作者感谢育种者和Boyé accouvage协会（法国La Boissière en Gâtine）提供受精卵。这项研究是在研究联盟APR-IA"INTEGRITY"（2017-2019）的主持下进行的，该联盟由法国卢瓦尔河谷地区中心资助。

动物护理程序是根据欧洲共同体理事会指令（86/609/EEC）和法国动物实验条例制定的准则进行的。
1. 隔离器准备
<ol><li>在正压下将必要的材料插入刚性隔离器:50 mL 管、5、10 和 25 mL 塑料移液器、辐照进料、高压灭菌水和无菌密封塑料容器。</li>
	<li>用2%季铵盐溶液填充转移杀菌阱。</li>
	<li>使用60 mL福尔马林（24%甲醛）加入30g高锰酸钾（m 3），用甲醛蒸气灭菌隔离器三次。在每次灭菌之间移动隔离器内的材料，以确保所有接触面的灭菌。</li>
	<li>在引入卵子之前，将隔离器温度设置为37°C至少2天。在引入当天将湿度计设置为相对湿度（RH）的65-70%。</li>
</ol>2. 种蛋收集和孵化
<ol><li>根据蛋鸡的良好孵化率（在产蛋高峰期至少80%）及其良好的卫生状况（即没有常见的家禽病原体和羊群中没有疾病）从选择的农场收集鸡蛋。在跑步机上直观地选择干净无瑕的鸡蛋。</li>
	<li>通过在室温下将其浸入1.5%过氧乙酸溶液中浸渍5分钟，立即对卵表面进行去污。</li>
	<li>使用用甲醛蒸气净化的盒子将鸡蛋运输到实验设施。</li>
	<li>将鸡蛋在4°C下储存24小时。 重复步骤2.2中描述的去污过程，并将其置于孵化箱（第0天）中19天。</li>
</ol>3. 孵化
<ol><li>在第19天，通过在无菌条件下使用轻鸡蛋蜡烛将它们倒入来验证生育能力，活力（运动性）和胚胎发育。仅将活胚胎卵引入无菌孵化隔离器中。</li>
	<li>通过喷洒1.5%过氧乙酸溶液30秒或直到每个鸡蛋的整个表面被覆盖来对选定的鸡蛋进行净化。在转移到隔离器的过程中，卵子将与喷雾保持16分钟和30秒的接触。</li>
	<li>将卵转移到无菌孵化隔离器中，并用无菌软化水冲洗，然后将其放入孵化空间。 注意:动物在无菌隔离器中孵化和饲养。他们 随意 喂食通过伽马辐照消毒的商业饮食，并用饮水机提供的高压灭菌的自来水浇水。</li>
</ol>4. 细菌学状态分析
<ol><li>孵化后一天，直接从不同雏鸡的直肠中取出粪便样本，并将其池化在灭菌玻璃管中，以使该样本代表给定隔离器内的所有动物。</li>
	<li>将粪便样品或多或少地是液体，用利沙林向9mL硫代甘氨酸盐肉汤中加入相当于1mL的粪便。将剩余的粪便样品加入9mL脑心输液（BHI）中，并将管子在37°C下孵育18至48小时而不摇动。这将允许广泛的有氧，兼性有氧和非快速厌氧物种的生长。 注意:含有刃天青的硫代乙酸盐肉汤用于检测非快速厌氧细菌，但它也可以检测好氧细菌。该培养基符合欧洲，美国和日本药典的无菌测试18，19，20。</li>
	<li>目视观察孵育18小时后生长培养基是否有任何修饰。48小时后，从BHI粪汤培养基中滴出一滴，将其放在载玻片上，并在显微镜下观察（40X放大倍率）是否没有或存在细菌。</li>
	<li>如果怀疑细菌的存在，请从BHI培养物中取样并将其播种到BHI琼脂平板上。在37°C下孵育18至48小时。</li>
	<li>如果存在菌落，则进行高通量技术，例如MALDI-TOF质谱法，以进行精确的微生物鉴定。 注意:72小时后，如果细菌学分析为阴性，则宣布动物无菌。</li>
</ol>

<ol>
	<li>Shang, Y., Kumar, S., Oakley, B., Kim, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chicken+microbiota:+importance+and+detection+echnology.">Chicken microbiota: importance and detection echnology.</a> Frontiers in Veterinary Science. 23, 5 (2018).</li><li>Grover, M., Kashyap, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ-free+mice+as+a+model+to+study+effect+of+gut+microbiota+on+host+physiology.">Germ-free mice as a model to study effect of gut microbiota on host physiology.</a> Neurogastroenterology &amp; Motility. 26 (6), 745-748 (2014).</li><li>Umesaki, Y. <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+gnotobiotic+mice+to+identify+and+characterize+key+microbes+responsible+for+the+development+of+the+intestinal+immune+system.">Use of gnotobiotic mice to identify and characterize key microbes responsible for the development of the intestinal immune system.</a> Proceedings of the Japan Academy, Ser. B, Physical and Biological Sciences. 90 (9), 313-332 (2014).</li><li>Carrasco, J. M., Casanova, N. A., Fern&#225;ndez, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiota,+gut+health+and+chicken+productivity:+what+is+the+connection?.">Microbiota, gut health and chicken productivity: what is the connection?.</a> Miyakawa. Microorganisms. 7 (10), 374 (2019).</li><li>Lee, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+microbial+communities+in+the+chicken+oviduct+and+the+origin+of+chicken+embryo+gut+microbiota.">Characterization of microbial communities in the chicken oviduct and the origin of chicken embryo gut microbiota.</a> International Journal of Scientific Reports. 9 (1), 6838 (2019).</li><li>Hincke, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+structural+barriers+and+innate+immune+components+during+incubation+of+the+avian+egg:+critical+interplay+between+autonomous+embryonic+development+and+maternal+anticipation.">Dynamics of structural barriers and innate immune components during incubation of the avian egg: critical interplay between autonomous embryonic development and maternal anticipation.</a> Journal of Innate Immunity. 11 (2), 111-124 (2019).</li><li>Le Bars, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demonstration+of+a+protocol+for+obtaining+germ-free+chickens.">Demonstration of a protocol for obtaining germ-free chickens.</a> Annals of Veterinary Research. 7, 383-396 (1976).</li><li>Lafont, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+study+of+some+factors+limiting+'competitive+exclusion'+of+Salmonella+in+chickens.">Experimental study of some factors limiting 'competitive exclusion' of Salmonella in chickens.</a> Research in Veterinary Science. 34 (1), 16-20 (1983).</li><li>Br&#233;e, A., Dho, M., Lafont, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+infectivity+for+axenic+and+specific-pathogen-free+chickens+of+O2+Escherichia+coli+strains+with+or+without+virulence+factors.">Comparative infectivity for axenic and specific-pathogen-free chickens of O2 Escherichia coli strains with or without virulence factors.</a> Avian Diseases. 33 (1), 134-139 (1989).</li><li>Dozois, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+colonization+and+in+vivo+expression+of+F1+(type+1)+fimbrial+antigens+in+chickens+experimentally+infected+with+pathogenic+Escherichia+coli.">Bacterial colonization and in vivo expression of F1 (type 1) fimbrial antigens in chickens experimentally infected with pathogenic Escherichia coli.</a> Avian Diseases. 38 (2), 231-239 (1994).</li><li>Schouler, C., Taki, A., Chouikha, I., Moulin-Schouleur, M., Gilot, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genomic+island+of+an+extraintestinal+pathogenic+Escherichia+coli+strain+enables+the+metabolism+of+fructooligosaccharides,+which+improves+intestinal+colonization.">A genomic island of an extraintestinal pathogenic Escherichia coli strain enables the metabolism of fructooligosaccharides, which improves intestinal colonization.</a> Journal of Bacteriology. 191 (1), 388-393 (2009).</li><li>Porcheron, G., Chanteloup, N., Trotereau, A., Br&#233;e, A., Schouler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+fructooligosaccharide+metabolism+on+chicken+colonization+by+an+extra-intestinal+pathogenic+Escherichia+coli+strain.">Effect of fructooligosaccharide metabolism on chicken colonization by an extra-intestinal pathogenic Escherichia coli strain.</a> PLoS One. 7 (4), 35475 (2012).</li><li>Kraimi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+a+gut+microbiota+transfer+on+emotional+reactivity+in+Japanese+quails+(Coturnix+japonica).">Effects of a gut microbiota transfer on emotional reactivity in Japanese quails (Coturnix japonica).</a> of Experimental Biology. 222, (2019).</li><li>Stanley, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+microbiota+associated+with+differential+feed+conversion+efficiency+in+chickens.">Intestinal microbiota associated with differential feed conversion efficiency in chickens.</a> Applied microbiology and biotechnology. 96, 1361-1369 (2012).</li><li>Han, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+the+gut+microbiota+composition+on+Campylobacter+jejuni+colonization+in+chickens.">Influence of the gut microbiota composition on Campylobacter jejuni colonization in chickens.</a> Infection and Immunity. 85 (11), 0038017 (2017).</li><li>Thomas, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut+microbial+dynamics+during+conventionalization+of+germ-free+chicken.">Gut microbial dynamics during conventionalization of germ-free chicken.</a> mSphere. 4 (2), 00035-00119 (2019).</li><li>Langhout, D. J., Schutte, J. B., de Jong, J., Sloetjes, H., Verstegen, M. W., Tamminga, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+viscosity+on+digestion+of+nutrients+in+conventional+and+germ-free+chicks.">Effect of viscosity on digestion of nutrients in conventional and germ-free chicks.</a> British Journal of Nutrition. 83 (5), 533-540 (2000).</li><li>European Pharmacopoeia. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> European Pharmacopoeia (Ph. Eur.): Supplement 6.3, Sterility. , (2009).</li><li>Japanese Pharmacopoeia, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Japanese Pharmacopoeia (JP): The 4.06 Sterility Test as it appeared in the partial revision of the JP 15th edition. , (2009).</li><li>United States Pharmacopeia (USP). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> United States Pharmacopeia (USP): Sterility Tests as presented in Pharmacopeial Forum, Interim Revision Announcement No. 6. 34 (6), (2008).</li><li>Harrison, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+germ-free+chicks:+a+comparison+of+the+hatchability+of+eggs+sterilized+externally+by+different+methods.">Production of germ-free chicks: a comparison of the hatchability of eggs sterilized externally by different methods.</a> Laboratory Animals. 3 (1), 51-59 (1969).</li><li>Yokota, H., Furuse, M., Okumura, J., Iwao Task, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+production+and+rearing+of+the+germ-free+chick.">A simple method for production and rearing of the germ-free chick.</a> J-STAGE home Nihon Chikusan Gakkaiho. 55 (8), (1984).</li><li>Ding, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inheritance+and+establishment+of+gut+microbiota+in+chickens.">Inheritance and establishment of gut microbiota in chickens.</a> Frontiers in Microbiology. 8, 1967 (2017).</li></ol>

作者没有什么可透露的。

以前已经描述了几种产生无菌鸡的方法7，21，22。简单的方法，例如这里介绍的方法，使用不同的消毒剂来减少卵子表面和隔离器中的细菌负荷。最常用的消毒剂是氯化汞、季铵盐、碘仿、次氯酸钠和二氧化氯溶液。结果往往令人满意。然而，尽管这些方法易于获得，但很少有结构可以应用该方法并大规模饲养动物，因此使得利用无菌鸡成为一种相对罕见的方法，仅用于解决非常具体的科学问题。这里描述的方法，材料和设备允许无菌肉鸡的高效孵化比例，这些肉鸡保持健康和无菌至少3周（生产它们的科学实验的持续时间）。
结果表明，将该方案改编为从商业家禽养殖场收集的鸡蛋中生产无菌雏鸡是成功的。SPF产蛋经验表明，孵化效率主要取决于蛋鸡的年龄和收集的蛋的质量。在选择农场和收集种蛋时，这两个参数都被考虑在内。使用相同的方法，无菌肉鸡的平均孵化率远远优于我们工厂上次生产SPF蛋鸡（79%对35%），而不影响动物不育（87%对83%）。这些差异可能与鸟类的遗传背景（肉鸡与蛋鸡）以及蛋壳的质量有关，蛋壳在SPF动物的蛋中可能更脆弱，在封闭繁殖中保持了40多年。此外，我们还表明，超过2小时的运输（从养殖场到实验设施）不会影响孵化效率和质量。
虽然用于孵化隔离器的灭菌过程和鸡蛋去污方案得到了优化，但大约10%的隔离器不是无菌的。为了了解污染的来源，在引入卵子之前对孵化的隔离器进行常规的无菌控制非常重要。
关于鸟类的不育状态，我们试图应用允许粪便样品中需氧和非快速厌氧细菌生长的方法，从而确保我们检测到活的活细菌。这些方法符合国际无菌测试药典，代表了常规使用的快速，简单和降低成本的技术。然而，分子生物学技术，如16S rRNA基因测序，虽然没有提供细菌活力的信息，但可以应用于确认不可培养细菌的存在。事实上，最近的一项研究表明，母体输卵管微生物群的一些细菌似乎通过蛋清转移到胚胎上，后来构成了大部分胚胎肠道细菌种群5。此外，另一项研究表明，早期胚胎中携带的部分微生物定殖者是从母鸡遗传的，肠道微生物的丰度和多样性后来受到发育过程中环境因素和宿主遗传学的影响23。然而，这些研究的结果是基于DNA序列分析，其中大量这些细菌可能在蛋清中死亡或不可复制（大量含有抗菌分子）。Thomas和16 名合作者通过粪便滴落在BHI平板上评估可培养的需氧和兼性需氧细菌，进行了无菌测试，从而突出了标准细菌学方法在无菌无菌控制方面的效率。此外，在提出的方案中，我们使用乙酰草素在硫代乙酸盐肉汤中的生长监测，以便能够检测非快速厌氧菌的生长。
该方案已经用于生产无菌鹌鹑和鸡，可适应大多数筑巢鸟类的无菌动物的生产，并为研究微生物群对这些动物的生理学贡献提供了视角。除了使用该模型来研究家禽肠道中的宿主 - 微生物群相互相互作用外，它还可用于应用研究。例如，它可用于评估来自鸡肠道共生微生物的益生菌的安全性和有效性，以改善动物的健康和健壮性。

关于肠道微生物群对动物健康的贡献的科学和大众兴趣急剧增加。微生物群由栖息在动物肠道不同生态位的细菌，病毒，真菌和古菌组成，直接或间接地与炎症，传染病和代谢性疾病的调节有关，这些疾病不仅影响哺乳动物物种，还影响牲畜，如家禽1。开发了几种动物模型，以更好地研究肠道微生物群对健康和疾病的贡献。例如，无菌和无菌动物允许分别在感染的物理病理学上研究完全没有微生物或已知微生物群2，3。然而，产生和维护这些动物需要专门的技术和设施，而维护它们所需的成本，劳动力和技能限制了它们与许多研究人员的联系。事实上，必须使用细菌培养方法、显微镜检查、血清学、大体形态学和基于测序的检测技术的组合，定期监测无菌动物是否存在可能的污染。类似的程序也适用于其他物种，如牲畜，其中动物通常体型较大，需要更大的设施进行繁殖和维护，这可能在一定程度上阻碍对微生物群的研究。
家禽，更具体地说是鸡，是全球畜牧业生产的基石，每年的家禽数量可能超过400亿只。它是世界上最重要的动物蛋白来源（http://www.fao.org/poultry-production-products/en/）。此外，没有与养鸡或食用鸡有关的文化或宗教禁忌。家禽肠道微生物群参与动物的生长、饲料转化率、免疫力、病原体抗性等诸多营养、生理或病理过程4。因此，无菌鸡的产生对于强调微生物群与其宿主之间的对话是必不可少的4。即使微生物群落栖息在鸡的输卵管5中，健康母鸡新鲜产下的蛋的含量也大多不含微生物，蛋壳和膜具有机械屏障，以避免微生物入侵4。此外，雏鸡在没有亲戚的情况下很容易饲养，与哺乳动物不同，这允许在无菌条件下没有父母抚养的情况下生产无菌动物。
实验设施"农场，模型和野生动物的感染学"（PFIE，UE-1277，法国努齐利卢瓦尔河谷中心，https://doi.org/10.15454/1.5572352821559333e12）是法国国家基础设施网络EMERG'IN（https://www.emergin.fr/）的一部分。PFIE已经掌握了无菌鸡的生产，进行了40多年的各种实验研究7，8，9，10，11。这些动物是由自20世纪70年代以来在封闭育种中饲养的白色腿角产蛋中产生的特定无病原体（SPF）卵产生的。主要用于微生物研究7，8，9，无菌鸟类正在经历兴趣的复苏，例如肠道微生物群对行为13，营养利用14，免疫发育15和内分泌活性的贡献等问题。即使一些研究已经使用无菌肉鸡系16发表，与使用实验层系的研究相比，这些研究仍然代表性不足。在家禽健康和福利中，微生物群与其宿主之间的串扰的科学问题演变促使我们调整我们的历史协议，以生产Ross PM3系列的无菌肉鸡，这是世界上使用最多的肉鸡系列。

<table border="0" cellpadding="0" cellspacing="0" style="width:904px;" width="904">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">2 mL sterile plastic pipettes</td>
			<td style="width:153px;">Starsted</td>
			<td style="width:119px;">86.1252.001</td>
			<td style="width:389px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">50 mL tubes</td>
			<td style="width:153px;">Falcon</td>
			<td style="width:119px;"></td>
			<td style="width:389px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">BHI agar plates</td>
			<td style="width:153px;">Thermo fisher diagnostic</td>
			<td style="width:119px;">PO1198A</td>
			<td style="width:389px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Brain Heart Infusion broth</td>
			<td style="width:153px;">Thermo fisher diagnostic</td>
			<td style="width:119px;">CM1135</td>
			<td style="width:389px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Glass tubes with 9 mL BHI broth</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td style="width:389px;">home made and sterilized by autoclaving</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Glass tubes with 9 mL thioglycolate broth with resazurin</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td style="width:389px;">home made and sterilized by autoclaving</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Hatching incubator</td>
			<td style="width:153px;">Fieme</td>
			<td style="width:119px;">MG 576</td>
			<td style="width:389px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Incubator</td>
			<td style="width:153px;">Memmert</td>
			<td style="width:119px;"></td>
			<td style="width:389px;">for bacteriological culture, 37 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Irradiated feed</td>
			<td style="width:153px;">Safe</td>
			<td style="width:119px;">U8983G10R</td>
			<td style="width:389px;">40 kG irradiated</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:243px;">Isolators</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td style="width:389px;">home made. 1 m3 rigid isolator under positive pressure</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Microbiological safety cabinet</td>
			<td style="width:153px;">thermon electron corporation</td>
			<td style="width:119px;"></td>
			<td style="width:389px;">model: Hera Safe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Microscope Visiscope series 300</td>
			<td style="width:153px;">VWR</td>
			<td style="width:119px;"></td>
			<td style="width:389px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Pipette aid</td>
			<td style="width:153px;">Drummond</td>
			<td style="width:119px;"></td>
			<td style="width:389px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Plastic pipettes</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td style="width:389px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Sterile sealed boxes</td>
			<td style="width:153px;">Tuperware</td>
			<td style="width:119px;"></td>
			<td style="width:389px;">diameter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Sterilized glass tube</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td style="width:389px;">&#34;sovirel&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Thioglycolate Broth with Resazurin</td>
			<td style="width:153px;">Merck</td>
			<td style="width:119px;">90404-500G</td>
			<td style="width:389px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">Water bath</td>
			<td style="width:153px;">Fisher scientific</td>
			<td style="width:119px;"></td>
			<td style="width:389px;">model: polystat 36, used to incubate 10 min at 100 &#176;C the glass tubes with 9 mL thioglycolate broth with resazurin in order to regenerate the medium</td>
		</tr>
	</tbody>
</table>

production of germ-free fast-growing broilers from a commercial line for microbiota studies

肠道微生物群对宿主生理学和免疫能力的贡献的研究通过无菌动物模型的可用性得到促进，这被认为是黄金标准。筑巢鸟类是生产无菌动物的理想模型，因为不需要在无菌条件下饲养亲戚。无菌鸡主要由无特异性病原体（SPF）实验品系产生，这些试验品系在商业鸡系中代表性较差。这里提出的方法允许从快速生长的肉鸡生产线Ross PM3生产无菌鸡，这是家禽业常用的。在肉鸡饲养场产卵后，迅速收集了鸡蛋。它们经历了严格的去污过程，从收集到引入无菌蛋孵化隔离器。雏鸡已经孵化并保存在这些无菌隔离器中，以控制其不育所必需的时间。该方案最初是为实验性SPF白叉角线开发的，不仅适用于Ross PM3肉鸡系，也适用于鹌鹑。因此，它代表了对其他家禽物种和具有经济，生物或生态相关性的筑巢鸟类的强大且易于适应的程序。

使用来自两个不同法国农场的Ross PM3卵进行了六次无菌雏鸡的生成（表1）。共收集了853枚卵，经过两个去污步骤和19天的孵化，86.40%是可行的。这些活蛋中有490个经过了第三个去污步骤，并被引入各种孵化隔离器中，平均孵化率为79.80%。这表示与收集的初始种蛋数量相比，孵化率为68.94%。
然而，孵化结果根据所进行的系列而有很大差异:与收集的种蛋数量相比，存活雏鸡的41.67%至88.16%。在同一实验中，在不同的舱口中也观察到了这些变化。由于并非所有隔离器都用于所有运行，因此很难排除隔离器相关效应。然而，这种批次效应与蛋鸡的年龄直接相关（图1），其中来自年长母鸡的鸡蛋不太可行。
六次运行是使用四种不同的孵化隔离器进行的。经过细菌学控制，16个隔离器中的14个动物被确认为无菌。这相当于87.5%的成功率。剩下的两个隔离器被一种环境和非致病性细菌污染。
所有用于科学实验的动物在孵化后至少三周内保持无菌状态，直到研究结束。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61148/61148fig1v2.jpg"> 图1:母鸡年龄对孵化率的影响 本图突出显示了根据蛋鸡群的年龄观察到的孵化率，以产蛋周数表示。 <a href="https://www.jove.com/files/ftp_upload/61148/61148fig1v2large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<table border="1" fo:font-size="5px" fo:keep-together.within-page="1"><tbody>
<tr>
<td></td>
			<td>收集的鸡蛋</td>
			<td>D19 活卵</td>
			<td>D19 活卵/收集的卵子</td>
			<td>将卵子转入隔离器 1</td>
			<td>将卵子转入隔离器 2</td>
			<td>将卵子转入隔离器 3</td>
			<td>将卵子转入隔离器 4</td>
			<td>转移的卵子总数</td>
			<td>孵化成隔离器1的活雏鸡</td>
			<td>孵化成隔离器2的活雏鸡</td>
			<td>孵化成隔离器3的活雏鸡</td>
			<td>孵化成隔离器的活雏鸡 4</td>
			<td>存活的孵化雏鸡总数</td>
			<td>活孵化/转移的种蛋分离器1</td>
			<td>活体孵化/转移的种蛋隔离器2</td>
			<td>活孵化/转移的种蛋隔离器3</td>
			<td>活孵化/转移的种蛋隔离器4</td>
			<td c="">全球可行的孵化/转移种蛋</td>
		</tr>
<tr>
<td>运行 1</td>
			<td>101</td>
			<td>93</td>
			<td>92.08%</td>
			<td>23</td>
			<td>24</td>
			<td>-</td>
			<td>-</td>
			<td>47</td>
			<td>22</td>
			<td>23</td>
			<td>-</td>
			<td>-</td>
			<td>45</td>
			<td>95.65%</td>
			<td>95.83%</td>
			<td></td>
			<td></td>
			<td>95.74%</td>
		</tr>
<tr>
<td>运行 2</td>
			<td>130</td>
			<td>117</td>
			<td>90.00%</td>
			<td>25</td>
			<td>25</td>
			<td>25</td>
			<td></td>
			<td>75</td>
			<td>20</td>
			<td>24</td>
			<td>18</td>
			<td>-</td>
			<td>62</td>
			<td>80.00%</td>
			<td>96.00%</td>
			<td>72.00%</td>
			<td></td>
			<td>82.67%</td>
		</tr>
<tr>
<td>运行 3</td>
			<td>132</td>
			<td>97</td>
			<td>73.48%</td>
			<td>26</td>
			<td>-</td>
			<td>26</td>
			<td>45</td>
			<td>97</td>
			<td>14</td>
			<td>-</td>
			<td>13</td>
			<td>28*</td>
			<td>27</td>
			<td>53.85%</td>
			<td></td>
			<td>50.00%</td>
			<td>62.22%</td>
			<td>56.70%</td>
		</tr>
<tr>
<td>运行 4</td>
			<td>130</td>
			<td>116</td>
			<td>89.23%</td>
			<td>36</td>
			<td>35</td>
			<td>-</td>
			<td>-</td>
			<td>71</td>
			<td>33</td>
			<td>34</td>
			<td>-</td>
			<td>-</td>
			<td>67</td>
			<td>91.67%</td>
			<td>97.14%</td>
			<td></td>
			<td></td>
			<td>94.37%</td>
		</tr>
<tr>
<td>运行 5</td>
			<td>180</td>
			<td>148</td>
			<td>82.22%</td>
			<td>30</td>
			<td>30</td>
			<td>30</td>
			<td>30</td>
			<td>120</td>
			<td>26*</td>
			<td>25</td>
			<td>17</td>
			<td>24</td>
			<td>66</td>
			<td>86.67%</td>
			<td>83.33%</td>
			<td>56.67%</td>
			<td>80.00%</td>
			<td>76.67%</td>
		</tr>
<tr>
<td>运行 6</td>
			<td>180</td>
			<td>166</td>
			<td>92.22%</td>
			<td>-</td>
			<td>40</td>
			<td>40</td>
			<td>-</td>
			<td>80</td>
			<td>-</td>
			<td>35</td>
			<td>35</td>
			<td>-</td>
			<td>70</td>
			<td>86.67%</td>
			<td>87.50%</td>
			<td>87.50%</td>
			<td></td>
			<td>87.50%</td>
		</tr>
<tr>
<td>总</td>
			<td>853</td>
			<td>737</td>
			<td>86.40%</td>
			<td>140</td>
			<td>154</td>
			<td>121</td>
			<td>75</td>
			<td>490</td>
			<td>89</td>
			<td>141</td>
			<td>83</td>
			<td>24</td>
			<td>337</td>
			<td>82.14%</td>
			<td>91.56%</td>
			<td>68.60%</td>
			<td>69.33%</td>
			<td>79.80%</td>
		</tr>
</tbody></table>表1:不同孵化实验的技术结果。 本表突出显示了进行6系列无菌孵化的结果:收集的种蛋数量，19天胚胎的存活率以及各种隔离器中存活的孵化雏鸡。

Watch this Scientific Journal Video about Production of Germ-Free Fast-Growing Broilers from a Commercial Line for Microbiota Studies at JoVE.com

Production of Germ-Free Fast-Growing Broilers from a Commercial Line for Microbiota Studies

在这里，我们描述了一种从商业肉鸡系的鸡蛋Ross PM3中生成无菌雏鸡的方法。这种方法可以适用于从其他家禽物种中产生无菌动物。

从用于微生物群研究的商业生产线上生产无菌快速生长的肉鸡

Studies of the gut microbiota contribution to the host physiology and immunocompetence are facilitated by the availability of germ-free animal models, ...

production-germ-free-fast-growing-broilers-from-commercial-line-for

Research

JoVE Journal

Immunology and Infection

3.0K 次观看.  INRAE, PFIE, UE-1277. 在这里，我们描述了一种从商业肉鸡系的鸡蛋Ross PM3中生成无菌雏鸡的方法。这种方法可以适用于从其他家禽物种中产生无菌动物。

视频: Production of Germ-Free Fast-Growing Broilers from a Commercial Line for Microbiota Studies

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

生产的灭鼠疟疾寄生虫的遗传交叉议定书"

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

科研

免疫与感染

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4
 Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism.
 In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7
 Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12.

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.

在无菌无鼠标进定植评估肝代谢的变化 1 H核磁共振光谱

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

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 ...

Simple and Efficient Production and Purification of Mouse Myelin Oligodendrocyte Glycoprotein for Experimental Autoimmune Encephalomyelitis Studies

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses targeting myelin. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model of CNS autoimmune disease, and is typically induced via immunization with short peptides representing immunodominant CD4+ T cell epitopes of myelin proteins. However, B cells recognize unprocessed protein directly, and immunization with short peptide does not activate B cells that recognize the native protein. As recent clinical trials of B cell-depleting therapies in MS have suggested a role for B cells in driving disease in humans, there is an urgent need for animal models that incorporate B cell-recognition of autoantigen. To this end, we have generated a new fusion protein containing the extracellular domain of the mouse version of myelin oligodendrocyte glycoprotein (MOG) as well as N-terminal fusions of a His-tag for purification purposes and the thioredoxin protein to improve solubility (MOGtag). A tobacco etch virus (TEV) protease cleavage site was incorporated to allow the removal of all tag sequences, leaving only the pure MOG1-125 extracellular domain. Here, we describe a simple protocol using only standard laboratory equipment to produce large quantities of pure MOGtag or MOG1-125. This protocol consistently generates over 200 mg of MOGtag protein. Immunization with either MOGtag or MOG1-125 generates an autoimmune response that includes pathogenic B cells that recognize the native mouse MOG.

We describe a simple protocol using only basic lab equipment to generate and purify large quantities of a fusion protein that contains mouse Myelin Oligodendrocyte Glycoprotein. This protein can be used to induce experimental autoimmune encephalomyelitis driven by both T and B cells.

简单而高效的生产和鼠标髓鞘少突胶质糖蛋白的纯化的实验性自身免疫性脑脊髓炎研究

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS), thought to occur as a result of autoimmune responses ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

人雌性生殖道树突状细胞表型鉴定的分离及功能研究

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

一种研究人体 T 细胞召回反应的吸水泡协议

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

Production and Characterization of Human Macrophages from Pluripotent Stem Cells

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key players in health and disease, acting as phagocytes during immune defense and mediating trophic, maintenance, and repair functions. Although it has been possible to study some of the molecular processes involved in human macrophage function, it has proved difficult to apply genetic engineering techniques to primary human macrophages. This has significantly hampered our ability to interrogate the complex genetic pathways involved in macrophage biology and to generate models for specific disease states. An off-the-shelf source of human macrophages that is amenable to the vast arsenal of genetic manipulation techniques would, therefore, provide a valuable tool in this field. We present an optimized protocol that allows for the generation of macrophages from human induced pluripotent stem cells (iPSCs) in vitro. These iPSC-derived macrophages (iPSC-DMs) express human macrophage cell surface markers, including CD45, 25F9, CD163, and CD169, and our live-cell imaging functional assay demonstrates that they exhibit robust phagocytic activity. Cultured iPSC-DMs can be activated to different&#160;macrophage states that display altered gene expression and phagocytic activity by the addition of LPS and IFNg, IL4, or IL10. Thus, this system provides a platform to generate human macrophages carrying genetic alterations that model specific human disease and a source of cells for drug screening or cell therapy to treat these diseases.

This protocol describes the robust generation of macrophages from human induced pluripotent stem cells, and methods for their subsequent characterization. Cell surface marker expression, gene expression, and functional assays are used to assess the phenotype and function of these iPSC-derived macrophages.

多能干细胞人类巨噬细胞的产生与表征

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key ...

Production of Human CRISPR-Engineered CAR-T Cells

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.

生产人类CRISPR工程的CAR-T细胞

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with ...

Production of Adeno-Associated Virus Vectors in Cell Stacks for Preclinical Studies in Large Animal Models

Adeno-associated virus (AAV) vectors are among the most clinically advanced gene therapy vectors, with three AAV gene therapies approved for humans. Clinical advancement of novel applications for AAV involves transitioning from small animal models, such as mice, to larger animal models, including dogs, sheep, and nonhuman primates. One of the limitations of administering AAV to larger animals is the requirement for large quantities of high-titer virus. While suspension cell culture is a scalable method for AAV vector production, few research labs have the equipment (e.g., bioreactors) or know how to produce AAV in this manner. Moreover, AAV titers are often significantly lower when produced in suspension HEK 293 cells as compared to adherent HEK293 cells. Described here is a method for producing large quantities of high-titer AAV using cell stacks. A detailed protocol for titering AAV as well as methods for validating vector purity are also described. Finally, representative results of AAV-mediated transgene expression in a sheep model are presented. This optimized protocol for large-scale production of AAV vectors in adherent cells will enable molecular biology laboratories to advance the testing of their novel AAV therapies in larger animal models.

Here we provide a detailed procedure for large-scale production of research-grade AAV vectors using adherent HEK 293 cells grown in cell stacks and affinity chromatography purification. This protocol consistently yields &#62;1 x 1013 vector genomes/mL, providing vector quantities appropriate for large animal studies.

在细胞堆中生产腺相关病毒载体，用于大型动物模型的临床前研究

Adeno-associated virus (AAV) vectors are among the most clinically advanced gene therapy vectors, with three AAV gene therapies approved for humans. ...

An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for maintaining local and systemic homeostasis; changes to gut microbiota composition (dysbiosis) associate with a wide array of human diseases. Methods for dissecting host-microbiota interactions encompass an inherent tradeoff among preservation of physiological tissue structure (when using in vivo animal models) and the level of control over the experiment factors (as in simple in vitro cell culture systems). To address this tradeoff, Yissachar et al. recently developed an intestinal organ culture system. The system preserves a naive colon tissue construction and cellular mechanisms and it also permits tight experimental control, facilitating experimentations that cannot be readily performed in vivo. It is optimal for dissecting short-term responses of various gut components (such as epithelial, immunological and neuronal elements) to luminal perturbations (including anaerobic or aerobic microbes, whole microbiota samples from mice or humans, drugs and metabolites). Here, we present a detailed description of an optimized protocol for organ culture of multiple gut fragments using a custom-made gut culture device. Host responses to luminal perturbations can be visualized by immunofluorescence staining of tissue sections or whole-mount tissue fragments, fluorescence in-situ hybridization (FISH), or time-lapse imaging. This system supports a wide array of readouts, including next-generation sequencing, flow cytometry, and various cellular and biochemical assays. Overall, this three-dimensional organ culture system supports the culture of large, intact intestinal tissues and has broad applications for high-resolution analysis and visualization of host-microbiota interactions in the local gut environment.

This article presents a unique method for analyzing host-microbiome interactions using a novel gut organ culture system for ex vivo experiments.

分析宿主-微生物群相互作用的肠道肠道器官培养系统

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for ...

从用于微生物群研究的商业生产线上生产无菌快速生长的肉鸡

摘要

探索更多视频

此视频中的章节