Name: development of a negative selectable marker for entamoeba histolytica
Uploaded: 2010-12-12T17:30:00
Duration: 6 min 5 s
Description: Watch this Scientific Journal Video about Development of a Negative Selectable Marker for Entamoeba histolytica at JoVE.com

我们要感谢为我们提供FCU1基因序列与菲利普Erbs博士。这项工作是支持由美国国立卫生研究院授予的AI 26649。

1。风机盘管阿米巴负选择系统<ol><li>矢量控制线（HM1 / pKT3M）和实验线（HM1/pKT3M-FCU1）产生先前所描述的技术1,2。种子行股票管到的T - 25培养瓶，正在研究并保持缇S - 33 3培养基中加入适当的抗生素（12微克/毫升G418）的选择。烧瓶应后16-18小时，增长70％至80％汇合，在37 ° C。 </li><li>准备在温暖缇S - 33中的5 - 氟胞嘧啶（5 - FC，Sigma公司）的新鲜50MM原液。涡严格几分钟完全解散5 - FC。通过一个0.22微米的过滤器，过滤，消毒原液。 </li><li>收获的烧瓶放置在冰3-5分钟，收集细胞，离心5分钟，在室温在200xg滋养体。重悬沉淀在新鲜缇介质和细胞计数。 </li><li>一个典型的实验设计采用在最终体积为1 mL 0.5 × 10 4％和48孔板的细胞，一式三份重复，并为每个测试条件。此外，为方便荧光测量，一个单独的板块是用来为每人时间点的压力。 </li><li>种子0.5 × 10 4细胞的每个测试的5 - FC浓度，一式三份，每口井。保持在缇介质的抗生素选择压力。现在加入5 - FC股票的解决方案，并每口井滋养体，所需金额。 </li><li>在厌氧袋板在37 ° C。 </li></ol> 2。选择效率的测定<ol><li>准备2毫克/毫升细胞跟踪绿色CMFDA（Invitrogen公司）在DMSO原液。为了使工作液稀释1000倍，在无血清缇中等的股票。 </li><li>取出介质完全从滋养体培养板和150μL无血清缇含有CMFDA更换。继续孵化，在37 ° C为1小时。 </li><li>从水井中取出的染料溶液和Spectramax M2微孔板分光光度计读取的板块。应设置激发波长在492 nm处，荧光光谱在517 nm处读取。 </li><li>比较与测试转染对照组细胞的荧光值。 </li><li>继续阅读在每个时间间隔板块。在每盘使用适当的阳性（缇只）和负（25微克/ mL潮霉素）控制井。 </li></ol> 3。代表性的成果的大肠杆菌阿米巴转染密码子优化重组FCU1（图1）显示了强劲的表达，当这个协议是正确遵循E.选择性消除它的结果阿米巴细胞表达为0.5毫米5 -氟胞嘧啶存在FCU1。控制细胞，继续在对比正常生长至5毫米5 - FC浓度，达到72和96小时之间的汇合（图2 - A）。 FCU1进行48小时的细胞完全裂​​解处理井时，在显微镜下看。这些也显示下降的荧光时至关重要的，这是涉及大量的样品（图2 - B）的定量研究中使用的染料CMFDA染色。 FCU1/5-FC系统是一个有效和强大的的的E.负选择工具阿米巴滋养体。 <img src="/files/ftp_upload/2410/2410fig1.jpg" alt="图1" /> 图1。生成的E.阿米巴 HM1表达负选择标记的转染 （ 一 ）FCU1重组蛋白被发现使用抗c Myc抗体（顶部面板）在西部印迹。正如预期的那样，可以检测到一个42kDa带，在从FCU转染的总裂解（箭头所示），但没有单独的矢量控制转染。底部面板上显示相同的印迹与抗肌动蛋白抗体检测作为加载控制。 （二）定量实时PCR结果显示FCU1具体归lgl4控制的mRNA表达。 <img src="/files/ftp_upload/2410/2410fig2.jpg" alt="图2" /> 图2。 体外E.药物的敏感性阿米巴表达负选择标记的转染 （ 一 ）控制转染及（b）FCU1表达转染48孔板培养的生存曲线。细胞生长浓度的增加，存在的前体药物5 - 氟胞嘧啶; CMFDA染色细胞的存活在每个时间间隔量化X轴表示。 Y轴代表荧光单位，是一个幸存的细胞群的直接反映。请注意FCU1表达转染表明，在0.5mm的前体药物浓度显着的灵敏度比对照。没有观察细胞在这些井为COM 120H时间点相比汇合增长在显微镜下相应的控制井（数据未显示）看到。湿度计表示25μg/ mL潮霉素作为阴性对照。

<ol>
	<li>Olvera, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+transfection+of+Entamoeba+histolytica+trophozoites+by+lipofection.">Stable transfection of Entamoeba histolytica trophozoites by lipofection.</a> Arch. Med. Res. , 49-51 (1997).</li><li>Saito-Nakano, Y., Yasuda, T., Nakada-Tsukui, K., Leippe, M., Nozaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab5-associated+vacuoles+play+a+unique+role+in+phagocytosis+of+the+enteric+protozoan+parasite+Entamoeba+histolytica.">Rab5-associated vacuoles play a unique role in phagocytosis of the enteric protozoan parasite Entamoeba histolytica.</a> J. Biol. Chem. 279, 49497-49507 (2004).</li><li>Diamond, L. S., Harlow, D. R., Cunnick, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+medium+for+the+axenic+cultivation+of+Entamoeba+histolytica+and+other+Entamoeba.">A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba.</a> Trans. R. Soc. Trop. Med. Hyg. 72, 431-432 (1978).</li><li>Clark, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anaerobic parasitic protozoa : genomics and molecular biology. , (2010).</li><li>Linford, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+hairpin+RNA-mediated+knockdown+of+protein+expression+in+Entamoeba+histolytica.">Short hairpin RNA-mediated knockdown of protein expression in Entamoeba histolytica.</a> BMC Microbiol. 9, 38-38 (2009).</li><li>Solis, C. F., Guill&#233;n, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+genes+by+RNA+interference+in+the+protozoan+parasite+Entamoeba+histolytica.">Silencing genes by RNA interference in the protozoan parasite Entamoeba histolytica.</a> Methods Mol. Biol. 442, 113-128 (2008).</li><li>Solis, C. F., Santi-Rocca, J., Perdomo, D., Weber, C., Guill&#233;n, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+bacterially+expressed+dsRNA+to+downregulate+Entamoeba+histolytica+gene+expression.">Use of bacterially expressed dsRNA to downregulate Entamoeba histolytica gene expression.</a> PLoS ONE. 4, e8424-e8424 (2009).</li><li>MacFarlane, R. C., Singh, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+dsRNA-based+gene+silencing+in+Entamoeba+histolytica:+implications+for+approaches+to+genetic+analysis.">Loss of dsRNA-based gene silencing in Entamoeba histolytica: implications for approaches to genetic analysis.</a> Exp. Parasitol. 119, 296-300 (2008).</li><li>Dhar, S. K., Vines, R. R., Bhattacharya, S., Petri, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosomal+DNA+fragments+enhance+the+stability+of+transfected+DNA+in+Entamoeba+histolytica.">Ribosomal DNA fragments enhance the stability of transfected DNA in Entamoeba histolytica.</a> J. Eukaryot. Microbiol. 45, 656-660 (1998).</li><li>Erbs, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+cancer+gene+therapy+by+adenovirus-mediated+transfer+of+a+bifunctional+yeast+cytosine+deaminase/uracil+phosphoribosyltransferase+fusion+gene.">In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene.</a> Cancer Res. 60, 3813-3822 (2000).</li><li>Braks, J. A. M., Franke-Fayard, B., Kroeze, H., Janse, C. J., Waters, A. 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生产此视频，从而产生一些本文中使用的试剂由Sigma Aldrich公司，公司赞助。

虽然已经有阿米巴的分子生物学工具的重大进展，有没有可用的电流的方法，删除或破坏基因4。特异表达的基因敲除已取得使用发夹RNA的5，小干扰RNA 6和细菌表达的dsRNA 7。然而，这些方法有效，同时为各自的靶基因，有一些局限性，包括昂贵的化学品的使用，对抗生素的连续选择和需要不断验证，由于高发病率的回归，随着时间的推移发生（艾丽西亚林福德，个人通信）8。 质粒可以复制在溶有显然不是一个严格的时序要求的DNA复制的起源9，所以一旦转，它通常是难以治愈的质粒。这限制了可能使用完整的质粒在重组和基因敲除实验。负选择标记基因的定位方法，因为它们可以被用来消除重组亚群的关键组成部分。我们已经成功地表达了密码子优化FCU1 阿米巴的基因，并测试其潜在的负选择标记。此融合基因已被用于人类肿瘤细胞的自杀基因治疗和代谢成有毒下游产品造成抑制RNA和DNA的合成 10的前体药物5 - FC 。 FCU1最近一直使用负选择标记，另一种原生动物寄生虫疟原虫疟原虫细胞表达FCU1被发现近1000倍更加敏感， 在体外和体内治疗药物前体5 - FC超过99.9％的死亡重组寄生虫感染疟疾11红细胞阶段的小鼠模型。 E. 阿米巴细胞表达FCU1表明在P 中观察到的敏感性只有30倍的前体药物5 - FC的敏感性增加疟原虫 。可能的原因可能与有毒代谢产物的竞争中成长中等核苷酸的大型游泳池。 在两个体育疟原虫和P.恶性疟原虫的FCU1标记已被用于有针对性的基因破坏。研究11,12。我们的目标是操纵阿米巴基因组同源重组。验证FCU1/5-FC负选拔制度是实现这一目标迈出的重要一步。

<table border="1">
<tr>
<td>Material Name</td>
<td>Type</td>
<td>Company</td>
<td>Catalogue Number</td>
<td>Comments</td>
</tr>
<tr>
<td>TYI-S-33 medium</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>&nbsp;</td>
<td>Reference included in the text</td>
</tr>
<tr>
<td><a href="http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=F7129|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC">5-Fluorocytosine</a></td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F7129</td>
<td>&nbsp;</td>
<tr>
<td>Cell tracker green CMFDA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>C2925</td>
<td>&nbsp;</td>
<tr>
<td>Spectra Max M2 plate-reader</td>
<td>&nbsp;</td>
<td>Molecular Devices</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		
</table>

development of a negative selectable marker for entamoeba histolytica

阿米巴的阿米巴病的病原体和感染世界人口的10 ％。已启用上和基因表达调节的分子生物学技术，依靠稳定保持质粒转染。虽然这些都增加了阿米巴致病因素的认识，能力整合到基因组，这将使反向遗传学实验，将在研究这种寄生虫的一个显着的优势外源DNA。这个有机体提出的挑战，包括无法选择同源重组事件和难以治愈episomal质粒DNA转染滋养体。后来的结果在一个高的背景外源DNA，在重大问题确定一个真正的基因整合事件发生滋养体。我们的报告基于转基因表达的酵母胞嘧啶脱氨酶和尿嘧啶磷酸核糖转移酶嵌合体（FCU1）和前体药物5 - 氟胞嘧啶（5 - FC）选择的负选择系统的发展。 FCU1酶转换成有毒的5 -氟尿嘧啶和5 -氟- 5&#39; -磷酸。E.的无毒的5 - FC 阿米巴线表示FCU1被发现30倍以上敏感的前体药物的对照菌株相比。

Watch this Scientific Journal Video about Development of a Negative Selectable Marker for Entamoeba histolytica at JoVE.com

Development of a Negative Selectable Marker for Entamoeba histolytica

我们的报告负选择系统的发展<em&gt; E。阿米巴</em&gt;基于嵌合蛋白的转基因表达（FCU1）和前体药物5 - 氟胞嘧啶的选择。 FCU1蛋白是一种融合酵母胞嘧啶脱氨酶和尿嘧啶phosphoribosyltransferase。 FCU1表达导致增加<em&gt; E。阿米巴</em&gt;对5 - 氟胞嘧啶的敏感性。

一个负选择标记的发展阿米巴</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

Research

JoVE Journal

Immunology and Infection

Development of a Negative Selectable Marker for Entamoeba histolytica

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 (Ehmeth) Enzyme

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 Ehmeth Enzyme

在体外的tRNA与阿米巴 DNA和tRNA甲基Dnmt2（Ehmeth）酶甲基化含量

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of ...

科研

免疫与感染

Examination of Thymic Positive and Negative Selection by Flow Cytometry

考试流式细胞仪检测胸腺阳性和阴性选择

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

革兰从血培养液使用MALDI-TOF质谱阴性细菌快速鉴定

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

上皮损伤制作分析<em&gt;阿米巴</em&gt;感染

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Detection of Human Leukocyte Antigen Biomarkers in Breast Cancer Utilizing Label-free Biosensor Technology

在乳腺癌检测人类白细胞抗原生物标志物的利用无标记生物传感技术

According to the American Cancer Society, more than 200,000 women will be diagnosed with invasive breast cancer each year and approximately 40,000 will ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

可视化痰生物膜发展影响使用腔室盖玻片型号

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

乙型肝炎病毒记者系统的开发监控复制周期的早期阶段

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

利用胶囊对生物达标中病毒样品的阴性染色

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

Simultaneous Distinction of Monospecific and Mixed DFS70 Patterns During ANA Screening with a Novel HEp-2 ELITE/DFS70 Knockout Substrate

Monospecific 和混合 DFS70 模式在 HEp-2 ELITE/DFS70 击倒底物筛选中的同时鉴别

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies ...