这项工作是支持的NIDDK KO1 DK075386授予和美国癌症协会RSG - 09 - 075 - 01 - MBC孙军。

该协议包括三部分：细菌培养，小鼠灌胃，检测和沙门氏菌 。 1。 沙门氏菌的生长条件。 <ol><li>准备沙门氏菌卢里亚Bertani肉汤（LB）板，37℃过夜孵育。 </li><li>从LB平板克隆选择和投入7毫升LB培养基，在12毫升管，并在37 ° C为5小时左右摇晃。 </li><li>固定相的文化和​​孵化用0.05毫升50毫升LB培养基，接种于37 ° C，不摇晃时间约18小时。 </li><li>自旋在室温下过夜的细菌培养，10分钟6000转，暂停使用比率100:3（LB：HBSS）的HBSS的细菌。例如： 每50毫升LB培养将暂停在1.5毫升的HBSS。 </li><li>对于动物灌胃，进一步在1:10的比例稀释的细菌培养。例如： 每1.5毫升LB培养将暂停在15毫升的HBSS。 </li></ol> 2。 沙门氏菌感染模型*. <ol><li>准备链霉素溶液。每个鼠标将在100μL的HBSS 7.5毫克链霉素。例如，准备10只小鼠在1.2毫升的HBSS总链霉素90毫克。准备链霉素溶液时，总是有一些额外的体积。 </li><li>水和食物4小时之前撤回灌胃治疗。 </li><li>链霉素（100μL对照组小鼠的HBSS）7.5毫克链霉素灌胃的小鼠。牢牢抓住用大拇指和中指在鼠标肩的皮肤，舒展的头部和颈部，用食指伸直，使食道。直接喂养针沿口的屋顶，向咽后部的右侧球头，然后轻轻地向下传递到食道，并注入100μL解决方案。应该感到无电阻。 </li><li>链霉素治疗后，被撤回的水和食物与细菌感染小鼠前再次的20小时。 </li><li>灌胃100μL悬浮在HBSS中每个鼠标或用无菌的HBSS（对照组）灌胃治疗。灌胃过程是相同的2.3）灌胃小鼠，用链霉素。 </li></ol> *动物实验，通过使用无特定病原体的雌性C57BL / 6小鼠（泰康利，哈德森，NY）6-7周龄，正如先前所描述的1。该协议被批准，由美国罗切斯特大学委员会的动物资源（UCAR）大学。 3。肠道沙门氏菌检测。 <ol><li>收集小鼠粪便（约100毫克）。 </li><li>粪便样品转移到1.5微离心管中，用1ml PBS和涡大力。 </li><li>离心10分钟800转。上清液转移到一个干净的离心管。 </li><li>离心5分钟转速6000。丢弃上清液200μL的PBS添加到颗粒和振荡。 </li><li>数的200μLPBS与一次性细胞吊具上BBL CHROMagar板检测沙门氏菌沙门氏菌出现紫红色（上升到紫色，参见图1）。如果200μL产量上盘太多的殖民地，使用100μL或50μL的连胜纪录。 </li></ol> 4。代表性的成果。 鼠伤寒沙门氏菌的定植是正确的协议时，可以在小鼠小肠中检测到6个月以上的沙门氏菌可通过粪便文化检测6个月以上（图1 ）。作为一个典型的出这种模式，车身重量损失和死亡，感染后4个星期内发生。依赖感染沙门氏菌株，有些老鼠可能无法存活超过6个月。 <img src="/files/ftp_upload/1947/1947fig1.jpg" alt="图1" /> 图1。肠道沙门氏菌沙门氏菌出现紫红色上升到紫色的颜色，由于代谢差异的存在，在选定chromogens的。是抑制其他细菌或产生蓝绿色或无色菌落。 <img src="/files/ftp_upload/1947/1947fig2.jpg" alt="图2" /> 图2。感染沙门氏菌突变株PhoP C，PhoP彗星的小鼠的存活比例阿夫拉， 和 PhoP彗星AvrA-/AvrA + PhoP彗星阿夫拉4周（28天）。 表1。 沙门氏菌感染4周的小鼠的体重。 <table border="1"><tbody><tr><td></td><td> 0 </td><td> 1个星期</td><td> 2个星期</td><td> 3个星期</td><td> 4周</td></tr><tr><td>控制</td><td> 16.78 ± 1.05 </td><td> 16.91 ± 1.28 </td><td> 18.26 ± 1.23 </td><td> 19.31 ± 1.26 </td><td> 20.26 ± 1.15 </td></tr><tr><td> PhoP 彗星 </TD&gt; <td> 16.89 ± 1.03 </td><td> 17.14 ± 1.19 </td><td> 17.43 ± 1.63 * </td><td> 18.68 ± 1.78 </td><td> 20.05 ± 1.11 </td></tr><tr><td> PhoP 彗星阿夫拉， </td><td> 16.91 ± 1.12 </td><td> 16.96 ± 1.39 </td><td> 17.06 ± 2.14 ** </td><td> 18.71 ± 2.18 </td><td> 20.15 ± 1.56 </td></tr><tr><td> PhoP 彗星 AvrA-/AvrA + </td><td> 16.94 ± 0.96 </td><td> 17.17 ± 1.02 </td><td> 17.63 ± 1.42 * </td><td> 18.44 ± 2.03 </td><td> 20.09 ± 1.17 </td></tr></tbody></table> *与对照组比较P &lt;0.05 **相比，对照组P &lt;0.01 表2。 沙门菌在这项研究中使用。 <table border="1"><tbody><tr><td>名称</td><td>说明</td><td>参考或来源</td></tr><tr><td> 沙门氏菌 14028s </td><td>野生型S.鼠伤寒沙门氏菌 </td><td> ATCC </td></tr><tr><td> PhoP 彗星 </td><td>非致病性的复杂的调节突变</td><td>米勒等人，1990年</td></tr><tr><td> PhoP 彗星阿夫拉， </td><td>阿夫拉的突变</td><td>科利尔- Hyams等，2002年</td></tr><tr><td> PhoP 彗星 AvrA-/AvrA + </td><td> PhoP 彗星阿夫拉补充质粒编码阿夫拉</td><td>科利尔- Hyams等，2002年</td></tr></tbody></table>

<ol>
	<li>Duan, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-Catenin+activity+negatively+regulates+bacteria-induced+inflammation.">beta-Catenin activity negatively regulates bacteria-induced inflammation.</a> Lab Invest. 87 (6), 613-624 (2007).</li><li>Barthel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pretreatment+of+mice+with+streptomycin+provides+a+Salmonella+enterica+serovar+Typhimurium+colitis+model+that+allows+analysis+of+both+pathogen+and+host.">Pretreatment of mice with streptomycin provides a Salmonella enterica serovar Typhimurium colitis model that allows analysis of both pathogen and host.</a> Infection and immunity. 71 (5), 2839-2858 (2003).</li><li>Miller, S. I., Mekalanos, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+expression+of+the+phoP+regulon+attenuates+Salmonella+virulence+and+survival+within+macrophages.">Constitutive expression of the phoP regulon attenuates Salmonella virulence and survival within macrophages.</a> Journal of bacteriology. 172 (5), 2485-2490 (1990).</li><li>Valdez, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nramp1+expression+by+dendritic+cells+modulates+inflammatory+responses+during+Salmonella+Typhimurium+infection.">Nramp1 expression by dendritic cells modulates inflammatory responses during Salmonella Typhimurium infection.</a> Cell Microbiol. 10 (8), 1646-1661 (2008).</li><li>Woo, H., Okamoto, S., Guiney, D., Gunn, J. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humane+endpoints+for+infectious+disease+animal+models.">Humane endpoints for infectious disease animal models.</a> ILAR J. 41 (2), 99-104 (2000).</li><li>Ye, Z., Petrof, E. O., Boone, D., Claud, E. C., Sun, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+effector+AvrA+regulation+of+colonic+epithelial+cell+inflammation+by+deubiquitination.">Salmonella effector AvrA regulation of colonic epithelial cell inflammation by deubiquitination.</a> Am J Pathol. 171 (3), 882-892 (2007).</li><li>Grassl, G. A., Valdez, Y., Bergstrom, K. S., Vallance, B. A., Finlay, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+enteric+salmonella+infection+in+mice+leads+to+severe+and+persiste+nt+intestinal+fibrosis.">Chronic enteric salmonella infection in mice leads to severe and persiste nt intestinal fibrosis.</a> Gastroenterology. 134 (3), 768-780 (2008).</li><li>Sukupolvi, S., Edelstein, A., Rhen, M., Normark, S. J., Pfeifer, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+murine+model+of+chronic+Salmonella+infection.">Development of a murine model of chronic Salmonella infection.</a> Infection and immunity. 65 (2), 838-842 (1997).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biosafety in Microbiological and Biomedical Laboratories (BMBL). , (2007).</li><li>Wu, S., Lu, R., Zhang, Y., Sun, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+effects+of+a+Salmonella+type+III+secretion+effector+protein+AvrA+in+vivo.">Chronic effects of a Salmonella type III secretion effector protein AvrA in vivo.</a> PLoS ONE. , (2010).</li></ol>

使用这个系统，它的研究人员，以了解如何灌胃动物是必要的。我们详细准备细菌培养和灌胃的小鼠的一种方法。我们还展示了如何监测在胃肠道（GI） 的沙门氏菌持久。在这个协议中的关键步骤包括： <ol><li>链霉素预处理：链霉素预处理可以摆脱一些共生的肠道菌群，使小鼠受到沙门氏菌 感染 2 。 </li><li> 沙门氏菌灌胃：对于初学者来说，灌胃可以是具有挑战性?...

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<td>HBSS</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>ABCD1234</td>
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<td>Luria-Bertani broth</td>
<td>&nbsp;</td>
<td>BD</td>
<td>244610</td>
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<td>Feeding needle</td>
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<td>Popper &amp; Sons, Inc</td>
<td>7920</td>
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<td>Streptomycin </td>
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<td>MP biomedicals, LLC</td>
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chronic salmonella infected mouse model

细菌感染的小鼠模型是一个强大的模型系统研究，如感染，炎症，免疫学，信号转导和肿瘤等领域。许多研究人员都采取诱导的结肠炎的优势<em&gt;沙门氏菌</em&gt;鼠伤寒沙门氏菌为炎症和感染的早期阶段的研究。然而，只有少数报告的慢性感染<em&gt;在体内</em&gt;。与小鼠<em&gt;沙门氏菌</em&gt;在胃肠道的持续存在，让我们去探索长期的宿主细菌的相互作用，信号转导和肿瘤。我们建立了一种慢性细菌感染小鼠模型<em&gt;沙门氏菌</em在小鼠小肠的鼠伤寒沙门氏菌的定植，6个月以上。使用这个系统，它为研究员，以了解如何准备的细菌培养和灌胃动物是必要的。我们详细准备细菌培养和灌胃小鼠的一种方法。我们还展示了如何检测<em&gt;沙门氏菌</em&gt;在胃肠道的持久性。总体而言，这一协议将有助于研究人员利用细菌感染小鼠模型，以从根本上解决重要的生物学和微生物学的问题。

Watch this Scientific Journal Video about Chronic Salmonella Infected Mouse Model at JoVE.com

Chronic Salmonella Infected Mouse Model

建立一个持久的慢性细菌感染的小鼠模型<em&gt;沙门氏菌</em&gt; 27周鼠伤寒肠道定植。

慢性沙门氏菌感染小鼠模型

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and ...

Research

JoVE Journal

Biology

Chronic Salmonella Infected Mouse Model

17.2K 次观看.  University of Rochester. 建立一个持久的慢性细菌感染的小鼠模型沙门氏菌 27周鼠伤寒肠道定植。

视频: Chronic Salmonella Infected Mouse Model

Orthotopic Mouse Model of Colorectal Cancer

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature cancer cells growing in their natural location, replicate human disease with high fidelity. Two techniques can be used to establish this model. Both techniques are similar and require mouse anesthesia and laparotomy for exposure of the cecum. One technique involves injection of a colorectal cancer cell suspension into the cecal wall. Cancer cells are first grown in culture, harvested when subconfluent and prepared as a single cell suspension. A small volume of cells is injected slowly to avoid leakage. The other technique involves transplantation of a piece of subcutaneous tumor onto the cecum. A mouse with a previously established subcutaneous colorectal tumor is euthanized and the tumor is removed using sterile technique. The tumor piece is divided into small pieces for transplantation to another mouse. Prior to transplantation, the cecal wall is lightly damaged to facilitate tumor cell infiltration. The time to developing primary tumors and liver metastases will vary depending on the technique, cell line, and mouse species used. This orthotopic mouse model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

Two techniques can be used to establish this model: injection of a cancer cell suspension into the cecal wall or transplantation of a piece of subcutaneous tumor onto the cecum. This model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

结直肠癌原位小鼠模型

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature ...

科研

生物学

Laser-Induced Chronic Ocular Hypertension Model on SD Rats

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

激光诱导SD大鼠慢性高眼压高压模型

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular ...

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, suprabasal, and stratum corneum layers. The precise localization within the epidermis of a particular protein lends clues toward its functional role within the epidermis. In this chapter, we describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory. Paraffin sections generally provide much better morphology, hence, superior results and photographs; however, not all antibodies will work with the harsh fixation and treatment involved in their processing. Therefore, the protocol for frozen sectioning is also included. Within paraffin sectioning, two fixation protocols are described (Bouin's and paraformaldehyde); the choice of fixative will be directly related to the antibody specifications and may require another fixing method.

We describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory.

使用荧光抗体对小鼠表皮石蜡切片的免疫组织化学

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, ...

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 ...

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 ...

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, chikungunya and Zika. Wolbachia-infected mosquitoes can be released into the field to either reduce population sizes through incompatible matings or to transform populations with mosquitoes that are refractory to virus transmission. For these strategies to succeed, the mosquitoes released into the field from the laboratory must be competitive with native mosquitoes. However, maintaining mosquitoes in the laboratory can result in inbreeding, genetic drift and laboratory adaptation which can reduce their fitness in the field and may confound the results of experiments. To test the suitability of different Wolbachia infections for deployment in the field, it is necessary to maintain mosquitoes in a controlled laboratory environment across multiple generations. We describe a simple protocol for maintaining Ae. aegypti mosquitoes in the laboratory, which is suitable for both Wolbachia-infected and wild-type mosquitoes. The methods minimize laboratory adaptation and implement outcrossing to increase the relevance of experiments to field mosquitoes. Additionally, colonies are maintained under optimal conditions to maximize their fitness for open field releases.

Maintaining Aedes aegypti Mosquitoes Infected with Wolbachia

Aedes aegypti mosquitoes infected with Wolbachia are being released into natural populations to suppress the transmission of arboviruses. We describe methods to rear Ae. aegypti with Wolbachia infections in the laboratory for experiments and field release, taking precautions to minimize laboratory adaptation and selection.

维护伊蚊蚊蚊子感染沃尔巴克

Aedes aegypti mosquitoes experimentally infected with Wolbachia are being utilized in programs to control the spread of arboviruses such as dengue, ...

The Murine Choline-Deficient, Ethionine-Supplemented (CDE) Diet Model of Chronic Liver Injury

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual inflammation, progressive destruction and regeneration of the hepatic parenchyma, liver progenitor cell proliferation, and fibrosis. The end-stage of every chronic liver disease is cirrhosis, a major risk factor for the development of hepatocellular carcinoma. To study processes regulating disease initiation, establishment, and progression, several animal models are used in laboratories. Here we describe a six-week time course of the choline-deficient and ethionine-supplemented (CDE) mouse model, which involves feeding six-week old male C57BL/6J mice with choline-deficient chow and 0.15% DL-ethionine-supplemented drinking water. Monitoring of animal health and a typical body weight loss curve are explained. The protocol demonstrates the gross examination of a CDE-treated liver and blood collection by cardiac puncture for subsequent serum analyses. Next, the liver perfusion technique and collection of different hepatic lobes for standard evaluations are shown, including liver histology assessments by hematoxylin and eosin or Sirius Red stainings, immunofluorescent detection of hepatic cell populations as well as transcriptome profiling of the liver microenvironment. This mouse model is suitable for studying inflammatory, fibrogenic, and liver progenitor cell dynamics induced through chronic liver disease and can be used to test potential therapeutic agents that may modulate these processes.

The Murine Choline-Deficient, Ethionine-Supplemented CDE Diet Model of Chronic Liver Injury

Here we describe a common method to induce chronic liver injury in mice by feeding of a choline-deficient and ethionine-supplemented (CDE) diet. We demonstrate health monitoring, liver perfusion, isolation, and preservation. A time course of six weeks can inform about liver injury, pathohistology, fibrosis, inflammatory, and liver progenitor cell responses.

慢性肝损伤小鼠胆碱缺乏、氨基补充的饮食模型

Chronic liver diseases, such as viral hepatitis, alcoholic liver disease, or non-alcoholic fatty liver disease, are characterized by continual ...

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 ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;

食品和环境样品中沙门氏菌的准总体基因体分析

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, ...

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.