这项工作得到了退伍军人事务优异奖 (D.A.H.) 和国家卫生补助金 R01 AI 034431 (D.A.H.) 的支持。

注意:钩端螺旋体病株) 的致病菌株必须在生物安全2级 (BSL-2) 控制程序下处理。必须佩戴适当的个人防护装备 (PPE)。第二类生物安全柜必须用于致病性钩端螺旋体的所有操纵。
1. 创建转变种库15
<ol><li>
通过共轭 (图 1) 将转转换成钩端螺旋体许可证.
	<ol>
<li>将致病性钩端螺旋体的指数相培养量与 107细胞相对应, 注入10毫升的 Ellinghausen----约翰逊-哈里斯介质 (EMJH)18,19。孵育在30° c 与 150 rpm 晃动, 直到密度达到 2-8 x 108细胞/毫升。 注意: 致病钩的加倍时间是12到 24 h, 这取决于菌株。</li>
		<li>接种50µL 的供体大肠杆菌菌株β216320携带动员转质粒 (pCjTKS2)16到5毫升 Luria 肉汤 (LB) 补充0.3 毫米 26-diaminopimelicacid (磷酸二铵), 50 µg/毫升卡那霉素 (公里) 和50µg/毫升的霉 (Spc) 和地方过夜在一个37° c 孵化器在 255 rpm。</li>
		<li>将60µL 的大肠杆菌细胞接种到3毫升的 EMJH, 辅以0.3 毫米的磷酸二铵 (EMJH + 磷酸二铵)。孵育在37° c 在 255 rpm 鼓动为 3-4 h 由 OD600nm≈0.3。</li>
		<li>要装配过滤单元 (图 1), 将底座放在125毫升侧臂锥形烧瓶上, 将醋酸纤维素过滤器 (孔径 0.1 mm; 直径 25 mm) 放置在底座上, 然后将漏斗夹在底座上。将过滤装置连接到真空系统。</li>
		<li>添加5毫升的钩端螺旋体。培养和0.5 毫升的大肠杆菌文化进入漏斗。通过过滤器将液体真空。</li>
		<li>将过滤器与细菌表面上的 EMJH + DAPplate。在30° c 的条件下, 在夜间孵化。</li>
		<li>将过滤器放入15毫升管中, 其中含有1毫升的 EMJH 和漩涡, 十年代将细菌释放到介质中。使用 10-15 1 毫米无菌玻璃珠或无菌一次性吊具, 将悬浮液的200µL 分布在 5 EMJH platescontaining 50 µg/毫升上。用膜把盘子包起来, 在30° c 的时候把它们倒挂在3至4周, 直到菌落可见。</li>
		<li>转移殖民地单独地入3毫升 EMJH 包含50µg/毫升公里 (EMJH + 公里) 在30° c 之下在 150 rpm 鼓动7到10天直到文化到达密度≈ 108/ml。 注: 培养物可贮存在-80 ° c 或液氮 (含4% 甘油)。</li>
	</ol>
</li>
	<li>
通过嵌套 PCR 识别转插入站点 (图 2)
	<ol>
<li>溶解50µL 的每转突变体 PCR 管由孵育在95° c 为 15 min。 注意: dna 可以被纯化, 使用 dna 提取试剂盒。</li>
		<li>根据表 1, 用 Deg1 和 Tnk1 底漆 (表 3) 准备 PCR 混合。转移23.7 µL 的混合物到每个 PCR 管, 并增加1.3 µL 的裂解细胞。运行程序:95 5 分钟;40周期:95 ° c 为十五年代, 40 ° c 为 1 min, 72 ° c 为 2 min;72° c 为 10 min。</li>
		<li>根据表 2, 使 PCR 与标记和 TnkN1 底漆 (表 3) 混合。将24.2 µL 的混合物转移到每个 pcr 管中, 并添加0.8 µL pcr #1 反应。运行程序:95 ° c 为5分钟;35周期:95 ° c 为十五年代, 55 ° c 为三十年代, 72 ° c 为 2 min;72° c 为 10 min。</li>
		<li>在10-15 伏/cm (图 2B) 的1% 琼脂糖凝胶上运行3µL 的 PCR 产品。</li>
		<li>用 pcr 纯化试剂盒提纯阳性样品的 pcr 产物。洗 dna 与试剂盒所允许的最低体积, 以最大限度地提高 dna 的浓度恢复。</li>
		<li>使用 TnkN1 底漆 (表 3) 发送用于桑格测序的纯化 PCR 产品。</li>
		<li>通过 BLASTN 分析 (http://blast.ncbi.nlm.nih.gov/) 或使用 SpiroScope 数据库 (http://www.genoscope.cns.fr/agc/mage)21, 通过将结果序列与父级应变的基因组序列进行比较来识别插入点。</li>
		<li>用引物退火法对侧翼宿主序列进行转的插入点确认。 注意: 转通过≈ 2 kb 增加了野生类型序列的大小。</li>
	</ol>
</li>
</ol>2. 动物实验 (图 3)
<ol><li>
钩端螺旋体变种的区域性
	<ol>
<li>分别生长在10毫升 EMJH + 公里在30° c 在 150 rpm 鼓动到密度 107-108钩/毫升的转突变体。</li>
		<li>用暗场显微镜和 Petroff-豪塞尔计数器或米勒23所描述的钩计数。</li>
		<li>将 EMJH 中的每个区域性稀释为相同的密度, 例如 106单元格/mL。</li>
		<li>要装配输入池, 将稀释的区域性混合在一起。 注意: 在输入池中加入控件, 添加已知的适应缺陷的变种, 如loa2217、24和不变的适应性的变种, 如ligB17,25,分别.突变体的池可以在-80 ° c 或液氮中储存4% 的甘油。</li>
	</ol>
</li>
	<li>
挑战 注: 这里描述的方法由退伍军人事务大洛杉矶机构动物保护和使用委员会 (协议 #09018-14) 批准。<ol>
<li>注射腹腔1毫升的输入池的每一个动物与胰岛素注射器 U-100 与 26G x ½ "针。 注意: 在试验中, 8 只动物被挑战1毫升的输入池, 即 106细菌总数17。这种感染可以在安乐死前4天进行。</li>
		<li>收集10毫升的输入池, 并旋转20分钟在 3220 x g. 小心清除上清, 而不干扰颗粒。储存细胞颗粒在80˚C 直到使用 (步骤 3.1.3)。</li>
		<li>每天监视动物, 直到它们在预定终点终止。每天称仓鼠, 寻找终点标准: 食欲不振、步态或呼吸困难、虚脱、皱毛或 > 10% 的最大重量损失。</li>
	</ol>
</li>
	<li>
体外实验
	<ol>
<li>在挑战日, 接种3瓶25毫升的 EMJH + 公里与5毫升的输入池。增长文化在30° c 在 150 rpm 鼓动之下。</li>
		<li>使用节2.1.2 的方法之一计算钩的每日计数。当密度达到≈ 1 x 108/毫升, 旋转下来的每一个暂停20分钟, 在 3220 x g。</li>
		<li>将细胞小球储存在-80 ° c 直到使用。</li>
	</ol>
</li>
	<li>
组织的收获和储存
	<ol>
<li>用异氟醚吸入安乐, 其次是双侧胸切口26。</li>
		<li>立即收集1至2毫升的血液心脏穿刺与3毫升注射器和 25G x 5/8 "针。将血液转移到含有 EDTA 的试管中。混合由反演, 5 到6次。</li>
		<li>收集一个肾脏和⅓到½腹正中叶的肝 cryotubes。</li>
		<li>在-80 ° c 贮存组织直至使用。</li>
	</ol>
</li>
</ol>3. 构建用于高通量排序的基因组库 (图 4)
<ol><li>
DNA 提取
	<ol>
<li>从血液中提取 DNA<ol>
<li>将100µL 的血液从 EDTA 管转移到离心管。</li>
			<li>使用 dna 提取试剂盒净化 dna。按照制造商的说明进行操作。</li>
		</ol>
</li>
		<li>从组织中提取 DNA<ol>
<li>使用手术刀和剪刀, 骰子每个器官50至80毫克的小块 (1 毫米 x 1 毫米), 并转移到一个无菌的螺旋帽干管。用精确的天平测量组织的重量。</li>
			<li>在试管中加入500µL 的无菌 PBS。</li>
			<li>质样品, 使用干扰1分钟, 在5运动每秒。</li>
			<li>使用以下等式计算与25毫克组织相对应的体积: <img alt="Equation 1" src="/files/ftp_upload/56442/56442eq1.jpg">
</li>
			<li>将计算出的体积转移到 DNA 提取试剂盒中。按照制造商的指示进行 DNA 纯化。</li>
		</ol>
</li>
		<li>从输入池和体外培养中提取 DNA。<ol>
<li>常温解冻细菌颗粒5-10 分钟。</li>
			<li>按照随试剂盒的指示进行 DNA 提取。</li>
		</ol>
</li>
		<li>将 DNA 储存在-80 ° c 直到使用。</li>
	</ol>
</li>
	<li>
剪切 DNA (图 5)
	<ol>
<li>转移50µL 的提取 DNA 到1.5 毫升离心管。</li>
		<li>把管子放入 sonicator 杯垫的架子上, 装满冷水 (4 ° c)。</li>
		<li>运行 sonicator 3 分钟, 在80% 强度与十年代脉冲和 5 s 关闭脉冲。 注意: 戴上耳罩或耳塞以保护听觉。</li>
		<li>在2% 琼脂糖凝胶上运行2.5 µL 的剪切 dna, 以确认大部分的 dna 是 < 600 bp 的大小。</li>
	</ol>
</li>
	<li>
加法 C 尾巴
	<ol>
<li>用小体积分光光度计测量 DNA 浓度。</li>
		<li>使用以下等式计算与 500 ng DNA 对应的体积: <img alt="Equation 2" src="/files/ftp_upload/56442/56442eq2.jpg">
</li>
		<li>准备尾矿反应 (表 4)。在37° c 下孵育1小时;在75° c 下钝化20分钟。 注: 对于需要调整到大于14.5 µL 的体积的样品, 将反应的最终体积增加到40µL, 并相应地扩大剩余的元件。</li>
		<li>用 PCR 纯化试剂盒清洗样品。用12µL 洗脱缓冲液洗 DNA。</li>
	</ol>
</li>
	<li>
巢式 PCR
	<ol>
<li>PCR #1<ol>
<li>根据表 3和5准备 PCR 混合。转移22µL 的图书馆混合到每个 PCR 管和添加3µL 纯化 DNA。与控制组合进行类似的操作。 注: 底漆 TnkN317特定于转, 而底漆 olj3766是特定于 C 尾的。控制组合缺乏 TnkN3 底漆, 专门针对转。</li>
			<li>运行以下程序:95 ° c 为2分钟;24周期:95 ° c 为三十年代, 60 ° c 为三十年代, 72 ° c 为 2 min;72° c 为 2 min。</li>
		</ol>
</li>
		<li>PCR #2。<ol>
<li>根据表 3和6准备第二个 PCR 混合。将49µL 的文库混合到每个 pcr 管中, 并添加1µL 的 pcr #1 文库反应。将控制组合的24.5 µL 转移到每个 pcr 管上, 并添加0.5 µL pcr #1 控制反应。 注: 底漆 pMargent2 特定于转, 而 IP 引物6包含六基对条码和由下一代测序平台识别的特定序列。</li>
			<li>运行以下程序:95 ° c 为2分钟;18周期:95 ° c 为三十年代, 60 ° c 为三十年代, 72 ° c 为 2 min;72° c 为 2 min。</li>
		</ol>
</li>
		<li>在2% 琼脂糖凝胶上运行3µL。图书馆应该显示一个涂片以信号的多数在200到 600 bp (图 6) 和没有放大为控制反应。</li>
	</ol>
</li>
	<li>
PCR 产物的纯化。 注意: 按照制造商的说明, 用 PCR 纯化试剂盒清洁基因组库。用30µL 洗脱缓冲液洗 DNA。</li>
	<li>用荧光测量 DNA 浓度。平均2到3读。</li>
	<li>使用下面的等式计算每个库的容量, 相当于15或 20 ng: <img alt="Equation 3" src="/files/ftp_upload/56442/56442eq3.jpg">
</li>
	<li>
根据以前的计算将所有库混合在一起.用以下网站的公式确定 DNA 的摩尔浓度: http://www.molbiol.edu.ru/eng/scripts/01_07.html 注意: DNA 浓度和体积要求取决于测序平台。</li>
</ol>4. 高通量测序和数据分析
<ol><li>序
	<ol>
<li>序列库作为 64 bp 单读取使用自定义测序底漆 pMargent3 和标准的商业测序底漆。测序平台将为您提供 FastQ 的文件和所有的顺序读取。</li>
	</ol></li>
	<li>分析与银河软件
	<ol><li>下载基因组序列文件<ol>
<li>在 SpiroScope 数据库主页 (参见步骤1.2.7 为链接), 选择用于实验的有机体并且点击 "装载入基因组浏览器"。</li>
			<li>在工具栏 (位于主页顶部附近), 选择 "搜索/导出 > 下载数据", 在 "序列 (fasta)" 行中, 单击 "基因组" 下载序列。</li>
			<li>打开文件与记事本 (PC) 或 "(Mac) 和重命名的染色体 (例如" 上帝 ")。保持 fasta 格式。</li>
			<li>按照相同的步骤下载染色体 II. 序列通过复制和粘贴将两个染色体序列组合成一个 .txt 文件。 注意: 还可以从 NCBI 网站 (https://www.ncbi.nlm.nih.gov) 下载染色体序列, 并将其合并为单个 .txt 文件。</li>
		</ol></li>
		<li>将文件上载到 Galaxy 服务器 注: Galaxy 是一个开源的网络平台, 用于管理数据密集型的生物信息学工作流27,28,29,30 , 可以在 https://usegalaxy.org/访问。<ol>
<li>在 "工具" 菜单中, 选择 "从计算机获取数据 > 上传文件"。将由测序平台生成的. fastq 文件拖放到窗口中, 然后单击 "开始"。</li>
			<li>按照相同的步骤上载钩端螺旋体基因组序列. txt 文件。</li>
		</ol></li>
		<li>新郎阅读<ol>
<li>从 "工具" 菜单中选择 "NGS: QC 和操作 > FASTQ 美容师"。</li>
			<li>在 "文件到新郎" 旁边, 选择 "步骤4.2.2 中上载的库"。在输入 FASTQ 质量分数类型中, 选择适当的测序系统。对于高级选项, 请保留 "隐藏高级 Office 选定"。</li>
			<li>单击 "执行"。</li>
		</ol></li>
		<li>删除序列工件<ol>
<li>选择 "NGS: QC 和操作 > 删除序列工件"。在要筛选的库旁边, 选择在步骤4.2.3 中生成的整理文件。单击 "执行"。</li>
		</ol></li>
		<li>删除 C 尾序列 注意: 重复这些步骤一两次, 以确保所有的 C 尾被删除。<ol>
<li>选择 "NGS: QC 和操作 > 剪辑适配器序列"。</li>
			<li>选择或输入以下内容: 库到剪辑: 选择在步骤4.2.4 中生成的文件。 最小序列长度:15 来源: 输入自定义序列 输入自定义剪切序列: CCCCCCC 输入非零值以保持适配器序列和跟随它的 x 基地: 0 丢弃具有未知 (N) 基的序列: 是 输出选项: 输出剪切和 non-clipped 序列</li>
			<li>单击 "执行"。</li>
		</ol></li>
		<li>删除适配器序列<ol>
<li>选择 "NGS: QC 和操作 > 剪辑适配器序列"。</li>
			<li>选择或输入以下内容:</li>
			<li>库到剪辑: 选择在步骤4.2.5 中生成的文件。</li>
			<li>最小序列长度:15</li>
			<li>来源: 输入自定义序列</li>
			<li>输入自定义剪切序列: CGTATGCCGTCTTCTGCTTG</li>
			<li>输入非零值以保持适配器序列和跟随它的 x 基地: 0</li>
			<li>丢弃具有未知 (N) 基的序列: 是</li>
			<li>输出选项: 输出剪切和 non-clipped 序列</li>
			<li>单击 "执行"。</li>
		</ol></li>
		<li>基于其质量的筛选器读取<ol>
<li>选择 "NGS: QC 和操作 > 按质量过滤"。 注意: 此工具根据质量分数选择读取。</li>
			<li>选择以下内容: 要筛选的库: 选择在步骤4.2.6 中生成的文件。 质量截止值:20 必须有质量等于/大于截止值的序列的基数百分比:95</li>
			<li>单击 "执行"。 注: 与这些设置, 读短于20核苷酸或以质量比分20或较少为95% 的周期被摒弃。调整你的实验的严谨设置。</li>
		</ol></li>
		<li>映射读取32<ol>
<li>选择 "NGS: 映射 > Bowtie2"32。</li>
			<li>在主窗口的字段中选择以下内容: 这是单个的还是配对的库: 单 FASTQ 文件: 从步骤4.2.7 中选择过滤质量的库。 写入齐 (以 fastq 格式) 读取到单独的文件 (s): 否 写对齐 (以 fastq 格式) 读取到单独的文件: 否 您是否会从历史记录中选择一个参考基因组或使用内置索引？: 使用历史记录和生成索引中的基因组 选择参考基因组: 选择在步骤4.2.2 中上传的钩端螺旋体 genome.txt 文件。 设置读取组信息: 不设置 选择分析模式: 1: 仅限默认设置 是否要使用预设？: 否, 仅使用默认值 将 bowtie2 映射统计信息保存到历史记录中: 否 作业资源参数: 使用默认作业资源参数</li>
			<li>单击 "执行" 以将读取与基因组对齐。</li>
		</ol></li>
		<li>转换文件<ol>
<li>选择 "NGS: SAMtools > BAM-to-SAM 转换 BAM 为 SAM"。</li>
			<li>选择以下内容: 要转换的 BAM 文件: 从步骤4.2.8 中选择映射库。 标题选项: 在 SAM 输出中包含页眉 (-h)</li>
			<li>单击 "执行"。</li>
		</ol></li>
		<li>转换文件<ol>
<li>选择 "NGS: SAMtools > 转换 SAM 为间隔"。</li>
			<li>选择以下内容: 选择要转换的数据集: 选择在步骤4.2.9 中生成的 SAM 映射库文件。 全部打印: 是</li>
			<li>单击 "执行"。</li>
		</ol></li>
		<li>排序读取<ol>
<li>选择 "按升序或降序对数据进行筛选和排序 > 排序"。</li>
			<li>选择以下内容: 排序数据集: 选择在步骤4.2.10 中生成的间隔文件。 上一栏: 2 具有风味: 数字顺序 所有内容: 升序排列</li>
			<li>单击 "执行"。</li>
		</ol></li>
		<li>选择读取匹配的染色体 I<ol>
<li>选择 "筛选并排序 > 选择与表达式匹配的行"。</li>
			<li>选择以下内容: 选择行: 选择在步骤4.2.11 中生成的排序读取的文件。 : 匹配 模式: 输入在步骤4.2.1.3 中确定的 I 染色体的名称 (例如, "上帝")。</li>
			<li>单击 "执行"。</li>
		</ol></li>
		<li>选择读取匹配的染色体 II 继续下面的步骤4.2.12。</li>
		<li>组读取根据插入点的染色体 I<ol>
<li>选择 "联接、减去和分组 > 按列对数据进行分组, 并对其他列执行聚合操作"。</li>
			<li>选择以下内容: 选择数据: 从步骤4.2.12 中选择结果文件。 按列分组: 2 分组时忽略大小写: 否 忽略以下列字符开头的行: Ø 操作 > + 插入操作 类型: 计数 上一栏: 2 舍入结果为最接近的整数: 否</li>
			<li>单击 "执行"。</li>
		</ol></li>
		<li>根据第二号染色体的插入部位进行组读 继续下面的步骤4.2.14。</li>
		<li>在 I 染色体上排序插入点<ol>
<li>选择 "按升序或降序对数据进行筛选和排序 > 排序"。</li>
			<li>选择以下内容: 排序数据集: 从步骤4.2.14 中选择文件。 上一栏: 1 具有风味: 数字顺序 所有内容: 升序排列</li>
			<li>单击 "执行"。</li>
		</ol></li>
		<li>在第二号染色体上排序插入点 继续下面的步骤4.2.16。</li>
	</ol></li>
	<li>统计分析
	<ol>
<li>通过从步骤4.2.16 复制和粘贴两列, 将 Galaxy 数据传输到电子表格文件。和4.2.17。到 Excel 文件中。 注意: 第一列是转插入站点的核苷酸坐标, 第二列是每个插入站点上的读取数。</li>
		<li>使用表中提供的核苷酸坐标来识别窝藏转的基因。 注: 例如, 在 i 染色体的核苷酸 #30718 中插入的转位于LIC10024基因中, 它在染色体 i 中跨越核苷酸29263-31539。</li>
		<li>计算每个组织中的每个突变体的相对频率 (F), 然后在下面的等式中输入池: <img alt="Equation 4" src="/files/ftp_upload/56442/56442eq4.jpg">
</li>
		<li>使用下面的等式计算每个突变体和组织的输出/输入比率 (R): <img alt="Equation 5" src="/files/ftp_upload/56442/56442eq5.jpg">
</li>
		<li>魏氏签名测试<ol>
<li>通过将每种动物组织中的中位比率设置为 1.0, 使所有的输出/输入比率正常化。1.0 的比率是中性的, > 1.0 是不利的, < 1.0 是有利的33。</li>
			<li>使用魏氏秩测试与 P 值 < 0.05 进行比较, 将输出/输入比率与 1.0 (中性适应) 进行对比。</li>
		</ol>
</li>
	</ol></li>
</ol>

<ol><li>Murray, G. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide+transposon+mutagenesis+in+pathogenic+Leptospira+species%5BTitle%5D)+AND+%22Infect+Immun%22%5BJournal%5D)">Genome-wide transposon mutagenesis in pathogenic Leptospira species.</a> Infect Immun. 77 (2), 810-816 (2009).</li>
<li>Adler, B., Lo, M., Seemann, T., Murray, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pathogenesis+of+leptospirosis%3A+the+influence+of+genomics%5BTitle%5D)+AND+%22Vet+Microbiol%22%5BJournal%5D)">Pathogenesis of leptospirosis: the influence of genomics.</a> Vet Microbiol. 153 (1-2), 73-81 (2011).</li>
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<li>van Opijnen, T., Bodi, K. L., Camilli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tn-seq%3A+high-throughput+parallel+sequencing+for+fitness+and+genetic+interaction+studies+in+microorganisms%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms.</a> Nat Methods. 6 (10), 767-772 (2009).</li>
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<li>Johnson, R. C., Harris, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differentiation+of+pathogenic+and+saprophytic+letospires.+I.+Growth+at+low+temperatures%5BTitle%5D)+AND+%22J+Bacteriol%22%5BJournal%5D)">Differentiation of pathogenic and saprophytic letospires. I. Growth at low temperatures.</a> J Bacteriol. 94 (1), 27-31 (1967).</li>
<li>Demarre, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+family+of+mobilizable+suicide+plasmids+based+on+broad+host+range+R388+plasmid+%28IncW%29+and+RP4+plasmid+%28IncPa%29+conjugative+machineries+and+their+cognate+Escherichia+coli+host+strains%5BTitle%5D)+AND+%22Res+Microbiol%22%5BJournal%5D)">A new family of mobilizable suicide plasmids based on broad host range R388 plasmid (IncW) and RP4 plasmid (IncPa) conjugative machineries and their cognate Escherichia coli host strains.</a> Res Microbiol. 156 (2), 245-255 (2005).</li>
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<li>Miller, J. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aMiller%2C+J+%22Spirochetes+in+body+fluids+and+tissues+manual+of+investigative+methods%22">Spirochetes in body fluids and tissues manual of investigative methods</a>. , 22-23 (1971).</li>
<li>Ristow, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+OmpA-like+protein+Loa22+is+essential+for+leptospiral+virulence%5BTitle%5D)+AND+%22PLoS+Pathog%22%5BJournal%5D)">The OmpA-like protein Loa22 is essential for leptospiral virulence.</a> PLoS Pathog. 3 (7), (2007).</li>
<li>Croda, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+mutagenesis+in+pathogenic+Leptospira+species%3A+disruption+of+the+LigB+gene+does+not+affect+virulence+in+animal+models+of+leptospirosis%5BTitle%5D)+AND+%22Infect+Immun%22%5BJournal%5D)">Targeted mutagenesis in pathogenic Leptospira species: disruption of the LigB gene does not affect virulence in animal models of leptospirosis.</a> Infect Immun. 76 (12), 5826-5833 (2008).</li>
<li>Haake, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hamster+model+of+leptospirosis%5BTitle%5D)+AND+%22Curr+Protoc+Microbiol%22%5BJournal%5D)">Hamster model of leptospirosis.</a> Curr Protoc Microbiol. 12, 2(2006).</li>
<li>Giardine, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Galaxy%3A+a+platform+for+interactive+large-scale+genome+analysis%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">Galaxy: a platform for interactive large-scale genome analysis.</a> Genome Res. 15 (10), 1451-1455 (2005).</li>
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<li>McCoy, K. M., Antonio, M. L., van Opijnen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MAGenTA%3B+a+Galaxy+implemented+tool+for+complete+Tn-Seq+analysis+and+data+visualization%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">MAGenTA; a Galaxy implemented tool for complete Tn-Seq analysis and data visualization.</a> Bioinformatics. , 1367(2017).</li>
<li>Langmead, B., Trapnell, C., Pop, M., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultrafast+and+memory-efficient+alignment+of+short+DNA+sequences+to+the+human+genome%5BTitle%5D)+AND+%22Genome+Biol%22%5BJournal%5D)">Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.</a> Genome Biol. 10 (3), R25(2009).</li>
<li>Kamp, H. D., Patimalla-Dipali, B., Lazinski, D. W., Wallace-Gadsden, F., Camilli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+fitness+landscapes+of+Vibrio+cholerae+at+important+stages+of+its+life+cycle%5BTitle%5D)+AND+%22PLoS+Pathog%22%5BJournal%5D)">Gene fitness landscapes of Vibrio cholerae at important stages of its life cycle.</a> PLoS Pathog. 9 (12), e1003800(2013).</li>
<li>Poggi, D., Oliveira de Giuseppe, P., Picardeau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antibiotic+resistance+markers+for+genetic+manipulations+of+Leptospira+spp%5BTitle%5D)+AND+%22Appl+Environ+Microbiol%22%5BJournal%5D)">Antibiotic resistance markers for genetic manipulations of Leptospira spp.</a> Appl Environ Microbiol. 76 (14), 4882-4885 (2010).</li>
<li>van Opijnen, T., Camilli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transposon+insertion+sequencing%3A+a+new+tool+for+systems-level+analysis+of+microorganisms%5BTitle%5D)+AND+%22Nat+Rev+Microbiol%22%5BJournal%5D)">Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms.</a> Nat Rev Microbiol. 11 (7), 435-442 (2013).</li>
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<li>Koskiniemi, S., Sun, S., Berg, O. G., Andersson, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selection-driven+gene+loss+in+bacteria%5BTitle%5D)+AND+%22PLoS+Genet%22%5BJournal%5D)">Selection-driven gene loss in bacteria.</a> PLoS Genet. 8 (6), e1002787(2012).</li>
<li>Troy, E. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+Tn-seq+analysis+of+carbohydrate+utilization+and+vertebrate+infectivity+of+Borrelia+burgdorferi%5BTitle%5D)+AND+%22Mol+Microbiol%22%5BJournal%5D)">Global Tn-seq analysis of carbohydrate utilization and vertebrate infectivity of Borrelia burgdorferi.</a> Mol Microbiol. 101 (6), 1003-1023 (2016).</li>
<li>Ramsey, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+high-throughput+genetic+screen+identifies+previously+uncharacterized+Borrelia+burgdorferi+genes+important+for+resistance+against+reactive+oxygen+and+nitrogen+species%5BTitle%5D)+AND+%22PLoS+Negl+Trop+Dis%22%5BJournal%5D)">A high-throughput genetic screen identifies previously uncharacterized Borrelia burgdorferi genes important for resistance against reactive oxygen and nitrogen species.</a> PLoS Negl Trop Dis. 13 (2), e1006225(2017).</li>
</ol>

作者没有什么可透露的。

虽然我们的试验结果为仓鼠挑战腹腔与 42 L. 螺旋体突变体提出了17, 我们预计, 更大的突变池可以筛选的 Tn-Seq。由于 transconjugants 的频率较低 (100-200 transconjugants/交配), 因此需要数交配来产生足够数量的突变体进行大规模的 Tn-Seq 实验。在液态文化中维持大量的变种人, 这就带来了必须解决的后勤挑战。文化可以在深96好的板块中孵化。在 420 nm 的分光光度计上, 可以通过光学...

由于可用的遗传工具数量有限, 某些细菌 (如钩端螺旋体) 的毒力基因鉴定很困难。一个常用的方法是创建一个突变体的随机转诱变后, 在每个突变体的插入点的识别和毒性测试的个别转突变动物模型。这种方法费时费钱, 需要大量的动物。
当随机诱变首次为病原体钩端螺旋体螺旋体时, 通过在动物模型1中测试个体突变株来鉴定致病基因。突变体的选择依据的标准, 如他们的潜在作用, 信号或运动或他们预测的外膜或表面位置。由于大多数端基因编码未知函数的假设蛋白质 2, 选择基于这些标准的突变体限制了发现新的端毒力基因的能力。
最近, L. 螺旋体转突变体的池在仓鼠和鼠模型3中被筛选出具有传染性。每只动物都被一个多达10变种人的水池所挑战。从血液和肾脏获得的培养物 PCR 检测出突变体的传染性。pcr 测试是费力的, 因为它需要一个单独的 pcr 反应为每个突变池。由于不同文化中每个突变体的频率没有被量化, 这种方法偏向于识别高度减毒突变体。
我们描述一个转测序 (Tn-Seq) 技术, 作为一种策略, 更有效地筛选毒力基因。Tn-seq 是由转诱变的变种人库的创建, 其次是大规模的并行排序4,5,6。简单地, 转突变体被汇集, 接种入动物, 并且以后从不同的器官恢复了 (输出水池)。从输出池的 DNA 提取和消化限制性酶或被超声。针对转插入点的交界处进行两轮 PCR。此步骤允许添加用于排序的必要适配器。通过高通量测序分析产生的 PCR 产物, 以确定池中每个突变体的转插入点及其相对丰度, 与突变体池的初始组成进行比较。
这种方法的主要优点是能够同时筛选大量的突变体和少量的动物。Tn-Seq 不需要事先知道的转插入点, 这增加了发现新的钩端螺旋体特定基因的机会, 涉及的毒力较少的时间和更高的效率。由于端在组织中的负担是相对较高的啮齿动物模型易受致命感染 (通常 104到 108细菌/g 的组织)7,8,9以及在水库主机10,11, 可以直接分析组织, 而无需培养, 减少由于体外生长而引起的偏见。
在迄今所描述的大多数细菌病原体的 Tn-Seq 研究中, 插入诱变的高频率允许感染的大池, 其中包含了变种, 在每个基因中都有多个紧密间隔的转插入物4 ,12,13,14。还开发了一种细菌, 其诱变频率远低于6。使用钩端螺旋体, 转突变体的库可以通过结合 Slamti et al15所描述的共轭动员质粒引入转的方法生成。然而, 转突变的频率在L. 螺旋体是低的。当在共轭质粒上引入Himar1转时, 报告的 transconjugant 频率仅为每个接收单元的 8.5 x 10-8 , 且其 Lai 应变为L. 螺旋体16 , 很可能是同样贫困与大多数其他菌株的L. 螺旋体。这里描述的协议部分基于为螺旋体螺旋体开发的, 其中转插入诱变的频率也是低的6。
对于我们的试验与协议17, 我们进行了转诱变与L. 螺旋体螺旋体 Manilae 菌株 L495 由于其他组的成功隔离转插入突变体在应变沿其低LD50 (致死剂量) 为毒力1。我们筛选了42变种人的 Tn, 并确定了几个突变候选者的毒力缺陷, 包括两个插入在候选腺苷酸苷基因。对仓鼠的两个突变体进行的单独检测证实它们的毒力不足17。

<table><tbody><tr><td>Kanamycin sulfate from &lt;em&gt;Streptomyces kanamyceticus&lt;/em&gt;</td><td>Sigma-Aldrich</td><td>K4000</td><td></td></tr><tr><td>2,6-diaminopimelic acid</td><td>Sigma-Aldrich</td><td>D1377</td><td></td></tr><tr><td>Spectinomycin dihydrochloride pentahydrate</td><td>Sigma-Aldrich</td><td>S4014</td><td></td></tr><tr><td>Axio Lab A1 microscope with a darkfieldcondenser</td><td>Zeiss</td><td>490950-001-000</td><td></td></tr><tr><td>DNeasy blood and tissue kit</td><td>Qiagen</td><td>69504/69506</td><td></td></tr><tr><td>MinElute PCR Purification</td><td>Qiagen</td><td>28004/28006</td><td></td></tr><tr><td>QIAquick PCR purification kit</td><td>Qiagen</td><td>28104/28106</td><td></td></tr><tr><td>Model 505 Sonic Dismembrator</td><td>Fisher Scientific</td><td>FB-505</td><td></td></tr><tr><td>2.5&quot; Cup horn</td><td>Fisher Scientific</td><td>FB-4625</td><td></td></tr><tr><td>Bead Ruptor 24</td><td>Omni International</td><td>19-010</td><td>Step 3.1.2.4</td></tr><tr><td>Terminal deoxynucleotidyl transferase</td><td>Promega</td><td>M828C</td><td></td></tr><tr><td>Master mix Phusion</td><td>Thermo Scientific</td><td>F531</td><td>Preparation of genomic libraries, step 3.4.</td></tr><tr><td>DreamTaq Master Mix</td><td>Thermo Scientific</td><td>K9011/K9012</td><td>Identification of the transposon insertion site, step 1.2.</td></tr><tr><td>dCTP</td><td>Thermo Scientific</td><td>R0151</td><td></td></tr><tr><td>ddCTP</td><td>Affymetrix/ USBProducts</td><td>77112</td><td></td></tr><tr><td>T100 Thermal cycler</td><td>BioRad</td><td>1861096</td><td></td></tr><tr><td>Qubit 2.0 fluorometer</td><td>Invitrogen</td><td>Q32866</td><td>step 3.6.</td></tr><tr><td>Qubit dsDNA HS assay kit</td><td>Invitrogen</td><td>Q32851/Q32854</td><td>step 3.6.</td></tr><tr><td>Qubit assay tubes</td><td>life technologies</td><td>Q32856</td><td>step 3.6.</td></tr><tr><td>PBS pH 7.2 (1X)</td><td>Gibco</td><td>20012-027 20012-050</td><td></td></tr><tr><td>Disposable scalpel No10</td><td>Feather</td><td>2975#10</td><td></td></tr><tr><td>Plastic K&lt;sub&gt;2&lt;/sub&gt; EDTA 2 ml tubes</td><td>BD vacutainer</td><td>367841</td><td></td></tr><tr><td>syringe U-100 with 26G x &amp;frac12;&amp;rdquo; needle</td><td>BD vacutainer</td><td>329652</td><td>IP challenge, step 2.2.1.</td></tr><tr><td>3 mL Luer-Lok tip syringe</td><td>BD vacutainer</td><td>309657</td><td>Cardiac puncture, step 2.4.2.</td></tr><tr><td>25G X 5/8&amp;rdquo; needle</td><td>BD vacutainer</td><td>305901</td><td>Cardiac puncture, step 2.4.2.</td></tr><tr><td>25 mm fritted glass base with stopper</td><td>EMD Millipore</td><td>XX1002502</td><td>Filtration unit system, step 1.1.7.</td></tr><tr><td>25 mm aluminum spring clamp</td><td>EMD Millipore</td><td>XX1002503</td><td>Filtration unit system, step 1.1.7.</td></tr><tr><td>15 ml borosilcate glass funnel</td><td>EMD Millipore</td><td>XX1002514</td><td>Filtration unit system, step 1.1.7.</td></tr><tr><td>125 ml side-arm Erlenmeyer flask</td><td>EMD Millipore</td><td>XX1002505</td><td>Filtration unit system, step 1.1.7.</td></tr><tr><td>Acetate-cellulose filter VVPP (pore size 0.1 mm; diameter 25 mm)</td><td>EMD Millipore</td><td>VVLP02500</td><td></td></tr></tbody></table>

high-throughput parallel sequencing to measure fitness of leptospira interrogans transposon insertion mutants during golden syrian hamster infection

在这份手稿中, 我们描述了一个转测序 (Tn) 技术, 以确定和量化的钩端螺旋体螺旋体突变在健康期间改变了金黄色的叙利亚仓鼠感染。将随机转突变与高通量测序技术结合起来。动物受到转突变体 (输入池) 的挑战, 然后在几天后采集血液和组织, 以确定和量化每个器官 (产出池) 的突变株数。将输出池与输入池进行比较, 以评估每个突变体的体内适用性。这种方法可以在数量有限的动物中筛选出一大批突变体。稍加修改, 本协议可以与任何动物模型的钩端螺旋体病, 水库宿主模型, 如老鼠和急性感染模型, 如仓鼠, 以及体外研究。Tn-Seq 提供了一个强大的工具, 筛选的变种人与在体内和体外健身缺陷。

在L. 螺旋体中创建转突变体库需要一个过滤单元, 如图 1所示。我们从每个交配中恢复了 100-200 transconjugants
在每个突变体中, 通过对随机 pcr 生成的 pcr 产物进行测序, 以转和相邻宿主序列的末端为目标, 转插入点被识别为15 (图 2A)。随机 PCR 结果的一个例子显?...

Watch this Scientific Journal Video about High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection at JoVE.c

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

我们在这里描述的技术, 结合转诱变和高通量测序, 以确定和量化转端突变体在组织后的挑战, 仓鼠。该协议可用于筛选动物生存和传播的变种人, 也可用于体外研究。

高通量并行测序在金黄叙利亚仓鼠感染期间测量钩端螺旋体螺旋体转插入突变体的适应性

In this manuscript, we describe a transposon sequencing (Tn-Seq) technique to identify and quantify Leptospira interrogans mutants altered in fitness ...

high-throughput-parallel-sequencing-to-measure-fitness-leptospira

Research

JoVE Journal

Immunology and Infection

High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

8.9K 次观看.  Veterans Affairs Greater Los Angeles Healthcare System. 我们在这里描述的技术, 结合转诱变和高通量测序, 以确定和量化转端突变体在组织后的挑战, 仓鼠。该协议可用于筛选动物生存和传播的变种人, 也可用于体外研究。

视频: High-throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants During Golden Syrian Hamster Infection

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

基于数字PCR的沙门氏菌健康高通量分析竞争指数

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...

科研

遗传学

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the strength of linkage and association mapping methods, which trace the relationship between DNA sequence variants and phenotype across recombinant progeny from matings between individuals of a species. These approaches, although powerful, are not well suited to trait differences between reproductively isolated species. Here we describe a new method for genome-wide dissection of natural trait variation that can be readily applied to incompatible species. Our strategy, RH-seq, is a genome-wide implementation of the reciprocal hemizygote test. We harnessed it to identify the genes responsible for the striking high temperature growth of the yeast Saccharomyces cerevisiae relative to its sister species S. paradoxus. RH-seq utilizes transposon mutagenesis to create a pool of reciprocal hemizygotes, which are then tracked through a high-temperature competition via high-throughput sequencing. Our RH-seq workflow as laid out here provides a rigorous, unbiased way to dissect ancient, complex traits in the budding yeast clade, with the caveat that resource-intensive deep sequencing is needed to ensure genomic coverage for genetic mapping. As sequencing costs drop, this approach holds great promise for future use across eukaryotes.

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

通过全基因组互惠性分析,对糖母菌菌种间热耐性差异的遗传图谱

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the ...

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome compared to matched normal tissue. Based on these analyses it is evident that there are many undiscovered genetic drivers of cancer1. Unfortunately these drivers are hidden within a much larger number of passenger anomalies in the genome that do not directly contribute to tumor formation. Another aspect of the cancer genome is that there is considerable genetic heterogeneity within similar tumor types. Each tumor can harbor different mutations that provide a selective advantage for tumor formation2. Performing an unbiased forward genetic screen in mice provides the tools to generate tumors and analyze their genetic composition, while reducing the background of passenger mutations. The Sleeping Beauty (SB) transposon system is one such method3. The SB system utilizes mobile vectors (transposons) that can be inserted throughout the genome by the transposase enzyme. Mutations are limited to a specific cell type through the use of a conditional transposase allele that is activated by Cre Recombinase. Many mouse lines exist that express Cre Recombinase in specific tissues. By crossing one of these lines to the conditional transposase allele (e.g. Lox-stop-Lox-SB11), the SB system is activated only in cells that express Cre Recombinase. The Cre Recombinase will excise a stop cassette that blocks expression of the transposase allele, thereby activating transposon mutagenesis within the designated cell type. An SB screen is initiated by breeding three strains of transgenic mice so that the experimental mice carry a conditional transposase allele, a concatamer of transposons, and a tissue-specific Cre Recombinase allele. These mice are allowed to age until tumors form and they become moribund. The mice are then necropsied and genomic DNA is isolated from the tumors. Next, the genomic DNA is subjected to linker-mediated-PCR (LM-PCR) that results in amplification of genomic loci containing an SB transposon. LM-PCR performed on a single tumor will result in hundreds of distinct amplicons representing the hundreds of genomic loci containing transposon insertions in a single tumor4. The transposon insertions in all tumors are analyzed and common insertion sites (CISs) are identified using an appropriate statistical method5. Genes within the CIS are highly likely to be oncogenes or tumor suppressor genes, and are considered candidate cancer genes. The advantages of using the SB system to identify candidate cancer genes are: 1) the transposon can easily be located in the genome because its sequence is known, 2) transposition can be directed to almost any cell type and 3) the transposon is capable of introducing both gain- and loss-of-function mutations6. The following protocol describes how to devise and execute a forward genetic screen using the SB transposon system to identify candidate cancer genes (Figure 1).

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

A method of identifying unknown drivers of carcinogenesis using an unbiased approach is described. The method uses the Sleeping Beauty transposon as a random mutagen directed to specific tissues. Genomic mapping of transposon insertions that drive tumor formation identifies novel oncogenes and tumor suppressor genes

鉴定<em&gt;睡美人</em&gt;转座子插入连接器介导的PCR实体瘤

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome ...

医学

Application of CRISPR Interference (CRISPRi) for Gene Silencing in Pathogenic Species of Leptospira

Leptospirosis is a global neglected zoonosis, responsible for at least 1 million cases per year and almost 60 thousand deaths. The disease is caused by pathogenic and virulent bacteria of the genus Leptospira, either by direct contact with the bacteria or indirectly by exposure to contaminated water or soil. Domestic and wild animals act as reservoir hosts of infection, shedding leptospires from colonized renal tubules of the kidney, via urine, into the environment. The generation of mutant strains of Leptospira is critical to evaluate and understand pathogenic mechanisms of infection. CRISPR interference (CRISPRi) has proven to be a straightforward, affordable, and specific tool for gene silencing in pathogenic Leptospira. Therefore, the methodological details of obtaining the plasmid constructs containing both dCas9 and guide RNA, delivery of plasmids to Leptospira by conjugation with the E. coli strain &#946;2163, and transconjugant recovery and evaluation, will be described. In addition, the recently described Hornsby-Alt-Nally (HAN) media allows for the relatively rapid isolation and selection of mutant colonies on agar plates.

Application of CRISPR Interference CRISPRi for Gene Silencing in Pathogenic Species of Leptospira

Here, the application of CRISPR interference (CRISPRi) for specific gene silencing in Leptospira species is described. Leptospira cells are transformed by conjugation with plasmids expressing dCas9 (catalytically "dead" Cas9) and a single-guide RNA (sgRNA), responsible for base pairing to the desired genomic target. Methods to validate gene silencing are presented.

CRISPR干扰（CRISPRI）在瘦素病原物种中基因沉默的应用

Leptospirosis is a global neglected zoonosis, responsible for at least 1 million cases per year and almost 60 thousand deaths. The disease is caused by ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

免疫与感染

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. The introduced genetic material may integrate into the embryonic genome and generate transgenic animals with foreign genetic information following transfer of the injected embryos to foster mothers. Following the success in mice, PN injection has been applied successfully in many other animal species. Recently, PN injection has been successfully employed to introduce reagents with gene-modifying activities, such as the CRISPR/Cas9 system, to achieve site-specific genetic modifications in several laboratory and farm animal species. In addition to mastering the special set of microinjection skills to produce genetically modified animals by PN injection, researchers must understand the reproduction physiology and behavior of the target species, because each species presents unique challenges. For example, golden Syrian hamster embryos have unique handling requirements in vitro such that PN injection techniques were not possible in this species until recent breakthroughs by our group. With our species-modified PN injection protocol, we have succeeded in producing several gene knockout (KO) and knockin (KI) hamsters, which have been used successfully to model human diseases. Here we describe the PN injection procedure for delivering the CRISPR/Cas9 complex to the zygotes of the hamster, the embryo handling conditions, embryo transfer procedures, and husbandry required to produce genetically modified hamsters.

Pronuclear (PN) injection of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system is a highly efficient method for producing genetically engineered golden Syrian hamsters. Herein, we describe the detailed PN injection protocol for the production of gene knockout hamsters with the CRISPR/Cas9 system.

CRISPR/Cas9 复合原注射液生产基因工程金黄叙利亚仓鼠

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. ...

生物工程

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below outlines a method for high efficiency transfer between bacterial strains of a plasmid harboring a transposon containing a kanamycin resistance marker. The plasmid-borne transposase is encoded by a variant tnp gene that inserts the transposon into the genome of the recipient strain with very low insertional bias. This method thus allows the creation of large mutant libraries in which transposons have been inserted into unique genomic positions in a recipient strain of either Escherichia coli or Shigella flexneri bacteria. By using bacterial conjugation, as opposed to other methods such as electroporation or chemical transformation, large libraries with hundreds of thousands of unique clones can be created. This yields high-density insertion libraries, with insertions occurring as frequently as every 4-6 base pairs in non-essential genes. This method is superior to other methods as it allows for an inexpensive, easy to use, and high efficiency method for the creation of a dense transposon insertion library. The transposon library can be used in downstream applications such as transposon sequencing (Tn-Seq), to infer genetic interaction networks, or more simply, in mutational (forward genetic) screens.

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

使用细菌共轭的 Enterobacterial 菌株 (如大肠杆菌或志贺菌福氏) 创建稠密的转插入库

Transposon mutagenesis is a method that allows gene disruption via the random genomic insertion of a piece of DNA called a transposon. The protocol below ...

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6K&#947; replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host&#8217;s genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli (E. coli) strain. The R6K&#947; replication origin allows the plasmid to replicate in pir+ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6K&#947; replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a wider variety of bacterial strains.

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Enterobacter sp. YSU grows in glucose minimal salts medium. Auxotrophs are generated by transforming it with a transposome which randomly inserts itself into the host genome. Mutants are found by replica plating from complex medium to minimal medium. Interrupted genes are identified by gene rescue and sequencing.

的产生<em&gt;肠杆菌</em&gt; SP。 YSU营养缺陷型使用转座子突变

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can ...

生物学

Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing

Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways that contribute to bacterial fitness under a wide range of environmental conditions. Tn-seq applications are extensive and have not only enabled examination of genotype-phenotype relationships at an organism level but also at the population, community and systems levels. Gram-negative bacteria are highly associated with antimicrobial resistance phenotypes, which has increased incidents of antibiotic treatment failure. Antimicrobial resistance is defined as bacterial growth in the presence of otherwise lethal antibiotics. The &#8220;last-line&#8221; antimicrobial colistin is used to treat Gram-negative bacterial infections. However, several Gram-negative pathogens, including Acinetobacter baumannii can develop colistin resistance through a range of molecular mechanisms, some of which were characterized using Tn-seq. Furthermore, signal transduction pathways that regulate colistin resistance vary within Gram-negative bacteria. Here we propose an efficient method of transposon mutagenesis in A. baumannii that streamlines generation of a saturating transposon insertion library and amplicon library construction by eliminating the need for restriction enzymes, adapter ligation, and gel purification. The methods described herein will enable in-depth analysis of molecular determinants that contribute to A. baumannii fitness when challenged with colistin. The protocol is also applicable to other Gram-negative ESKAPE pathogens, which are primarily associated with drug resistant hospital-acquired infections.

We describe a method to generate saturating transposon mutant libraries in Gram-negative bacteria and subsequent preparation of DNA amplicon libraries for high-throughput sequencing. As an example, we focus on the ESKAPE pathogen, Acinetobacter baumannii, but this protocol is amenable to a wide range of Gram-negative organisms.

在革兰氏负性细菌中生成跨波子插入库，用于高通量测序

Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways ...

Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a Burkholderia gladioli Symbiont of Lagria villosa Beetles

Inferring the function of genes by manipulating their activity is an essential tool for understanding the genetic underpinnings of most biological processes. Advances in molecular microbiology have seen the emergence of diverse mutagenesis techniques for the manipulation of genes. Among them, transposon-insertion sequencing (Tn-seq) is a valuable tool to simultaneously assess the functionality of many candidate genes in an untargeted way. The technique has been key to identify&#160;molecular mechanisms for the colonization of eukaryotic hosts in several pathogenic microbes and a few beneficial symbionts.
Here, Tn-seq is established as a method to identify colonization factors in a mutualistic Burkholderia gladioli symbiont of the beetle Lagria villosa. By conjugation, Tn5 transposon-mediated insertion of an antibiotic-resistance cassette is&#160;carried out at random genomic locations in B. gladioli. To identify the effect of gene disruptions on the ability of the bacteria to colonize the beetle host, the generated B. gladioli transposon-mutant library is inoculated on the beetle eggs, while a control is grown in vitro in a liquid culture medium. After allowing sufficient time for colonization, DNA is extracted from the in vivo and in vitro grown libraries. Following a DNA library preparation protocol, the DNA samples are prepared for transposon-insertion sequencing. DNA fragments that contain the transposon-insert edge and flanking bacterial DNA are selected, and the mutation sites are determined by sequencing away from the transposon-insert edge. Finally, by analyzing and comparing the frequencies of each mutant between the in vivo and in vitro libraries, the importance of specific symbiont genes during beetle colonization can be predicted.

Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a Burkholderia gladioli Symbiont of Lagria villosa Beetles

This is an adapted method for identifying candidate insect colonization factors in a Burkholderia beneficial symbiont. The beetle host is infected with a random mutant library generated via transposon mutagenesis, and library complexity after colonization is compared to a control grown in vitro.