我们感谢小肖在北卡罗来纳大学教堂山为我们提供了 pEMBL-CMV-GFP 质粒。我们感谢克里斯托弗和塞缪尔. 黄对手稿的批判性阅读。这项工作得到公共卫生服务补助金 (NIH R01 NS088399 和 T32 EY232113) 的支持。

注意:表 1提供了此协议所需的解决方案和缓冲区的配方。下面描述的协议是为副校长交叉互补斑点杂交试验研究的作用, 在衣壳组装的蛋白。对纯化 AAV 矢量滴定法进行了较为通用的定量斑点杂交试验, 并在代表性结果部分作了说明。
1. 构建 VP3、AAV2 和表达质粒的代表
<ol><li>
pCMV-AAVx-VP3 的构建 (x = 血清型)
	<ol>
<li>PCR-放大整个 VP3 ORF (1.6 kb) 使用高保真 DNA 聚合酶和以下底漆对: VP3 向前, CTAA-RE1-CACC-N25 (VP3 ORF 的前25核苷酸);VP3 反向, TCTT-RE2-N25 (最后25核苷酸 VP3 ORF)。 注: RE1 和 RE2 是用于克隆的限制酶 (REs) 的场所。CTAA 和 TCTT 是终端 5 ' 和 3 ' tetranucleotides 增加了促进限制酶消化接近末端的双链 DNA。CACC 是一个科扎克的共识序列。</li>
		<li>克隆在哺乳动物表达载体对应的 re 地点之间的重新消化 PCR 产物, 即使用人巨细胞病毒立即-早期 (CMV-IE) 增强剂-促进者为高层次表达。 注: 对于分子克隆, 在最佳温度下, 在 4 U/µg DNA 浓度为1小时, 消化5µg 的主干质粒 DNA 与限制酶 (s)。对于 PCR 片段, 增加使用的酶的单位 (例如, 10 U/µg 1.6 kb VP3 PCR 产品) 和一个更长的潜伏期 (例如, 4 h) 由于限制酶识别站点的数量增加每单位长度。分子生物学酶和克隆过程中有用的信息, 包括细菌转化, 可以在别处找到15,16,17。</li>
	</ol>
</li>
	<li>
pCMV 旗 AAPx (x = 血清型) 的构建
	<ol>
<li>PCR-放大整个 CTAA-RE1-CACCATGGACTACAAGGACGACGATGACAAA-N (0.6 kb) 除了第一个氨基酸使用高保真 DNA 聚合酶和以下底漆对: 前25 (25 核苷酸从第四核苷酸在ORF);反向, TCTT-RE2-N25 (最后25核苷酸的)。 注: GACTACAAGGACGACGATGACAAA 编码一个标志标记, 它已被证明没有有害影响4,5 , 但可以省略, 如果不必要的。</li>
		<li>克隆在哺乳动物表达载体的相应地点之间的重新消化 PCR 产物与巨细胞病毒-IE 增强器-启动子15,16,17。</li>
	</ol>
</li>
	<li>
建设 pHLP-代表
	<ol>
<li>消化5µg 的 pAAV-RC2 质粒 (7.3 kb) 与20单位每 Xho i 和 Xcm i, 并纯化 dna 使用商业 dna 纯化试剂盒或苯酚-氯仿萃取。 注意: 从 7.3 kb pAAV-RC2 质粒中删除 1.8 kb Xho i Xcm 片段会导致衣壳副总裁蛋白表达丢失, 同时保留代表蛋白表达式。</li>
		<li>钝 dna 末端与6个单位 T4 脱氧核糖核酸聚合酶, 琼脂糖凝胶-纯化 5.5 kb 脱氧核糖核酸片断, 并且自已结扎被纯化的片断使用50到 100 ng 脱氧核糖核酸和400个单位 T4 脱氧核糖核酸连接酶根据制造商的建议15。</li>
		<li>按照步骤1.1.2 说明中引用的标准细菌转换过程进行操作。</li>
	</ol>
</li>
	<li>通过限制酶消化和顺序15,18验证质粒结构。</li>
	<li>使用商用试剂盒进行质粒 minipreps 或 maxipreps, 以获得足够数量的质粒 DNA 进行下游 AAV 生产试验。</li>
</ol>2. 用质粒 DNA 转染 (交叉互补法) 生产 HEK 293 细胞中的 AAV
<ol><li>培养 HEK 293 细胞在 Dulbecco 的改性鹰培养基 (DMEM)-高葡萄糖 (4.5 克/升) 补充10% 胎牛血清 (血清), 1% 青霉素 & 链霉素混合, 1 毫米 l-谷氨酰胺, 在37摄氏度孵化器与5% 二氧化碳 (CO2)。 注: AAV 的滴度因 HEK 293 细胞的来源而有显著差异。 注意: 虽然 AAV 可以在生物安全级别 1 (BSL1) 控制中进行处理, 但建议 HEK 293 细胞工作时 BSL2 包容。</li>
	<li>在-1 天 (在转染之前24小时), 板材 6–7 x 105 HEK 293 个细胞/井在2毫升在台阶2.1 在6个好板材 (s) 描述的完全媒介。这通常达到90% 融合第二天。</li>
	<li>
0天: 转染
	<ol>
<li>DNA 转染的制备<ol>
<li>确保细胞达到了90% 融合。</li>
			<li>预热 DMEM 补充与1% 青霉素 & 链霉素混合和1毫米 l-谷氨酰胺, 但没有10% 的血清 (即无血培养基) 在37°c 水浴。</li>
			<li>允许聚乙烯亚胺 (PEI) 溶液达到室温。</li>
		</ol>
</li>
		<li>质粒 DNA 混合物的制备<ol>
<li>在96µL 磷酸盐缓冲盐水 (PBS) 中混合质粒, 不 CaCl2或氯化镁2 , 不育1.5 毫升离心管, 如表 2所示。DNA 的总数量是2µg/好。 注: 如果它们是标称的, 则可以忽略质粒 DNA 溶液的体积。如果质粒 DNA 溶液的总体积为≥10µL, 建议容积调整。</li>
		</ol>
</li>
		<li>裴转染<ol>
<li>将4µL 的裴溶液 (1 毫克/毫升) 添加到 PBS-质粒 DNA 组合中 (如上所述)。最后容量是大约100µL (文化媒介的5% 容量)。涡流管和孵化的 DNA: 裴混合物在室温下15分钟。</li>
			<li>在等待15分钟孵化完成时, 用步骤2.3.1.2 中描述的 prewarmed 无血清培养基取代培养基。</li>
			<li>一旦15分钟的 DNA 孵化: 裴合剂完成, 简要旋转管与离心收集液体到底部的管, 并添加 DNA: 裴混合物滴到培养基中的 HEK 293 培养板。轻轻搅动板, 并返回到37°c 孵化器与 5% CO2。</li>
			<li>保持这种转染培养基中的细胞, 直到5天的收获 (不需要中等变化)。</li>
		</ol>
</li>
	</ol>
</li>
	<li>在1和2天, 观察在倒置荧光显微镜下转染的 pCMV-GFP 质粒的细胞, 以评估染电效率。 注: 对于荧光显微术, 本文采用了 10 x/0.25 数值孔径物镜与10×目镜相结合的方法, 采用了450–490纳米励磁带通滤波器和515–565纳米带通发射滤波器。上述条件通常产生70% 以上的转染效率。细胞可能表现出一些形态学的变化 (如细胞变得苗条) 由于无血清的条件。</li>
	<li>在天 3–5, 继续培养转染细胞在37°c 孵化器与 5% CO2。</li>
	<li>在5 天, 将细胞和含有病毒的介质收集到15毫升聚丙烯锥形管中, 吹打上下或用刮刀刮。</li>
	<li>将样品存放在摄氏-80 摄氏度, 直到使用。</li>
</ol>3. 斑点杂交法测定 AAV 定量
<ol><li>病毒微粒的恢复
	<ol>
<li>在37摄氏度的水浴中迅速解冻冰冻的管子。将管子大力旋转1分钟, 以最大限度地从细胞中恢复 AAV。</li>
		<li>将细胞碎片通过离心在 3700 x g 在4°c 为10分钟。取200µL 的上清从每管和转移到一个标记的离心管与螺钉帽的斑点印迹化验。 注: 将剩余的上清整除为离心管, 贮存于-80 摄氏度冷冻, 供日后使用。在这里, 使用的聚丙烯离心管连接螺钉帽和 O 形环。为了防止 AAV 和苯酚-氯仿的溢出, 需要严密的密封。</li>
	</ol></li>
	<li>沙雷质限制性治疗
	<ol>
<li>准备混合 a 和混合 B 试剂 (表 3)。添加10µL 混合 a 和10µL 混合 B 到每个管。涡流管为 5 s, 简要地旋转管收集液体到管子的底部和孵化管子在37°c 至少为 1 h。 注: 混合含有氢氧化钠, 并优化 pH 值治疗质限制性。混合 B 补充镁。质限制性在商业上可用的酶种群中的浓度可能有所不同。质限制性的体积需要相应调整, 以使 200 U/毫升后加入混合 A 和混合 B 成管在步骤3.2.1。 注: 潜伏期延长4小时, 可减少背景信号。</li>
		<li>在孵化结束时, 用台式离心机简单地旋转管子, 从管子的顶端和两侧收集凝结和溶液。 注意: 协议可以在这里暂停。样品可以被存放冻结在-20 °c 或-80 °c。</li>
	</ol></li>
	<li>蛋白酶 K 治疗
	<ol>
<li>准备混合 C 试剂 (表 3)。在每管中加入180µL 的混合 C。涡流管 5 s, 简要旋转管和孵化管在55°c 1 小时。 注: EDTA 在混合 C 试剂中螯合游离镁离子和钝化质限制性。</li>
		<li>在孵化的最后, 允许样品达到室温, 并简要地旋转管与台式离心机收集冷凝和解决方案从顶部和两侧的管。 注意: 协议可以在这里暂停。样品可以被存放冻结在-20 °c 或-80 °c。要恢复该协议, 在55摄氏度处孵化出5至10分钟的管子, 完全溶解在缓冲器中的 SDS 晶体。</li>
	</ol></li>
	<li>苯酚-氯仿萃取与乙醇沉淀
	<ol>
<li>在样品中加入200µL 苯酚-氯仿, 并将其涡旋1分钟. ≥16,100 x g 离心中的样品在室温下≥5最小。 注意: 苯酚-氯仿应在化学油烟机中处理, 并配备适当的个人防护设备 (PPE;例如, 丁腈橡胶手套, 护目镜或面罩, 和长袖的实验室大衣)。</li>
		<li>将320µL 的水层 (160 µL 两次与 P200 吸管) 转移到一个新的标准离心管 (80% 的水中流体体积)。 注意: 在样品间取相同体积的水溶液是非常重要的, 否则化验结果就会失去定量的准确度。</li>
		<li>准备混合 D 试剂 (表 3)。在每管中添加833µL 的混合 D。涡流管 5 s, 并孵化管在-80 °c 为≥20分钟。 注: 混合 D 是乙醇、醋酸钠和糖原的混合物, 用于方便乙醇沉淀 DNA。在这个步骤中, 样品可以存储在摄氏-80 摄氏度, 并且该协议可以在以后恢复。</li>
		<li>离心样品与离心在≥16,100 x g 在4°c 为≥15极小. 在干净的纸巾上倒上清和涂抹一次。把大约500µL 70% 乙醇入每个管子, 漩涡管子为 5 s, 并且离心机管子与台式离心在≥16,100 x g 在4°c 为≥5分钟。</li>
		<li>在干净的纸巾上倒上上清和涂抹一次。干颗粒在65°c;颗粒可以完全干燥。 注意: 不要使用吸管去除过量的乙醇, 在印迹后仍然残留。颗粒也可以在室温下干燥一夜。该协议可以暂停在这里, 干燥的 DNA 小球可以储存在室温下数天与管盖关闭。</li>
	</ol></li>
	<li>病毒 DNA 在 TE 缓冲器中的泥沙
	<ol>
<li>通过在室温下将每管30分钟的震动, 将120µL 中的 DNA 小球分解成1小时。</li>
	</ol></li>
	<li>斑点印迹
	<ol>
<li>质粒 DNA 标准的制备<ol>
<li>稀释 AAV 载体基因组质粒 DNA 到水或三盐酸缓冲液中的10µL (10 毫米, pH 8.0)。采取25µL 的稀释和消化与适当的限制酶1小时进行线性化质粒 DNA, 在50µL 的反应量。 注意: 我们做了一组重复的消化 (见步骤 3.6.4.1)。适当的酶应该是切割斑点印迹探针结合区外的质粒 DNA 的一种。为方便起见, 稀释的质粒 DNA 可以 aliquoted (25 µL/管), 并储存在-20 摄氏度冷冻, 供将来使用。当试管在台阶3.5.1 时, 消化质粒 DNA。不要过度消化质粒 DNA 标准。</li>
			<li>将450µL 的水或 TE 添加到含有消化质粒 DNA 标准的管中, 并进行彻底混合。将这种混合物的70µL 转移到一个新的1.5 毫升离心管, 1330 µL 水或 TE, 使稀释质粒标准 (25 pg/µL)。</li>
			<li>按照表 4创建一组双折叠串行稀释 (600 µL/管)。将稀释彻底混合涡流5秒。</li>
		</ol>
</li>
		<li>标准和病毒 DNA 样本的变性<ol>
<li>添加600µL 的2x 碱性溶液, 以每个标准稀释。混合涡流5至十年代. 室温孵育10分钟。</li>
			<li>在每种病毒 DNA 样品中加入120µL 2x 碱性溶液。混合涡流5至十年代. 室温孵育10分钟。</li>
		</ol>
</li>
		<li>设置斑点印迹装置<ol>
<li>使用剪刀, 切割一个印迹 (如, 泽塔探针) 膜, 以适当的大小, 样本和标准的数量。将膜与水浸泡10分钟, 然后将其放置在斑点印迹装置上。覆盖膜设备上未使用的油井。 注: 用清洁镊子处理膜。为了覆盖空井, 可以使用与膜一起的浅蓝色保护片。在装订样品和标准之前, 不要让膜干燥。有关设备组装和使用的详细信息, 请参阅用户手册19。</li>
			<li>将水添加到要装载样品的水井中。使用真空和拉水通过水井检查错误和重新测试时, 固定。在使用真空时重新拧紧螺钉以确保密封紧密。 注: 不完全密封可能导致井间样品渗漏。</li>
			<li>一旦水完全通过, 完全释放真空 (即真空歧管应开放气压)。</li>
		</ol>
</li>
		<li>对斑点印迹装置的装载标准和样品<ol>
<li>将每个稀释质粒 DNA 标准的400µL 应用于各井, 并运行四道标准稀释。使用标准摘要的两个单独的整除数, 并将它们分别加载。应用每种病毒 DNA 样本的200µL/井。 注: 使用剩余的40µL 变性样品, 可以制备稀释的样品 (例如, 10 倍稀释样品使用20µL 样品加上180µL 1x 碱性溶液) 和涂抹, 如果必要的话。</li>
			<li>应用柔和的真空, 通过井中提取 DNA 溶液。 注: 真空压力需要通过部分打开三路阀门来调整, 以便将真空压力应用于斑点印迹装置以及大气层 (即, 旋塞阀臂位于近似45°角度, 在那里它发出更响亮的吸入噪音)。</li>
			<li>一旦所有的水井都清空, 通过调整三路阀门释放真空。添加400µL 的1x 碱性溶液, 每个井, 其中包含标准和样品。等待5分钟, 然后再应用真空, 以清空水井。</li>
			<li>用同样的方法重新应用真空 (见步骤 3.6.4.2)。</li>
			<li>在真空下拆卸斑点印迹装置, 取出膜, 并用 2x SSC 冲洗。将膜放在干净的纸巾上, 将 DNA 绑定面朝上移除多余的 2x SSC。</li>
			<li>用适当的紫外交联剂将涂抹的 DNA 涂到膜上;膜现在已准备好进行杂交。 注: 湿膜可用于 UV 交联。干燥的 UV 交联膜可在室温下贮存。在材料表中可以找到进一步的信息。</li>
		</ol>
</li>
	</ol></li>
	<li>杂交和洗涤
	<ol>
<li>在65摄氏度的水浴中预热杂交缓冲。</li>
		<li>酶标签一个 DNA 探针与放射性α-32P dCTP 和净化它使用商业可用的套件根据制造商的建议。 注意: 我们在病毒基因组中使用 0.5–2.0 kb 的长度的探针, 从增强启动子区域或蛋白质编码序列。尽管该协议使用32P 标记的放射性探针进行信号检测, 但也可以采用非放射性化学发光或荧光探针 (请参阅讨论部分)。</li>
		<li>将膜放在一个杂交瓶与 DNA 绑定的一侧向上, 冲洗膜与5毫升 prewarmed 杂交缓冲, 并丢弃缓冲区。然后添加10毫升的 prewarmed 杂交缓冲, 并把瓶子放在一个旋转杂交烤箱设置在65摄氏度。旋转≥5分钟。</li>
		<li>热变性20µL 10 毫克/毫升剪切鲱鱼或鲑鱼精子 DNA 溶液和32P 标记探针 (≥107 cpm) 为5分钟, 通过放置在一个热块设置在100°c。然后把它们放在冰上≥2分钟, 简单地旋转, 直到使用。 注意: 对于放射性 DNA 探针, 必须使用1.5 毫升的螺钉盖和 O 形环管。</li>
		<li>快速将变性精子 DNA 和放射性探针添加到杂交瓶中的杂交缓冲中, 并在十年代将瓶子摇匀。将瓶子返回到65摄氏度烤箱, 并在65摄氏度的≥4上孵化瓶子。</li>
		<li>一旦杂交完成, 停止旋转, 移除杂交瓶, 然后将放射性探针溶液倒入50毫升锥形管中, 并带有防漏塞密封盖。将探头存放在指定为放射性材料的冰箱中的适当容器中。 注: 使用的杂交缓冲器与探针存储在4°c 可以重新使用至少5次, 通过放置一个防漏插头密封盖, 其中包含杂交缓冲在100°c 水浴5分钟。</li>
		<li>用洗涤缓冲器冲洗膜, 加热到65摄氏度。将20至30毫升的洗涤缓冲液添加到杂交瓶中, 旋转5分钟. 重复此洗涤2次。 注: 测量洗涤液的放射性, 并在当地研究所要求时记录。</li>
		<li>洗膜时, 将荧光粉成像屏放在图像橡皮擦上5分钟。</li>
		<li>第三次冲洗后, 将膜从杂交瓶中取出, 用纸巾去除膜上多余的缓冲液, 并将膜放在透明的塑料纸夹中。使用盖格计数器检查膜上的放射性信号。根据信号强度, 将拭除的荧光粉成像屏暴露在膜上10分钟至一夜。</li>
		<li>使用荧光粉图像扫描系统扫描屏幕, 获取每个点的信号强度数据。</li>
	</ol></li>
	<li>数据分析
	<ol>
<li>使用电子表格和数据分析软件 (如Excel) 绘制标准曲线。 注: 对数对数线性回归用于绘制标准曲线。</li>
		<li>通过插值确定每个病毒 DNA 样本的纳克当量 (ng-eq)。ng eq 是用来绘制标准曲线的质粒 dna 的数量, 相当于病毒 dna 分子的数量。当质粒 dna 的长度为 8000 bp 时, 单链 AAV 病毒 dna 1 的 ng eq 对应 2.275 x 108粒子。 注: 可通过以下方程式将 ng eq 转化为病毒粒子的数量: <img alt="Equation 1" src="/files/ftp_upload/56766/56766eq1.jpg"> <img alt="Equation 2" src="/files/ftp_upload/56766/56766eq2.jpg"> AAV 粒子的数量通常被表达为 "vg" (向量基因组) 或 "DRP" (DNase 抗性粒子)。</li>
		<li>计算起始物料的 AAV 浓度。因为被涂抹的病毒 dna 代表<img alt="Equation 3" src="/files/ftp_upload/56766/56766eq3.jpg"> 66.7% 的病毒 dna 在起始材料, 1 ng eq 对应 1.7 x 109粒子/毫升<img alt="Equation 4" src="/files/ftp_upload/56766/56766eq4.jpg"> </img>。 注意: 如果起始材料中包含的所有病毒 DNA 都在无损耗的膜上涂抹, 则不需要进行这种校正 (见图 1)。</li>
	</ol></li>
</ol>

<ol>
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作者没有什么可透露的。

本文介绍了定量斑点杂交检测 AAV AAPs 的应用及其在衣壳装配中的作用。从这些研究中获得的知识可以对 AAV 衣壳组装过程中的先天差异和不同血清型间 AAPs 的功能作用提供详细的见解。在这方面, AAV VP3-AAP 交叉互补斑点杂交法揭示, 蛇 AAV VP3 显示了严格依赖于其同源的联合表达的衣壳组装和蛇, 不促进外源血清型衣壳组装.这一观察很有趣, 因为以前调查过的 AAV 血清型 (AAV1、2、3、6、7、8、9和 12) 都...

腺相关病毒 (AAV) 是一种小的、非封闭的单链 DNA 病毒, 其基因组约为 4.7 kilobases (kb)。AAV 基因组包含开放阅读框架 (码) 的代表和cap基因。在 2010年, 一个先前不明的结构性蛋白编码由 +1 帧转移的 ORF 在 AAV2帽基因中发现了报等, 并发现发挥关键作用, 在组装 AAV2 衣壳副总裁单体蛋白成病毒衣壳1。这种新的蛋白质被命名为组装活化蛋白 () 后, 它发挥作用, 促进衣壳组装1。
码 bioinformatically 在Dependoparvovirus的所有细小的基因组中被识别出来, 但不在细小病毒家族1、2的不同属的基因组中。本文首先从原型 AAV2 (AAP2) 对该蛋白的功能研究进行了初步研究, 确立了 AAP2 在靶向未组装 VP 蛋白对核仁的聚集和形成中的重要作用, 从而充分组装衣壳1,3,4。AAV2 衣壳无法在没有该表达式的情况下组装, 已由多个组独立确认, 包括我们的1、2、3、4、5.随后对 AAV 血清型1、8和9的研究证实了 AAPs 在衣壳组装中的关键作用, 因为 AAV1、8和9的 VP3 单体蛋白在没有联合表达的情况下无法形成完全组装的衣壳.
最近, 通过包括使用定量斑点杂交检测的方法, 我们研究了 AAV1 12 VP3 单体组装成衣壳的能力, 在缺乏表达和 AAP1 12 的能力, 以促进 VP3 单体组装异源血清型。这项研究表明, AAV4, 5 和 11 VP3 单体可以组装, 不用的。另外, 据发现, 我们研究的十二种血清蛋白中有八 (即除了 AAP4、5、11和 12) 显示了广泛的能力, 支持外源 AAV 血清型衣壳的衣壳组装, 而 AAP4, 5, 11 和12显示了极大的有限的能力在这方面6。这四种血清型是 phylogenetically 远离其他的儿科血清2,3。此外, 该研究发现不同 AAPs6的亚细胞定位有显著的异质性。此外, 研究还表明, 与组装的衣壳和核仁, AAV2 衣壳组装的标志紧密关联, 不一定可以扩展到其他血清型包括 AAV5, 8 和 9, 这显示核仁排除组装衣壳6。因此, 从研究任何特定的血清学杂志获得的信息并不广泛适用于所有的儿科生物学。此类令人费解的生物学特性突显出, 需要从规范和非规范的 AAV 血清型中研究每个标准的角色和功能。
通过测定人胚肾 (HEK) 293 细胞中所产生的完全包装的 AAV 病毒微粒滴度, 可以评估在衣壳组装中的生物作用, 其中最常用的细胞系为 AAV 向量生产, 有或没有蛋白表达。标准的 AAV 定量方法是定量 PCR (qPCR) 为基础的化验7,8和定量斑点杂交为基础的化验9。其他 AAV 病毒微粒定量的方法, 如酶联免疫吸附试验10,11或光学密度测量12不理想的样本来自许多不同的 AAV 血清学或样品被杂质污染 (粗裂解物或培养基), 通常是用于 AAV 研究的样品。目前, qPCR 被广泛应用于 AAV 定量;然而, 有必要承认 qPCR 的检测可能的警告, 因为该检测可能导致系统性错误和显著的效价变化13,14。基于 pcr 的化验受到一些潜在的混杂因素的影响, 例如在 pcr 模板中存在共价键闭合终端发夹, 抑制放大13。即使有经验的个体也可以在不知不觉的13中引入潜在的混杂因素到 qPCR 的检测中。相比之下, 定量斑点杂交检测是一种经典的分子生物学技术, 不涉及基因组扩增, 并使用一个简单的原理, 与 qPCR 的检测相比, 错误的风险最小。该方法在技术上缺乏挑战性;因此, 即使没有经验的人也能合理地重现化验结果。
在本报告中, 我们描述了一个定量斑点杂交检测的方法学细节, 我们经常使用 AAV 向量定量, 并提供了一个例子, 如何应用该方法来研究 AAPs 在共同的血清学 (AAV5 和 AAV9) 的组装促进作用和以前 uncharacterized 从蛇 AAV14。在自然界中, AAV vp 蛋白和蛋白的表达在 cis 从一个单一的基因 (即, 副总裁的 cis 互补), 而在这里描述的化验, vp 和儿科蛋白是从两个独立的质粒提供反相 (即, vp跨互补)。由于来自不同血清型的每种 vp 或蛋白都可以从每个独立的质粒中表达出来, 因此就有可能测试外源副总裁--在衣壳装配上的组合 (即, 副校长交叉互补)。简单地, AAV VP3 从不同的血清学表达在 HEK 293 细胞通过质粒 DNA 转染包裹一个 AAV 向量基因组在存在或没有同源血清学, 或在存在的异种血清学。继生产后, 培养基和细胞裂解物受到斑点印迹检测, 以量化在衣壳内的病毒基因组。斑点杂交法的第一步是用核酸酶处理样品, 去除污染的质粒 dna 和无包装的 AAV 基因组。如果不这样做, 将增加背景信号, 特别是当粗样品被检测。接下来是蛋白酶治疗, 以打破病毒衣壳和释放核酸酶病毒基因组的样本解决方案。其次, 病毒基因组变性, 在膜上涂抹, 并与病毒基因组特异的 DNA 探针杂交进行定量。在这里报告的例子化验中, 我们证明蛇 AAV VP3 需要蛇的电泳为衣壳装配, 并且蛇的附属程序不促进 AAV9 衣壳的汇编, 不同于由依赖于异源血清衣壳。最后, 我们报告了一个重要的告诫, qPCR 或斑点印迹为基础的 AAV 定量分析的选择核酸酶显着影响的化验结果。

<table>
	<tbody>
		<tr>
			<td>Benzonase</td>
			<td>MilliporeSigma</td>
			<td>1016970001</td>
			<td>Referred to as Serratia marcescen endonuclease in the main text.</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Roche</td>
			<td>4716728001</td>
			<td>Referred to as DNase I Enzyme A in the main text.</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Invitrogen</td>
			<td>18047019</td>
			<td>Referred to as DNase I Enzyme B in the main text.</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>New England Biolabs</td>
			<td>M0303L</td>
			<td>Referred to as DNase I Enzyme C in the main text.</td>
		</tr>
		<tr>
			<td>1.5 mL Attached O-Ring Screw Cap Microcentrifuge Tubes</td>
			<td>Corning</td>
			<td>430909</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Microcentrifuge Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Polypropylene Conical Tube</td>
			<td>Corning</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>3 M Sodium Acetate</td>
			<td>Teknova</td>
			<td>S0298</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Polypropylene Conical Tube</td>
			<td>Corning</td>
			<td>430291</td>
			<td></td>
		</tr>
		<tr>
			<td>AAV-293 Cells</td>
			<td>Agilent</td>
			<td>240073</td>
			<td>HEK-293 cell line optimized for the packaging of AAV virions.</td>
		</tr>
		<tr>
			<td>AccuGENE 0.5 M EDTA Solution</td>
			<td>Lonza</td>
			<td>51234</td>
			<td></td>
		</tr>
		<tr>
			<td>AccuGENE 1 M Tris-HCl pH 8.0</td>
			<td>Lonza</td>
			<td>51238</td>
			<td></td>
		</tr>
		<tr>
			<td>AccuGENE 10% SDS</td>
			<td>Lonza</td>
			<td>51213</td>
			<td></td>
		</tr>
		<tr>
			<td>AccuGENE 1X TE Buffer</td>
			<td>Lonza</td>
			<td>51235</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Dot Apparatus</td>
			<td>Bio-Rad</td>
			<td>1706545</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>MilliporeSigma</td>
			<td>A3294</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Lifter</td>
			<td>Corning</td>
			<td>3008</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>MilliporeSigma</td>
			<td>372978</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA Extractor Kit</td>
			<td>Wako Pure Chemical Industries</td>
			<td>295-50201</td>
			<td>This is used for quantitative dot blots on purified virus. We use only a half volume of all the reagents in each step recommended for the Protocol Scheme 2 in the manual provided by the manufacturer.</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium (DMEM)-high glucose (4.5 g/L)</td>
			<td>Lonza</td>
			<td>12-614F</td>
			<td></td>
		</tr>
		<tr>
			<td>ElectroMAX DH10B Cells</td>
			<td>Thermo Fisher Scientific</td>
			<td>18290015</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 Proof</td>
			<td>Decon Labs</td>
			<td>2716</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>VWR</td>
			<td>1500-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll 400</td>
			<td>Alfa Aesar</td>
			<td>B22095</td>
			<td>Referred to as Polysucrose 400.</td>
		</tr>
		<tr>
			<td>Fluorescence&#160;Microscope</td>
			<td>Zeiss</td>
			<td>Axiovert 40 CFL</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Roche</td>
			<td>10901393001</td>
			<td></td>
		</tr>
		<tr>
			<td>Herring Sperm DNA</td>
			<td>Invitrogen</td>
			<td>15634-017</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization Tubes</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-247-150</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageQuant TL</td>
			<td>GE Healthcare Life Sciences</td>
			<td>ImageQuant TL</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine 200 mM</td>
			<td>Lonza</td>
			<td>17-605E</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Choloride Hexahydrate</td>
			<td>MilliporeSigma</td>
			<td>M0250</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroPulser Electroporator</td>
			<td>Bio-Rad</td>
			<td>1652100</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Quick Spin DNA Columns</td>
			<td>MilliporeSigma</td>
			<td>11814419001</td>
			<td></td>
		</tr>
		<tr>
			<td>pAAV-RC2 Vector</td>
			<td>Cell Biolabs Inc</td>
			<td>VPK-422</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin 10K/10K</td>
			<td>Lonza</td>
			<td>17-602E</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Lonza</td>
			<td>17-516F</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphorimaging Exposure Cassette</td>
			<td>GE Healthcare Life Sciences</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphorimaging Screen</td>
			<td>GE Healthcare Life Sciences</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid Maxi Kit</td>
			<td>QIAGEN</td>
			<td>12162</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum Pfx DNA Polymerase</td>
			<td>Invitrogen</td>
			<td>11708013</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylenimine</td>
			<td>Polysciences Inc</td>
			<td>23966-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone</td>
			<td>MilliporeSigma</td>
			<td>P5288</td>
			<td></td>
		</tr>
		<tr>
			<td>Prime-It II Random Primer Labeling Kit</td>
			<td>Agilent Technologies</td>
			<td>300385</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer AAP Forward (N25 nucleotides from the 4th nucleotide in the AAP ORF, RE1 is a restriction enzyme site for cloning): CTAA-RE1-CACCATGGACTACAAGGACGACGATGACAAA-N25</td>
			<td>MilliporeSigma</td>
			<td>Custom Primer</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer AAP Reverse (N25 is the last 25 nucleotides of the AAP ORF, RE2 is a restriction enzyme site for cloning): TCTT-RE2-N25</td>
			<td>MilliporeSigma</td>
			<td>Custom Primer</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer VP3 Forward (N25 the first 25 nucleotides of the VP3 ORF, RE1 is a restriction enzyme site for cloning): CTAA-RE1-CACC-N25</td>
			<td>MilliporeSigma</td>
			<td>Custom Primer</td>
			<td></td>
		</tr>
		<tr>
			<td>Primer VP3 Reverse (N25 is the last 25 nucleotides of the AAP ORF, RE2 is a restriction enzyme site for cloning): TCTT-RE2-N25</td>
			<td>MilliporeSigma</td>
			<td>Custom Primer</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K Solution</td>
			<td>Invitrogen</td>
			<td>25530-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Restriction Enzymes</td>
			<td>New England Biolabs</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmon Sperm DNA</td>
			<td>Invitrogen</td>
			<td>15632011</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipettes</td>
			<td>Thermo Fisher Scientific</td>
			<td>13-678-11D / 13-678-11E / 13-678-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Thermo Fisher Scientific</td>
			<td>S271-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Citrate Tribasic Dihydrate</td>
			<td>MilliporeSigma</td>
			<td>C8532</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Polymerase</td>
			<td>New England Biolabs</td>
			<td>M0203L</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermal Cycler</td>
			<td>Eppendorf</td>
			<td>6321000515</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-culture Treated 6-well Plate</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Thermo Fisher Scientific</td>
			<td>BP152-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Typhoon FLA 7000</td>
			<td>GE Healthcare Life Sciences</td>
			<td>28955809</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Buffer-Saturated Phenol</td>
			<td>Invitrogen</td>
			<td>15513-047</td>
			<td></td>
		</tr>
		<tr>
			<td>UV Crosslinker</td>
			<td>Spectroline</td>
			<td>XLE-1000</td>
			<td>Optimal Crosslink mode is used, providing a UV energy dosage of 120 mJ/cm2.</td>
		</tr>
		<tr>
			<td>Zeta-Probe Membrane</td>
			<td>Bio-Rad</td>
			<td>162-0165</td>
			<td></td>
		</tr>
	</tbody>
</table>

a quantitative dot blot assay for aav titration and its use for functional assessment of the adeno-associated virus assembly-activating proteins

虽然腺相关病毒 (AAV) 被广泛接受为一种有吸引力的基因治疗载体, 但它也作为一种了解病毒生物学的模型病毒。在后者方面, 最近发现一种非结构化的 AAV 蛋白 (称为组装活化蛋白), 已经为病毒衣壳副总裁蛋白质组装过程中所涉及的进程注入了新的光。虽然许多 AAV 血清型需要用于程序集, 我们最近报告说, AAV4, 5 和11是这个规则的例外。此外, 我们还证明了不同血清型的 AAPs 和组装衣壳不同的亚细胞间隔。这种意想不到的异质性的生物学性质和功能作用的 AAPs 之间的不同 AAV 血清学强调了研究的重要性, AAPs 来自不同的血清型。本手稿详细介绍了 AAV 定量的直接斑点印迹法及其在衣壳体组装中对其依赖性和血清型特异性的评估。为了证明这种斑点杂交化验的效用, 我们着手描述了蛇 AAV, 以前的 uncharacterized 爬行动物 AAV, 以及 AAV5 和 AAV9 的衣壳组装和儿科的依赖性, 这些以前被证明是独立于血清型, 分别。该化验结果显示, 蛇 AAV 衣壳组件需要蛇的 AAPs, 不能由 AAV5 和 AAV9 促进。该检测还表明, 不同于许多常见的血清 AAPs, 促进异种衣壳组装交叉互补, 蛇的儿科学会不促进组装 AAV9 衣壳。此外, 我们还表明, 核酸酶的选择对斑点印迹检测的读数有显著影响, 因此, 选择最佳酶对 AAV 滴度的成功评估是至关重要的。

如图 1所示, 在大规模生产的纯化 AAV 矢量种群定量斑点印迹的一个代表性结果。用这种斑点杂交法测定了双绞股 AAV2G9-CMV-GFP 载体的滴度。用两轮氯化铯 (中海) 密度梯度离心和前述20的透析纯化了该载体。一般而言, 对于纯化的 AAV 向量股, 每 AAV 矢量股的三种不同体积 (例如, 0.3 µL、0.1 µL 和0.03 µL) 都受到上面描述的斑点杂交?...

Watch this Scientific Journal Video about A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins at JoVE.com

A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins

本手稿详细介绍了一种直接斑点印迹法定量的腺相关病毒 (AAV) 的效价及其应用研究组装活化蛋白 (AAPs) 的作用, 一种新型的非结构化病毒蛋白在所有 AAV 发现血清型, 促进衣壳的组装从同源和异源 AAV 血清型。

AAV 滴定定量斑点印迹法及其在腺相关病毒组装活化蛋白功能评价中的应用

While adeno-associated virus (AAV) is widely accepted as an attractive vector for gene therapy, it also serves as a model virus for understanding virus ...

a-quantitative-dot-blot-assay-for-aav-titration-its-use-for

Research

JoVE Journal

Biology

13.6K 次观看.  Oregon Health & Science University. 本手稿详细介绍了一种直接斑点印迹法定量的腺相关病毒 (AAV) 的效价及其应用研究组装活化蛋白 (AAPs) 的作用, 一种新型的非结构化病毒蛋白在所有 AAV 发现血清型, 促进衣壳的组装从同源和异源 AAV 血清型。

视频: A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

梯级步行任务：一个评分系统，其实际应用。

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

科研

生物学

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

微探针：操作和使用的局部表面处理

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

使用油脂介孔结构的测定结晶膜蛋白

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction followed by probe hybridization on a nylon membrane, which is re-usable. Probes are 5' amine modified to allow fixation to the membrane. Primers are 5' biotin modified which allows detection of hybridized PCR products using streptavidin-peroxidase and a chemiluminescent substrate via photosensitive film. With low setup and consumable costs, this technique is inexpensive (approximately US$2 per sample), high throughput (multiple membranes can be processed simultaneously) and has a short turnaround time (approximately 10 hours). 
The technique can be utilized in a number of ways. Multiple probes can be designed to detect sequence variation within a single amplified product, or multiple products can be amplified
simultaneously, with one (or more) probes used for subsequent detection. A combination of both approaches can also be used within a single assay. The ability to include multiple probes for a single target sequence makes the assay highly specific.
Published applications of mPCR/RLB include detection of antibiotic resistance genes1,2, typing of methicillin-resistant Staphylococcus aureus3-5 and Salmonella sp6, molecular serotyping of Streptococcus pneumoniae7,8, Streptococcus agalactiae9 and enteroviruses10,11, identification of Mycobacterium sp12, detection of genital13-15 and respiratory tract16 and other17 pathogens and detection and identification of mollicutes18. However, the versatility of the technique means the applications are virtually limitless and not restricted to molecular analysis of micro-organisms.
The five steps in mPCR/RLB are a) Primer and Probe design, b) DNA extraction and PCR amplification c) Preparation of the membrane, d) Hybridization and detection, and e) Regeneration of the Membrane.

Multiplex PCR and Reverse Line Blot Hybridization Assay mPCR/RLB

An inexpensive, high throughput method for simultaneous detection of up to 43 molecular targets is described. Applications of mPCR/RLB include microbial typing and detection of multiple pathogens from clinical samples.

多重PCR和反向线杂交检测（MPCR / RLB）

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

一个定量测定胰岛素表达细胞集落形成先觉

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

使用机器人在油脂中间相高通量膜蛋白的结晶

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

一种混合DNA提取方法细菌群落的定性和定量评估，从家禽生产样品

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

We provide a standardized protocol for the use of gene set enrichment analysis of transcriptomic data to identify an ideal mouse model for translational research. 
This protocol can be used with DNA microarray and RNA sequencing data and can further be extended to other omics data if data are available.

一个协议来使用基因集富集分析以确定合适的动物模型转化型研究

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison ...

Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are highly efficient at virus transmission plays a key role in virus epidemics in fields. Studying virus-insect vector interactions can advance our understanding of virus transmission and epidemics with the aim of designing novel strategies to control plant viruses and their vector insects. Immunofluorescence labeling has been widely used to analyze interactions between pathogens and hosts and is used here in the white-backed planthopper (WBPH, Sogatella furcifera), which efficiently transmits the southern rice black streaked dwarf virus (SRBSDV, genus Fijivirus, family Reoviridae), to locate the virions and insect proteins in the midgut epithelial cells. Using laser scanning confocal microscopy, we studied the morphological characteristics of midgut epithelial cells, cellular localization of insect proteins, and the colocalization of virions and an insect protein. This protocol can be used to study virus activities in insects, functions of insect proteins, and interactions between virus and vector insect.

This protocol for immunofluorescent labeling of both plant virus proteins and vector insect proteins in excised insect guts can be used to study interactions among virus and vector insects, insect protein functions and molecular mechanisms underlying virus transmission.

半翅目肠道中植物病毒和昆虫载体蛋白的免疫荧光标记

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are ...

Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System

Adeno-associated viruses (AAV) are promising vectors for gene therapy applications. Here, the AAV2 vector is produced by co-culture of Spodoptera frugiperda (Sf9) cells with Sf9 cells infected with baculovirus (BV)-AAV2-GFP (or therapeutic gene) and BV-AAV2-rep-cap in serum-free suspension culture. Cells are cultured in a flask in an orbital shaker or Wave bioreactor. To release the AAV particles, producer cells are lysed in buffer containing detergent, which is subsequently clarified by low-speed centrifugation and filtration. AAV particles are purified from the cell lysate using AVB Sepharose column chromatography, which binds AAV particles. Bound particles are washed with PBS to remove contaminants and eluted from the resin using sodium citrate buffer at pH 3.0. The acidic eluate is neutralized with alkaline Tris-HCl buffer (pH 8.0), diluted with phosphate-buffered saline (PBS), and further concentrated with tangential flow filtration (TFF). The protocol describes small-scale pre-clinical vector production compatible with scale-up to large-scale clinical-grade AAV manufacturing for human gene therapy applications.

Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System

In this protocol, AAV2 vector is produced by co-culturing Spodoptera frugiperda (Sf9) insect cells with baculovirus (BV)-AAV2-green fluorescent protein (GFP) or therapeutic gene and BV-AAV2-rep-cap infected Sf9 cells in suspension culture. AAV particles are released from the cells using detergent, clarified, purified by affinity column chromatography, and concentrated by tangential flow filtration.