作者感谢由德国研究基金会（DFG）通过合作研究中心SFB的578"从基因到产品"在德国不伦瑞克工业大学提供的财政支持。

1。栽培反应堆的安装和启动共4生物反应器栽培进行。 <ol><li> 答:种植3大号搅拌式生物反应器使用的2.2升工作体积尼日尔 SKAn1015。 </li><li>倒入到反应堆72.6克葡萄糖水合物，填补了1.9升去离子水。 </li><li>安装反应堆设备，如3挡板，两个六叶圆盘涡轮叶轮，pH电极，进气口与空气滤清器，冷却的手指，排气空气冷却器与空气过滤器，空气过滤器的无菌采样浸入管为培养基，接种量，酸，碱和进水软管。 </li><li>在121°C高压灭菌20分钟的反应堆。 </li><li>酸碱水库（2 M盐酸，氢氧化钠2米）和相应的泵连接反应堆和设立的pH值5.0±0.05，pH值控制单元。 </li><li>连接反应堆的冷却水系统，并加热夹克各地的反应堆压力容器。 </li><li>安装相应的控制单元温度传感器，并设立了37个温度±0.1°C的温度控制单元。 </li><li>适合搅拌电机之上反应堆，并把服务与搅拌速度为200分钟-1。 </li><li>最小的无菌培养基中11槽添加无菌进水软管（250毫升）。为中等准备所有组件在121℃高压灭菌20分钟，混在了一起。三栽培的组件包括滑石粉（1 g / L的浓度3MgO•4SiO 2·H 2 O）（反应堆2），3 g / L的（反应堆）和10 g / L的（反应堆）。在使用之前，重新挂起微粒在50毫米醋酸钠缓冲液（pH值6.5），并把它添加到无菌培养基。在控制文化（无颗粒）微颗粒悬浮液取代50毫米醋酸钠缓冲液（pH值6.5）。 </li><li>新增的孢子悬浮液（接种）准备投入Wucherpfennig等。人（2011）11槽无菌进水软管（50毫升）的之一，使1X10 6毫升-1孢子的浓度。接种标志着开始种植（H = 0 H）。 </li><li>启动速度的1.0升分钟-1曝气。 </li></ol> 2。无菌采样后24，48和栽培72小时<ol><li>成flacon管50毫升的无菌培养液。 </li><li>用于测定生物量干重，β-呋喃果糖苷酶的活性和微观分析的样本。 </li></ol> 3。测定生物量干重的24后，48和栽培72小时<ol><li>生物样品要至少一式两份。 </li><li>重量与微观尺度的纤维素过滤器后，在干燥器和干燥过滤器放置在一个连接的水喷射真空泵的布氏漏斗。 </li><li>过滤定义的样本量（如10毫升）ND用10 mL去离子水冲洗过滤器，从生物中取出介质的化合物。 </li><li>皱纹的过滤器，一旦在中间，放置在一个玻璃培养皿和投入舱室干衣机，直到体重恒定（至少24小时）。 </li><li>在干燥器中冷却过滤器，并测量体重。 </li><li>作为过滤器的重量之间的差异并没有干生物量除以所使用的样本量计算生物量干重。 </li></ol> 4。神/ POD-文章胞外β-呋喃果糖苷酶的活性测定，24后，48和栽培72小时与他们一起工作时存储在冰样本不断。 <ol><li>过滤器1.5毫升培养液按培养液用注射器通过过滤器进入反应管槽醋酸纤维素过滤。储存在-20°C，直到使用的反应管。 </li><li>反应混合物用20μL样品和添加200μL1.65米，0.05 M磷酸缓冲液（pH值5.4）溶解，启动反应葡萄糖蔗糖的蔗糖。出反应，至少在卡里复制。 </li><li>在40°C孵育反应混合物加热块为20分钟。停止反应，在95°C孵育10分钟加热块。冷却反应混合物由储存在冰和自旋向下13.000Ğ离心机10分钟在4°C冷凝水</li><li>为了帐户的pH值和温度依赖葡萄糖蔗糖裂解，开展使用20μL去离子水，而不是20μL样品空白值。 </li><li>要交代的培养液中的残糖，开展每个样品为阴性对照。为此，使用20μL的样品和灭活β-呋喃果糖苷酶在95℃加热10分钟前添加蔗糖和孵化步骤4.3中所述。 </li><li>稀释样品，例如，下面的测量ured吸附在标定值范围内。 </li><li>开展一式三份酶检测。适用于微滴度板以及2μL稀释后的样品。申请使用10个不同的知情浓度（从1毫米到15毫米），而不是2μL样品10个葡萄糖溶液2μL每一个微滴度板的校准标准范围。对于零点校准，使用2μL去离子水，而不是2μL样品。 </li><li>加入200μL试剂解决方案，以每口井，通过使用多吸管。 </li><li>孵育的混合物在室温为10分钟。 </li><li>微滴度板存放在6°，直到测量（最多几个小时）， </li><li>在96日出微板读者和麦哲伦数据检索软件的使用450纳米测量的吸收。设置搅拌时间为5秒。和1秒的休息时间。 </li><li>打开电子表格的结果图表和使用范围的标准校准线建设: Ğlucose浓度=一个X吸收+ B </li><li>计算活动: 活动= X稀释倍数的空白值（吸收）×A + B </li><li>通过计算样品的活动和适当的阴性对照之间的差异，找出β-呋喃果糖苷酶的活性。 </li><li>计算，考虑到生物量干重和β-呋喃果糖苷酶的活性11以下使用特定的生产力。 </li></ol> 5。栽培72小时后镜和自动图像分析<ol><li> 3毫升左右，在塑料培养皿地方文化的暂停和他们，直到分离的形态结构与生理氯化钠溶液稀释。 </li><li>位于培养皿，在显微镜下，它的特点是集成或连接相机。收购和储存约100个，每个样品的形态结构图像（ 图2）。注意，对每个图像至少一个对象被完全描绘。 </li><li>打开图像处理程序ImageJ的12对同一样品的所有图像。将图像转换为黑色和白色在使用过程中工具的"二进制"（ 图2）。申请命令，以全系列的图像，使用宏的代码如下。 运行（，二"） </li><li>打开ImageJ的再次加工的二进制图像。计算直径，形状因子FERET的投影面积，周长，圆，长宽比，圆度为每一个分析工具"设置测量"的形象UND坚固。一个严重申请的命令ES图像使用如下的宏代码。 运行（"8位"）; （"二进制"）; 运行（"集规模..."，"距离= X已知= 1000像素= 1单位=微米全球"）; 运行（"集测量..."，"区外围形状FERET的限制显示重定向=无小数= 3"）; （"分析粒子的......"，"大小= 10000无限圆= 0.00-1.00展=大纲显示"）; 决定兴建一条直线横跨比例尺与1000微米的像素数量X。关联的直线比例尺长度在微米的像素数目。 </li><li>打开的结果图表，包含形状因子值，为每一个电子表格的形象。计算如下为每个图像的形态数量。 <img alt="式（1）" src="/files/ftp_upload/4023/4023eq1.jpg" /></li><li>计算平均值和标准偏差为形态，考虑到一个样品的所有图像。 </l我&gt; <li>使用与特定的生产力形态的图形相关的图形和数据分析程序，并确定由数学回归的数学关系。 </li></ol> 6。代表结果除了 ​​通过滑石粉微粒A.尼日尔 SKAN 1015形态是一个真正的颗粒形态从分散甚至菌丝的形态改变。而在标准条件下表现出颗粒形态，菌丝形态与滑石微粒的10克/升（ 图4）。补充介质创建。同时β-呋喃果糖苷酶的活性增加约3倍3-5。一个补充的1或3克/ L的滑石粉导致分散的形态，用一倍的呋喃果糖苷酶的活性（ 图4）。 由铁道部，微观粒子的依赖形态可以全面描述phology数可以使用自动图像分析测定的参数计算。浑圆，光滑的小球会出现在显微图像完美的圆圈。对于这样的颗粒形态数量值1。菌丝形态的最小片段，可以简化为一维的形态产生了0号线。因此，所有像拉长的不规则颗粒或团块的中间形态的形式，将有0和1之间的值。高，真菌粒子具有较大的表面或拉长颗粒在一个相当低的形态11号，将导致相当大的颗粒。 在标准条件下反应堆1形态展品约0.8形态。约0.1锰的形态特征与滑石粉10 g / L的4反应堆。反应堆2和3与1和3 g / L时，滑石粉的浓度，形态介于这两个极端，展现分散形态logy。由于微观粒子的依赖形态是β-呋喃果糖苷酶生产力密切相关， 图5类似的形态数量和生产力的数学关系得到。 <img alt="图1" src="/files/ftp_upload/4023/4023fig1.jpg" /> 答:1。实验设计和分析过程的总体方案。 尼日尔是培养（带或不带微颗粒）在72小时搅拌式生物反应器3的大号。 24后，48和72公顷样品测定生物量干重和β-呋喃果糖苷酶的活性，而这又是用于计算的具体生产力。 48小时后，真菌形态学检查显微镜和数字图像分析为特色。相关参数的图像分析相结合，一个形态，这是数学与特定的生产力相关。 <imgALT ="图2"的src ="/ files/ftp_upload/4023/4023fig2.jpg"/&gt; 从 A微观形态结构生成的图像图像处理步骤图2。 尼日尔 。步骤1:图像采集用显微镜。第2步:如果有必要的形象得到改善。步骤3:图像二值化，黑色和白色（二进制）在ImageJ的图像。步骤4:二进制图像处理和清除不必要的对象。第5步:ImageJ的开源计划"分析粒子"功能进行形态分析。 <img alt="图3" src="/files/ftp_upload/4023/4023fig3.jpg" /> 图3。 答:不同形态的形式尼日尔对增值微粒浓度依赖性。随着微粒浓度的增加，颗粒大小可精确下降小核壳小球，小羊群，甚至自由分散的菌丝。砷的形态工程pergillus尼日尔SKAn1015深层培养的微观粒子的补充。没有微粒（一），10毫克/升（B）0.1 g / L的（C）0.2 g / L的（D）0.3 g / L的（E）为0.6 g / L（女），1.0克/升（G）1.5 g / L的（H）2.0 g / L的（一），（十）2.5克/ L，3.0 g / L的（金），3.5克/升（L），4.0克/升（男克/升），4.5（N）5.0 g / L的（O）10克/ L，（P），15 g / L的（Q）20克/升（R）为30 g / L（）和40-50克/升（T）培养72小时后光镜图像。 <img alt="图4" src="/files/ftp_upload/4023/4023fig4.jpg" /> 图4。呋喃果糖苷酶的活性依赖的滑石粉微粒浓度为1 g / L的（反应堆），3 g / L的（反应堆）和10 g / L的（反应堆）。反应堆1辅以微颗粒;这里种植的标准条件下进行。 <img alt="图5" src="/files/ftp_upload/4023/4023fig5.jpg" /> 图5。代表良好的相关性形态（R 2 = 0.91）数量和具体的生产力。绘制的形态数量（横坐标）对特定生产率（坐标）。非线性回归得到指数的相关性。

<ol>
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我什么都没有透露。

几十年来一直在生物技术的兴趣，因为真菌形态的修改。不同的研究都试图改变选定的工艺参数，如pH值，输入功率，温度，营养中等或接种浓度为1，但苦于相当不精确的和不完整的形态，控制高能源成本，抑制效果或产品不稳定，相比之下，微粒的补充，可以通过微调粒度和浓度变化的真菌形态的精密工程。这将打开新的可能性，以用于优化和度身订造的高产形态设计与A.生物技术生产的微型颗粒尼日尔和其他丝状微生物。 数字图像分析是一种简单的重复的方法来描述真菌的宏观形态。然而，各种大小，形状和表面字符的参数形态结构之三在文献中描述的真菌形态复杂的快速评估。相关参数的组合形态，避免了这方面的不足，不仅可以全面表征形态结构，也为数学与生产力直接相关。这再次呈现形态和过程形态的定制生产力的估计需要。 使用形态的号码，它是可以区分不同的沉淀和聚集形态4,5。为进一步发展形态数量的分形维数的审议似乎被看好。一个分形维数，给出了一个对象的填充属性13的复杂性和质量的测量，因此整体菌丝形态的表征豫定。 铬然而，eation一个高产的菌丝形态，可能会导致与工艺性能，尤其是在大规模种植的问题，菌丝生长形式，因为以前一直表现出更大的培养液粘度2。这将导致与传热传质问题，并形成停滞的非混合区，这需要更高的输入功率，使种植更昂贵的操作1。因此真菌的形态和培养液粘度之间的关系应被视为改变形态和进一步车型纳入。

Table of Equipment:
<table border="1">
	<tbody>
		<tr>
			<td>
			Equipment</td>
			<td>
			Company</td>
			<td>
			Catalogue Number/model</td>
		</tr>
		<tr>
			<td>
			autoclave</td>
			<td>
			Systec</td>
			<td>
			V150</td>
		</tr>
		<tr>
			<td>
			B&uuml;chner funnel (plastic)</td>
			<td>
			VWR</td>
			<td>
			-</td>
		</tr>
		<tr>
			<td>
			cellulose filter (for biomass dry weight)</td>
			<td>
			Sartorius Stedim Biotech</td>
			<td>
			Filter Discs Grade 389</td>
		</tr>
		<tr>
			<td>
			cellulose acetate filter (for air filtration at reactor)</td>
			<td>
			Sartorius stedim biotech</td>
			<td>
			Midisart 200 PTFE</td>
		</tr>
		<tr>
			<td>
			cellulose acetate filter (for enzyme activity)</td>
			<td>
			Sartorius Stedim Biotech</td>
			<td>
			Midisart NML</td>
		</tr>
		<tr>
			<td>
			centrifuge</td>
			<td>
			Eppendorf</td>
			<td>
			Centrifuge 5415R</td>
		</tr>
		<tr>
			<td>
			centrifuge</td>
			<td>
			Heraeus</td>
			<td>
			Biofuge fresco</td>
		</tr>
		<tr>
			<td>
			centrifuge</td>
			<td>
			Heraeus sepatech</td>
			<td>
			Varifuge 3.0R</td>
		</tr>
		<tr>
			<td>
			compartment dryer (105 &deg;C)</td>
			<td>
			Heraeus</td>
			<td>
			Kelvitran t</td>
		</tr>
		<tr>
			<td>
			control unit (temperature)</td>
			<td>
			Jumo</td>
			<td>
			Jumo iTron 08</td>
		</tr>
		<tr>
			<td>
			control unit (pH-value)</td>
			<td>
			meredos</td>
			<td>
			pH Control 2</td>
		</tr>
		<tr>
			<td>
			desiccator</td>
			<td>
			Duran</td>
			<td>
			Vacuum stable</td>
		</tr>
		<tr>
			<td>
			Falcon tubes</td>
			<td>
			Omnilab</td>
			<td>
			FALC352070</td>
		</tr>
		<tr>
			<td>
			heating block 40 &deg;C</td>
			<td>
			Biometra</td>
			<td>
			TB1 Thermoblock</td>
		</tr>
		<tr>
			<td>
			heating block 95 &deg;C</td>
			<td>
			HLC</td>
			<td>
			HBT 130</td>
		</tr>
		<tr>
			<td>
			micro plate reader</td>
			<td>
			Tecan</td>
			<td>
			Sunrise-Microplate-Reader</td>
		</tr>
		<tr>
			<td>
			micro scales</td>
			<td>
			Sartorius</td>
			<td>
			CP 225</td>
		</tr>
		<tr>
			<td>
			microscope (digital inverted)</td>
			<td>
			AMG</td>
			<td>
			EVOS xl</td>
		</tr>
		<tr>
			<td>
			micro pipettes and tips (different sizes)</td>
			<td>
			Omnilab</td>
			<td>
			5283303
			5283298 
			5283299 
			5283300</td>
		</tr>
		<tr>
			<td>
			micro titer plate</td>
			<td>
			Nunc</td>
			<td>
			MaxiSorp</td>
		</tr>
		<tr>
			<td>
			multi pipette and tips</td>
			<td>
			Eppendorf/ Omnilab</td>
			<td>
			5283611/ 5283611</td>
		</tr>
		<tr>
			<td>
			pH-electrode</td>
			<td>
			Schott</td>
			<td>
			pH-Meter CG840</td>
		</tr>
		<tr>
			<td>
			reaction tubes</td>
			<td>
			Roth</td>
			<td>
			E518.1</td>
		</tr>
		<tr>
			<td>
			scale</td>
			<td>
			Sartorius</td>
			<td>
			CP 3202 S</td>
		</tr>
		<tr>
			<td>
			stirred tank bioreactor with equipment</td>
			<td>
			Applikon Biotechnology</td>
			<td>
			2L Bioreactor set</td>
		</tr>
		<tr>
			<td>
			syringe</td>
			<td>
			Eppendorf</td>
			<td>
			Combitips Plus 5 mL</td>
		</tr>
	</tbody>
</table>
Table of Reagents:
<table border="1">
	<tbody>
		<tr>
			<td>
			Name of the reagent</td>
			<td>
			Company </td>
		</tr>
		<tr>
			<td>
			Acetic acid</td>
			<td>
			Roth</td>
		</tr>
		<tr>
			<td>
			Disodium hydrogen phosphate</td>
			<td>
			Merck</td>
		</tr>
		<tr>
			<td>
			Ethanol (95%)</td>
			<td>
			Roth</td>
		</tr>
		<tr>
			<td>
			Glucose monohydrate, (&alpha;-D-)</td>
			<td>
			Roth</td>
		</tr>
		<tr>
			<td>
			Glucose oxidase (Typ II from Aspergillus niger)</td>
			<td>
			Sigma</td>
		</tr>
		<tr>
			<td>
			Hydrochloride acid (37 % w/v)</td>
			<td>
			Fiedel-de Ha&euml;n</td>
		</tr>
		<tr>
			<td>
			Hydrous magnesium silicate</td>
			<td>
			Roth</td>
		</tr>
		<tr>
			<td>
			Monopotassium phosphate</td>
			<td>
			Merck</td>
		</tr>
		<tr>
			<td>
			o-dianoisidine dihydrochloride</td>
			<td>
			Sigma</td>
		</tr>
		<tr>
			<td>
			Peroxidase (Typ II from horseradish)</td>
			<td>
			Sigma</td>
		</tr>
		<tr>
			<td>
			Sodium acetate</td>
			<td>
			Roth</td>
		</tr>
		<tr>
			<td>
			Sodium hydroxide</td>
			<td>
			Merck</td>
		</tr>
		<tr>
			<td>
			Sucrose, D-(+)</td>
			<td>
			Fluka</td>
		</tr>
		<tr>
			<td>
			Water (deionized)</td>
			<td>
			-</td>
		</tr>
	</tbody>
</table>
Table of Solutions and Medium Composition:
<table border="1">
	<tbody>
		<tr>
			<td>
			Solution</td>
			<td>
			Components</td>
			<td>
			Amount</td>
		</tr>
		<tr>
			<td>
			50 mM sodium acetate buffer (pH 6.5)</td>
			<td>
			Sodium acetate
			Bring to volume with deionized water
			Adjust at pH 6.5 with acetic acid</td>
			<td>
			4.1 g L-1</td>
		</tr>
		<tr>
			<td>
			0.05 M monopotassium phosphate solution</td>
			<td>
			Monopotassium phosphate
			Bring to volume with deionized water</td>
			<td>
			6.805 g L-1</td>
		</tr>
		<tr>
			<td>
			0.05 M disodium hydrogen phosphate solution</td>
			<td>
			Disodium hydrogen phosphate
			Bring to volume with deionized water</td>
			<td>
			7.1 g L-1</td>
		</tr>
		<tr>
			<td>
			0.05 M phosphate buffer (pH 7.0)</td>
			<td>
			0.05 M disodium hydrogen phosphate solution
			Bring to volume with 0.05 M monopotassium phosphate solution</td>
			<td>
			61.2 mL</td>
		</tr>
		<tr>
			<td>
			0.05 M phosphate buffer (pH 5.4)</td>
			<td>
			0.05M disodium hydrogen phosphate solution
			Bring to volume with 0.05 M monopotassium phosphate solution</td>
			<td>
			3 mL</td>
		</tr>
		<tr>
			<td>
			1.65 M sucrose solution</td>
			<td>
			D-(+)-sucrose
			Bring to volume with phosphate buffer (pH 5.4)</td>
			<td>
			564.8 g L-1</td>
		</tr>
		<tr>
			<td>
			reagent solution</td>
			<td>
			o-Dianisidin-Dihydrochlorid
			Ethanol (95%)</td>
			<td>
			25 mg
			10 mL</td>
		</tr>
		<tr>
			<td>
			Glucose reagent solution</td>
			<td>
			Glucose oxidase
			Peroxidase
			Phosphate buffer (pH 7.0)
			reagent solution</td>
			<td>
			10.5 mg
			3 mg
			90 mL
			10 mL</td>
		</tr>
	</tbody>
</table>

customization of aspergillus niger morphology through addition of talc micro particles

丝状真菌A.尼日尔是一个在范围广泛，从食品到医药行业的工业生产过程中广泛使用的应变。这种丝状生物体中最引人注目的，往往不可控的特点之一是其复杂的形态。它的范围从密集的球形颗粒，粘稠的菌丝体（ 图1）。各种工艺参数和配料是众所周知的影响真菌的形态1。由于最佳生产力与具有特定形态的形式强烈，真菌形态，往往代表着生产力的工业生产中的瓶颈。 一个直线前进和优雅的方法，能够精确地控制形态形状是另外无机的不溶性微粒（如水合硅酸镁，三氧化二铝或钛硅酸盐氧化物）的培养基，有助于增加酶生产2-6。既然有1 OBV白条之间的依赖微观粒子的形态和产酶的相关性，这是可取的数学链接生产力和外观形态。因此，精确的定量和整体形态描述的目标。 因此，我们提出了一个方法来生成和表征微观粒子的依赖形态结构和相关真菌的形态与生产力（图1），这可能有助于更好地理解的丝状微生物形态。 重组菌株A。尼日尔 SKAn1015 3大号搅拌槽反应器中培养72小时。除了滑石粉微粒浓度1克/ L，3 g / L的10 g / L的接种前要通过各种不同的形态结构，再现产生。无菌抽取样本后24，48和72小时的成长进步和产生的酶的活性测定。 "形成的产品是高附加值的酶β-呋喃果糖苷酶，糖在食品或制药行业，其中除其他催化反应的蔗糖，葡萄糖7-9新形成的一个重要的生物催化剂。因此，量化后加入蔗糖葡萄糖意味着产β-呋喃果糖苷酶量。葡萄糖定量是由一个神/过氧化物酶的含量10，这是在96孔微滴度板的高通量分析修改。 72小时后真菌形态显微镜检查和数字图像分析为特色。这样做，宏真​​菌形态像FERET的直径，投影面积，周长，圆，长宽比，圆度和坚固的粒子形状因子的计算与开源图像处理程序ImageJ的。相关参数相结合，一个无量纲的形态数量（锰）11，从而使一个全面的表征真菌的形态。强调数学回归的形态数量和生产力密切相关。

Watch this Scientific Journal Video about Customization of Aspergillus niger Morphology Through Addition of Talc Micro Particles at JoVE.com

Customization of Aspergillus niger Morphology Through Addition of Talc Micro Particles

一个方法来精确地生成和全面表征丝状菌形态<em&gt;黑曲霉（Aspergillus niger）</em&gt;描述，它允许的外观形态和生产力的数学相关。

定制黑曲霉（Aspergillus niger）通过添加滑石粉微粒形态

The filamentous fungus A. niger is a widely used strain in a broad range of industrial processes from food to pharmaceutical industry. One of the most ...

customization-aspergillus-niger-morphology-through-addition-talc

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

Customization of Aspergillus niger Morphology Through Addition of Talc Micro Particles

19.0K 次观看.  Technische Universität Braunschweig. 一个方法来精确地生成和全面表征丝状菌形态黑曲霉（Aspergillus niger）描述，它允许的外观形态和生产力的数学相关。

视频: Customization of Aspergillus niger Morphology Through Addition of Talc Micro Particles

Packaging HIV- or FIV-based Lentivector Expression Constructs &amp; Transduction of VSV-G Pseudotyped Viral Particles

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient expression of effectors. Packaging lentiviral expression constructs into pseudoviral particles, however, enables up to 100% transduction, even with difficult-to-transfect cells, such as primary, stem, and differentiated cells. Moreover, the lentiviral delivery does not produce the specific cellular responses typically associated with chemical transfections, such as cell death resulting from toxicity of the transfection reagent 1, 2. When transduced into target cells, the lentiviral construct integrates into genomic DNA and provides stable expression of the small hairpin RNA (shRNA), cDNA, microRNA or reporter gene 3, 4. Target cells stably expressing the effector molecule can be isolated using a selectable marker contained in the expression vector construct such as puromycin or GFP. After pseudoviral particles infect target cells, they cannot replicate within target cells because the viral structural genes are absent and the long terminal repeats (LTRs) are designed to be self-inactivating upon transduction 5, 6.
There are three main components necessary for efficient lentiviral packaging 1, 5, 6, 7. 
1. The lentiviral expression vector that contains some of the genetic elements required for packaging, stable integration of the viral expression construct into genomic DNA, and expression of the effector or reporter. 
2. The lentiviral packaging plasmids that provide the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant pseudoviral particles. This protocol uses the pPACK plasmids (SBI) that encode for gag, pol, and rev from the HIV or FIV genome and Vesicular Stomatitis Virus g protein (VSV-G) for the viral coat protein. 
3. 293TN producer cells (derived from HEK293 cells) that express the SV40 large T antigen, which is required for high-titer lentiviral production and a neomycin resistance gene, useful for reselecting the cells for maintenance.
An overview of the viral production protocol can be seen in Figure 1. Viral production starts by co-transfecting 293TN producer cells with the lentiviral expression vector and the packaging plasmids. Viral particles are secreted into the media. After 48-72 hours the cell culture media is harvested. Cellular debris is removed from the cell culture media, and the viral particles are precipitated by centrifugation with PEG-it for concentration. Produced lentiviral particles are then titered and can be used to transduce target cells. Details of viral titering are not included in this protocol, but can be found at:
<a href="http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf" target="_blank">http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf</a> 8.
This protocol has been optimized using the specific products indicated. Other reagents may be substituted, but the same results cannot be guaranteed.

Packaging HIV- or FIV-based Lentivector Expression Constructs & Transduction of VSV-G Pseudotyped Viral Particles

Lentiviral expression vectors are the most effective vehicles for stably expressing different effector molecules or reporter constructs in dividing and non-dividing mammalian cells and whole organisms. Here we provide a protocol on how to package lentivector expression constructs in pseudoviral particles and to transduce target cells using the pseudoviral particles.

包装艾滋病毒或FIV的基于Lentivector表达结构与VSV-G假型病毒颗粒转导

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient ...

科研

免疫与感染

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or specific treatments for the disease caused by these viruses, and work with infectious Ebola viruses is restricted to biosafety level 4 laboratories, significantly limiting the research on these viruses. Lifecycle modeling systems model the virus lifecycle under biosafety level 2 conditions; however, until recently such systems have been limited to either individual aspects of the virus lifecycle, or a single infectious cycle. Tetracistronic minigenomes, which consist of Ebola virus non-coding regions, a reporter gene, and three Ebola virus genes involved in morphogenesis, budding, and entry (VP40, GP1,2, and VP24), can be used to produce replication and transcription-competent virus-like particles (trVLPs) containing these minigenomes. These trVLPs can continuously infect cells expressing the Ebola virus proteins responsible for genome replication and transcription, allowing us to safely model multiple infectious cycles under biosafety level 2 conditions. Importantly, the viral components of this systems are solely derived from Ebola virus and not from other viruses (as is, for example, the case in systems using pseudotyped viruses), and VP40, GP1,2 and VP24 are not overexpressed in this system, making it ideally suited for studying morphogenesis, budding and entry, although other aspects of the virus lifecycle such as genome replication and transcription can also be modeled with this system. Therefore, the tetracistronic trVLP assay represents the most comprehensive lifecycle modeling system available for Ebola viruses, and has tremendous potential for use in investigating the biology of Ebola viruses in future. Here, we provide detailed information on the use of this system, as well as on expected results.

Work with infectious Ebola viruses is restricted to biosafety level 4 laboratories. Tetracistronic minigenome-containing replication and transcription-competent virus like particles (trVLPs) represent a lifecycle modeling system that allows us to safely model multiple infectious cycles under biosafety level 2 conditions, relying exclusively on Ebola virus components.

造型埃博拉病毒的生命周期在生物安全二级条件下的病毒样颗粒包含Tetracistronic小基因组

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or ...

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

定量微中和试验的优化

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase reporter, using a simple transfection procedure of the widely available HEK-293T cell line. Once formed and released from producer cells, these pseudovirions incorporate a luciferase reporter gene. Since they only contain the heterologous coronavirus spike protein on their surface, the particles behave like their native coronavirus counterparts for entry steps. As such, they are the excellent surrogates of native virions for studying viral entry into host cells. Upon successful entry and infection into target cells, the luciferase reporter gets integrated into the host cell genome and is expressed. Using a simple luciferase assay, transduced cells can be easily quantified. An important advantage of the procedure is that it can be performed in biosafety level 2 (BSL-2) facilities instead of BSL-3 facilities required for work with highly pathogenic coronaviruses such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Another benefit comes from its versatility as it can be applied to envelope proteins belonging to all three classes of viral fusion proteins, such as the class I influenza hemagglutinin (HA) and Ebola virus glycoprotein (GP), the class II Semliki forest virus E1 protein, or the class III vesicular stomatitis virus G glycoprotein. A limitation of the methodology is that it can only recapitulate virus entry steps mediated by the envelope protein being investigated. For studying other viral life cycle steps, other methods are required. Examples of the many applications these pseudotype particles can be used in include investigation of host cell susceptibility and tropism and testing the effects of virus entry inhibitors to dissect viral entry pathways used.

Here, we present a protocol to generate pseudotyped particles in a BSL-2 setting incorporating the spike protein of the highly pathogenic viruses Middle East respiratory syndrome and severe acute respiratory syndrome coronaviruses. These pseudotyped particles contain a luciferase reporter gene allowing quantification of virus entry into target host cells.

在生物安全2级环境中制备假型颗粒研究高致病性冠状病毒

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase ...

Identification of Nucleolar Factors During HIV-1 Replication Through Rev Immunoprecipitation and Mass Spectrometry

The HIV-1 infectious cycle requires viral protein interactions with host factors to facilitate viral replication, packaging, and release. The infectious cycle further requires the formation of viral/host protein complexes with HIV-1 RNA to regulate the splicing and enable nucleocytoplasmic transport. The HIV-1 Rev protein accomplishes the nuclear export of HIV-1 mRNAs through multimerization with intronic cis-acting targets - the Rev response element (RRE). A nucleolar localization signal (NoLS) exists within the COOH-terminus of the Rev arginine-rich motif (ARM), allowing the accumulation of Rev/RRE complexes in the nucleolus. Nucleolar factors are speculated to support the HIV-1 infectious cycle through various other functions in addition to mediating mRNA-independent nuclear export and splicing. We describe an immunoprecipitation method of wild-type (WT) Rev in comparison to Rev nucleolar mutations (deletion and single-point Rev-NoLS mutations) in the presence of HIV-1 replication for mass spectrometry. Nucleolar factors implicated in the nucleocytoplasmic transport (nucleophosmin B23 and nucleolin C23), as well as cellular splicing factors, lose interaction with Rev in the presence of Rev-NoLS mutations. Various other nucleolar factors, such as snoRNA C/D box 58, are identified to lose interaction with Rev mutations, yet their function in the HIV-1 replication cycle remain unknown. The results presented here demonstrate the use of this approach for the identification of viral/host nucleolar factors that maintain the HIV-1 infectious cycle. The concepts used in this approach are applicable to other viral and disease models requiring the characterization of understudied pathways.

Here we describe Rev immunoprecipitation in the presence of HIV-1 replication for mass spectrometry. The methods described can be used for the identification of nucleolar factors involved in the HIV-1 infectious cycle and are applicable to other disease models for the characterization of understudied pathways.

通过转版免疫沉淀和质谱测定,在HIV-1复制过程中核子因子的识别

The HIV-1 infectious cycle requires viral protein interactions with host factors to facilitate viral replication, packaging, and release. The infectious ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

利用流细胞测定人类白细胞测定阿斯珀吉鲁斯紫锥体烟曲的噬菌体

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...

Analyzing the Permeability of the Blood-Brain Barrier by Microbial Traversal through Microvascular Endothelial Cells

The human blood-brain barrier (BBB) is characterized by a very low permeability for biomolecules in order to protect and regulate the metabolism of the brain. The BBB is mainly formed out of endothelial cells embedded in collagen IV and fibronectin-rich basement membranes. Several pathologies result from dysfunction of the BBB followed by microbial traversal, causing diseases such as meningitis. In order to test the effect of multiple parameters, including different drugs and anesthetics, on the permeability of the BBB we established a novel human cell culture model mimicking the BBB with human brain microvascular endothelial cells. The endothelial cells are grown on collagen IV and fibronectin-coated filter units until confluence and can then be treated with different compounds of interest. In order to demonstrate a microbial traversal, the upper chamber with the apical surface of the endothelial cells is inoculated with bacteria. After an incubation period, samples of the lower chamber are plated on agar plates and the obtained colonies are counted, whereby the number of colonies correlate with the permeability of the BBB. Endogenous cellular factors can be analyzed in this experimental set-up in order to elucidate basic cellular mechanisms of the endothelial cells contributing to the BBB. In addition, this platform allows performing a screen for compounds that might affect the permeability of the endothelial cells. Finally, bacterial traversal can be studied and linked to different pathologies, such as meningitis. It might be possible to extend the model and analyze the pathways of the bacteria through the BBB. In this article, we provide a detailed protocol of the described method to investigate the permeability of the BBB.

The human blood-brain barrier selectively prevents penetration of hydrophilic molecules and pathogens into the brain. Several pathologies, including meningitis and postoperative delirium, are associated with an increased permeability of the blood-brain barrier. Here, we describe an endothelial cell culture model to test the barrier permeability by microbial traversal.

微生物通过微血管内皮细胞对血脑屏障渗透性分析

The human blood-brain barrier (BBB) is characterized by a very low permeability for biomolecules in order to protect and regulate the metabolism of the ...

Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is responsible for altering infection for many of these viruses. Thus, characterizing the nature of virus-bacteria binding is a foundational step needed for elucidating the mechanism(s) by which bacteria alter viral infection. For human norovirus, commensal bacteria enhance B cell infection. The virus directly binds to these bacteria, indicating that this direct interaction is involved in the mechanism of infection enhancement. A variety of techniques can be used to quantify interactions between bacteria and viruses including scintillation counting of radiolabeled viruses and polymerase chain reaction (PCR). Both methods require the use of live virus, which may need to be generated in the laboratory. Currently, none of the established in vitro culture systems available for human norovirus are robust enough to allow for generation of highly concentrated viral stocks. In lieu of live virus, virus-like particles (VLPs) have been used to characterize the interactions between norovirus and bacteria. Herein a flow cytometry method is described with uses virus specific antibodies to quantify VLP binding to gram-negative and gram-positive bacteria. Inclusion of both bacteria only and isotype controls allowed for optimization of the assay to reduce background antibody binding and accurate quantification of VLP attachment to the bacteria tested. High VLP:bacterium ratios result in VLPs binding to large percentages of the bacterial population. However, when VLP quantities are decreased, the percent of bacteria bound also decreases. Ultimately, this method can be employed in future experiments elucidating the specific conditions and structural components that regulate norovirus:bacterial interactions.

The goal of this protocol is to quantify binding of the eukaryotic pathogen human norovirus to bacteria. After performing an initial virus-bacterium attachment assay, flow cytometry is used to detect virally-bound bacteria within the population.

使用流细胞测定量化人类诺如病毒样颗粒与准细菌结合

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is ...

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal drugs, IA can cause &#62;50% mortality in infected immunocompromised patients. It is crucial to determine both host and pathogen factors that contribute to infection susceptibility and low survival rates in infected patients in order to develop novel therapeutics. Innate immune responses play a pivotal role in recognition and clearance of Aspergillus spores, though little is known about the exact cellular and molecular mechanisms. Reliable models are required to investigate detailed mechanistic interactions between the host and pathogen. The optical clarity and genetic tractability of zebrafish larvae make them an intriguing model&#160;to study host-pathogen interactions of multiple human bacterial and fungal infections in a live and intact host. This protocol describes a larval zebrafish Aspergillus infection model. First, Aspergillus spores are isolated and injected into the zebrafish hindbrain ventricle via microinjection. Then, chemical inhibitors such as immunosuppressive drugs are added directly to the larval water. Two methods to monitor the infection in injected larvae are described, including the 1) homogenization of larvae for colony forming unit (CFU) enumeration and 2) a repeated, daily live imaging setup. Overall, these techniques can be used to mechanistically analyze the progression of Aspergillus infection in vivo and can be applied to different host backgrounds and Aspergillus strains to interrogate host-pathogen interactions.

Infection of Zebrafish Larvae with Aspergillus Spores for Analysis of Host-Pathogen Interactions

This protocol describes an Aspergillus infection model in zebrafish larvae. Aspergillus spores are microinjected into the hindbrain of larvae, and chemical treatment is used to induce immunosuppression. Infection progression is monitored via a daily imaging setup to monitor fungal growth and immune responses as well as enumeration of live spores by colony forming unit plating.

斑马鱼幼虫感染 阿斯珀吉卢斯 孢子分析宿主-病原体相互作用

Invasive aspergillosis (IA) is one of&#160;the most common fungal infections among immunocompromised individuals. Despite the availability of antifungal ...