Name: transformation of probiotic yeast and their recovery from gastrointestinal immune tissues following oral gavage in mice
Uploaded: 2016-02-08T14:00:00
Description: Watch this Scientific Journal Video about Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice at JoVE.com

作者承认通过授予特蕾西J.羔羊儿童中心免疫与疫苗和美国国立卫生研究院新的创新奖（1DP2AI112242-01）的资金。作者还感谢纳塔利娅·P. Degtyareva为RAD1 S的慷慨捐助酵母。

1. UV诱变产生营养缺陷型酵母菌株<ol><li>生成生存曲线，以确定所需剂量的紫外线照射的<ol><li>根据标准方法26制备的YPD（酵母提取物蛋白胨葡萄糖）的媒体，在表1中列出的其它试剂和接种单个菌落到5-10毫升的YPD培养基。孵育在辊筒培养在30℃的CO / N至饱和至少8小时。 </li><li>通过稀释细胞1:10在水中的塑料比色杯中测定使用分光光度计O / N培养物的细胞浓度。稀释细胞在20ml无...

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一起，这些协议本文描述所必需的开发和营养缺陷型益生菌酵母菌株的测试用于递送外源治疗性蛋白质的于肠的基本步骤。这种操纵和重组益生菌酵母的测试需要的技术和资源，与任何个人的实验室目前可能不熟悉。因此，尽管先前的许多研究已经说明上述协议多次酵母和小鼠品系，这些方法都没有作者的知识进行了详细，统一的形式提交。此外，本手稿特别强调适应当前标准化协议为益生菌酵?...

益生菌微生物是有效和经济地提供异源蛋白对胃肠道一个有趣的电位的装置。这些微生物能够通过胃肠道存活通路还没有殖它1，使控制剂量和限制性的药物表示曝光的。此外，为了容易工程师这些生物体产生大规模的异源蛋白质的能力使它们的经济替代合成递送颗粒。然而，这样的方法的开发，作为使用益生菌酵母布拉酵母菌 2的营养缺陷型菌株最近证明，需要的实验室技术在给定研究中传统上不结合，从酵母和分子生物学到动物处理技术和免疫学方法的知识。因此，尽管本文所描述的各个过程本身并不新颖实验室协议，本手稿的目标是提出一种统一介绍所需益生菌酵母的实验测试作为药物递送载体的鼠胃肠道的技术。提供是为必要的协议汇编：1）代的酵母的营养缺陷型突变株，可以很容易进行遗传操纵的; 2）酵母培养的改造，表达外源蛋白; 3）重组酵母经口服灌胃肠道的管理;和4）从它们的异源蛋白质表达的鼠肠和评估可行重组益生菌酵母的回收​​。 首先，虽然用于酵母物种的操作存在许多阳性和阴性选择方法，阴性选择如通过利用营养缺陷型标记的增大的效率和缓解与酵母可转化和选择。使用抗生素转化的积极选择，在合作ntrast，显著增加酵母操纵的成本。此外，酵母在含有抗生素的固体培养基的选择可允许相对于合成滴出固体介质（未发表的观测值）的营养缺陷型酵母的选择未转化的背景菌落增加的生长。营养缺陷型酵母是缺乏酶的必需氨基酸或尿嘧啶的合成关键菌株。如果补充丢失的代谢产物或代谢基因，从而使消极的选择，当酵母接种到合成辍学媒体缺乏必要的代谢产物如酵母只能生长。许多常用的酿酒酵母的实验室菌株中实际上已经营养缺陷型突变体3。工业，临床和益生菌酵母菌株，但是，通常是原养与合成所需的所有营养物质的能力。为了使这样的酵母更高效的基因操作，营养缺陷型的基因可以被选择性地定位产生能够无抗生素选择菌株。 6 -营养缺陷型的标记基因特异性靶向可通过PCR介导的基因破坏依靠同源重组或最近通过CRISPR / Cas9目标4来实现。可替代地，紫外诱变可以快速生成，即使在酵母菌株为哪些变换与多个质粒在技术上是困难7营养缺陷型突变体。虽然PCR定位和CRISPR / Cas9已经被广泛地描述在其他地方，部分提出这个手稿之一是描述紫外诱变的方法来创建营养缺陷型菌株，将允许阴性选择，而不是酵母转化的积极的抗生素选择一个详细的协议。 在使用为异源蛋白质的口服递送这样的营养缺陷型菌株的下一个必要的步骤是酵母转化用质粒DNA。由于赞成票的第一个成功转型在1978年8报道了酿酒酵母牛逼的球状体，大量的修改进行了表征，以提高效率和易用性与酵母菌种可遗传修饰。利用电对DNA的成功转型为S.酵母最早是在1985年9节 ，至今已通过加入1M山梨醇培养渗透到细胞支持10提高。电效率此外，已显示依赖于酵母种和菌株，细胞数和生长的阶段，电体积，电场强度，和特定的缓冲区11。醋酸锂（LiOAc）变换，最初由Ito等人12所述，是最常用的变换协议之间，因为它不需要特殊的设备。附加分析表明，LiOAc酵母转化效率时，细胞在数中期收集大大增加生长并热在聚乙二醇（PEG）和DNA的存在下，在42℃12震惊。用PEG整个完整的酵母的培养是高效转化必需的，有可能通过提高DNA与细胞膜的附着以及通过在膜13等效果。锂本身也增加了完整细胞14的渗透性。尽管大多数实验室S.酵母菌株可以很容易地使用LiOAc变换3被转化，其它酵母物种可更有效地使用替代的协议变换。 巴斯德毕赤酵母 ，例如，是通过电穿孔，而不是LiOAc变换13最有效地转化，这是至关重要的，因此，要测试多个改造和方法，试图在基因修改未鉴定的酵母菌株，优化培养时间和试剂浓度。这份手稿从而描述了两种LiOAc TRansformation和电穿孔作为营养缺陷型突变体和野生型S的转化技术布拉 。感兴趣的读者可以直接用于酵母转化，替代协议和行动13,15的可能机制进一步讨论演变的深入描述最近的评论。用质粒编码易于检测的蛋白质的酵母的转化是为了确保正确的表达和异源蛋白质的功能下游测试进一步必要的。无数的不同蛋白质可以根据治疗研究的最终目的和通过免疫印迹，ELISA和其它技术可用于蛋白质检测的抗体来选择。对这些技术的协议已被彻底别处16,17描述，并且可用于通过比较标准曲线，以确定从转化的酵母异源蛋白质的生产水平。为目的的示范的，并显示出成功地生产在酵母生物学一个很常用的蛋白质，这手稿呈现质粒编码绿色荧光蛋白（GFP），其允许使用荧光显微镜后续检测转变。 到生产表达异源蛋白质是适当管理和胃肠组织内的这些微生物的检测，如在第三和第四部分中描述的益生菌微生物的同样重要。通过口服管饲法重组酵母的施用允许递送的酵母的控制量直接进入胃，从中C57BL / 6小鼠是自然不能呕吐18。然而，不当处理动物灌胃，并可能导致食管损伤，穿孔，胃穿孔，气管管理和吸入性肺炎19,20。可怜的技术和缺乏经验可以进一步提高小鼠的免疫反应和实验地区环境部门一道变异LTS，已经口服管饲法21,22归因于动物的压力。在适当的技术实践可因此不仅衰减动物的不适，但也可以增加的实验结果的精度。这份手稿介绍并演示了动物处理和口服灌胃的掌握剂量重组酵母的管​​理。 最后，这是至关重要的，通过分析淋巴组织用于酵母和异源蛋白质的存在下，以确认重组酵母的成功递送。可以最容易地且可预测地检测为酵母的存在下消化道免疫组织是淋巴集结。淋巴集结沿小肠二级淋巴器官是粘膜免疫应答的诱导23关键点。从内腔抗原通过微皱transcellularly转印在上皮（M）的细胞和被释放到淋巴集结，从而暴露括D抗原呈递细胞肠肠腔内容物。虽然在整个肠上皮粒子的摄取也可以由杯状细胞来实现，这些细胞已被证明仅占用颗粒小于0.02微米的直径24。跨上树突从CD103 +树突状细胞（DC）也采取了从肠道小颗粒管腔25延伸;然而，目前还没有报告表明CD103 +的DC占用比细菌大颗粒。因此，完好的益生菌酵母，3-6微米直径之间的平均大小，最有可能由M细胞吸收并转移到淋巴集结。这里描述的是用于收集和淋巴集结为可行的重组酵母的筛选的协议，虽然这过程也可以容易地适应用于评估益生菌摄取。 综上所述，评估重组益生菌酵母的交付rapeutic蛋白质在肠道需要实验室技术跨越分子生物学动物处理和免疫能力。 1这里提出是协议），它可以很容易地无抗生素负选择的生成和营养缺陷型酵母菌株的筛选，2）替代协议来转化酵母，并启用异源蛋白质的表达，3）的适当的动物处理方法和用于口服管饲法示范胃内递送重组酵母的，4）协议淋巴集结解剖和筛选可行的重组酵母和功能性的异源蛋白质。相结合，这些协议将允许产生和能够提供异源的治疗性蛋白的胃肠道的益生菌酵母株的测试。

<table border="0" cellpadding="0" cellspacing="0" width="704">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">SmartSpec 3000 Spectrophotometer</td>
			<td style="width:107px;">BioRad</td>
			<td style="width:107px;">170-2501</td>
			<td style="width:275px;">Example of spectrophotometer for determining cell concentration and OD600 of yeast cultures</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">New Brunswick Roller Drum</td>
			<td style="width:107px;">Eppendorf</td>
			<td style="width:107px;">M1053-4004</td>
			<td style="width:275px;">Example of roller drum for yeast culture incubation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UV Stratalinker 2400</td>
			<td style="width:107px;">Stratagene</td>
			<td style="width:107px;">400075-03</td>
			<td style="width:275px;">Example stratalinker</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Stuart Colony Counter SC6PLUS</td>
			<td style="width:107px;">11983044</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td style="width:275px;">Plate stand with magnification records colony count upon sensing pressure from pen</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Scienceware Colony Counter&#160;</td>
			<td style="width:107px;">F378620002</td>
			<td style="width:107px;">Bel-Art Scienceware</td>
			<td style="width:275px;">Hand held colony counter pen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Replica plating device</td>
			<td style="width:107px;">Fisherbrand</td>
			<td>09-718-1</td>
			<td rowspan="2" style="width:275px;">Example of replica plating stand and pads&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Velveteen squares</td>
			<td style="width:107px;">Fisherbrand</td>
			<td>09-718-2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;L shaped sterile cell spreaders&#160;</td>
			<td style="width:107px;">Fisherbrand</td>
			<td style="width:107px;">14665230</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Deoxyribonucleic acid, single stranded from salmon testes</td>
			<td style="width:107px;">Sigma-Aldrich</td>
			<td style="width:107px;">D7656-1ML</td>
			<td style="width:275px;">Example carrier DNA for yeast LiOAc transformation</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">Gavage needles</td>
			<td style="width:107px;">Braintree Scientific</td>
			<td style="width:107px;">N-PK&#160;002</td>
			<td style="width:275px;">For mice 15-20 g, the suggested needle is a 22 gauge (1.25 mm ball), 1 in long, straight reusable gavage needle. For mice weighing greater than 20 g, 20 gauge or larger straight or curved gavage needles may be used</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1mL sterile slip-tip disposable tuberculin syringe&#160;</td>
			<td style="width:107px;">Becton Dickinson</td>
			<td style="width:107px;">BD 309659</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Blunt forceps such as Electron Microscopy Sciences 7&#34; (178 mm) serrated tip, broad grip forceps</td>
			<td style="width:107px;">Electron Microscopy Sciences</td>
			<td style="width:107px;">77937-28</td>
			<td style="width:275px;">Example of blunt forceps needed for dissection&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Straight and curved dissection scissors&#160;</td>
			<td style="width:107px;">Electron Microscopy Sciences&#160;</td>
			<td style="width:107px;">72966-02 and 72966-03</td>
			<td style="width:275px;">Examples of scissors needed for dissection&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">IMDM</td>
			<td style="width:107px;">Life technologies</td>
			<td style="width:107px;">12440053</td>
			<td style="width:275px;"></td>
		</tr>
	</tbody>
</table>

transformation of probiotic yeast and their recovery from gastrointestinal immune tissues following oral gavage in mice

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat gastrointestinal disorders. Adapting probiotic yeast in particular for this approach carries several advantages. These strains have not only the potential to synthesize a wide variety of complex heterologous proteins but are also capable of surviving and protecting those proteins during transit through the intestine. Critically, however, this approach requires expertise in many diverse laboratory techniques not typically used in tandem. Furthermore, although individual protocols for yeast transformation are well characterized for commonly used laboratory strains, emphasis is placed here on alternative approaches and the importance of optimizing transformation for less well characterized probiotic strains. Detailing these methods will help facilitate discussion as to the best approaches for testing probiotic yeast as oral drug delivery vehicles and indeed serve to advance the development of this novel strategy for gastrointestinal therapy.

以下UV照射一个存活曲线的产生需要稀释的酵母细胞，使得不同的集落形成单位（CFU）能够形成的镀覆。收集上述各500μl的样品含有约5×10 6个细胞;然而，每板大于100个菌落都难以准确区分。电镀未稀释样品以及照射细胞的连续1点10稀释从而确保了CFU可以在每个UV剂量可以列举， 如在图1表明该CFU计数，乘以稀释因子，然后通过的总数量除以原始照射细?...

Watch this Scientific Journal Video about Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice at JoVE.com

Transformation of Probiotic Yeast and Their Recovery from Gastrointestinal Immune Tissues Following Oral Gavage in Mice

这里介绍的是，可以用于开发，变换，管理，和测试益生菌酵母布拉酵母菌中异源蛋白质表达技术的统一描述。

益生菌酵母的转型及其胃肠道粘膜免疫组织恢复继小鼠口服灌胃

Development of recombinant oral therapy would allow for more direct targeting of the mucosal immune system and improve the ability to combat ...

Research

JoVE Journal

Immunology and Infection

Seven Steps to Stellate Cells

星状细胞的七个步骤

Hepatic stellate cells are liver-resident  cells of star-like morphology and are located in the space of Disse between  liver sinusoidal endothelial cells ...

科研

免疫与感染

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

口头传播的<em&gt;单核细胞增生李斯特氏菌</em小鼠通过食入被污染的食物

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

诱导小鼠肠道炎症由效应CD4的过继转移<sup&gt; +</sup&gt; CD45RB<sup&gt;高</sup&gt; T细胞免疫缺陷小鼠

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

原位检测使用地高辛标记的DNA探针石蜡包埋组织中的细菌靶向16S rRNA基因

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

分离，鉴定和牙龈免疫细胞网络的功能性考试

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

在免疫细胞在不同组织与疾病相关的分布和小鼠实验评价性自身免疫性脑脊髓炎的诱导

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

Protective Efficacy and Pulmonary Immune Response Following Subcutaneous and Intranasal BCG Administration in Mice

保护疗效及肺部免疫反应皮下和鼻内BCG管理在小鼠

Despite global coverage of intradermal BCG vaccination, tuberculosis remains one of the most prevalent infectious diseases in the world. Preclinical data ...

Probiotic Studies in Neonatal Mice Using Gavage

新生儿小鼠益生菌的研究

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult ...