of L3&#8211;L5 spinous processes along with supra- and inter-spinous ligaments and detachment of paraspinous muscles.

腰椎不稳定的鼠标模型

Intervertebral disc degeneration (IDD) is a common pathological change leading to low back pain. Appropriate animal models are desired for understanding ...

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport,&#160;antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

Here, we describe the preparation of human organoid-derived intestinal epithelial monolayers for studying intestinal barrier function, permeability, and transport. As organoids represent original epithelial tissue response to external stimuli, these models combine the advantages of expandability of cell lines and the relevance and complexity of primary tissue.

类器官来源的上皮单层:临床相关的肠道屏障功能体外模型

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...

Partial Hepatectomy in Adult Zebrafish

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy livers are capable of regenerating from toxic damage, but in advanced liver disease, the natural ability of the liver to regenerate is impaired. Zebrafish have emerged as a powerful experimental system for studying regeneration. They are an ideal model for studying liver regeneration from partial hepatectomy, a procedure with direct clinical relevance in which part of the liver is surgically removed, leaving the rest intact. There is no standard protocol for partial hepatectomy; previous studies using this model have used slightly different protocols and reported disparate results. Described here is an efficient, reproducible protocol for performing a partial hepatectomy in adult zebrafish. We use this technique to demonstrate that zebrafish are capable of epimorphic regeneration of the resected lobe. This protocol can be used to further interrogate the mechanisms required for liver regeneration in zebrafish.

This protocol describes the procedure for removing the ventral lobe of the liver in adult zebrafish to enable the study of liver regeneration.

成人斑马鱼的部分肝切除术

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy ...