Name: constitutive and inducible systems for genetic in vivo modification of mouse hepatocytes using hydrodynamic tail vein injection
Uploaded: 2018-02-02T13:00:00.000+00:00
Duration: 9 min 35 s
Description: Watch this Scientific Journal Video about Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection at JoVE.com

这项工作得到德国德意志 Krebshilfe (111289 号至 ue)、露西尔的儿童健康基金会的支持 (欧内斯特和阿米莉亚-奖授予博士后奖学金-UL1 RR025744 至 ue)。我们感谢马克. 凯博士的向量结构和实验建议和朱利安博士鼠和实验支持。

所有动物实验都是根据实验室动物的护理和使用指南进行的, 并得到主管当局的批准 (Regierung Oberbayern、慕尼黑、德国和斯坦福机构动物保育和使用委员会,斯坦福, 加利福尼亚, 美国)。在补充表 S1 中提供了所有用于克隆的质粒的清单 (步骤1至 4)。
1. 本构基因表达的转基因克隆
<ol><li>设计引物为转基因放大13,14。</li>
	<li>将总署 (TTAATTAA) 的限制站点添加到前引物的5末端。将 AscI (GGCGCGCC) 或 FseI (GGCCGGCC) 的限制站点添加到反向底漆的5端。</li>
	<li>利用优化的条件为所需的转基因14放大转基因。使用商业上可用的 DNA 纯化试剂盒进行净化。 注: 退火时间取决于在步骤1.1 中设计的底漆, 伸长时间取决于构造的长度。例如, 为 1000 bp 长度的结构使用六十年代伸长时间。</li>
	<li>本构基因表达的消化转基因和载体15与各自的制约核酸 (参见步骤 1.2) 和缓冲在总容量50µL 在37° c 隔夜。例如, 在适当的情况下, 5 单元总署和5单元 FseI 的摘要构造 (参见步骤 1.2)。</li>
	<li>凝胶净化消化载体和插入使用商业凝胶提取试剂盒根据制造商的指示。在14° c 下使用 400 U T4 连接, 在65° c 下执行一个标准结扎 100 ng 向量和 100–1,000 ng, 16 h。</li>
	<li>使用标准的热休克协议16转换合格的细菌。</li>
	<li>琼脂板上含有100µg/毫升氨苄西林。孵育在30° c 为24小时。</li>
	<li>选择单一的殖民地, 纯化质粒 DNA 使用商业 miniprep 套件根据制造商的说明和验证序列的桑格排序17 (测序底漆: 5 "TGCTGGAGTTCTTCGCC 3")。</li>
	<li>使用阳性菌落的最大规模扩增的质粒 DNA 和纯化使用无内毒素质粒准备试剂盒18根据制造商的说明。</li>
	<li>构造已准备好注入, 从而继续执行步骤5。</li>
</ol>2. 转基因诱导基因表达的克隆
<ol><li>设计引物为转基因放大13,14。</li>
	<li>将倡议 (GAGCTC)、SpeI (ACTAGT) 或 KpnI (GGTACC) 的限制站点添加到前引物的5末端。将 NotI (GCGGCCGC) 或 XhoI (CTCGAG) 的限制站点添加到反向底漆的5端。</li>
	<li>利用优化的条件为所需的转基因14放大转基因。使用商业 DNA 纯化试剂盒进行净化。</li>
	<li>消化纯净的转基因和4µg 词条传染媒介 (pEN_TTmcs-传染媒介, 看见材料表)19分开地与适当的制约核酸 (参见步骤 2.2) 和缓冲在总容量50µL 在37° c 隔夜。</li>
	<li>凝胶净化消化载体和插入使用商业凝胶提取试剂盒根据制造商的指示。使用 400 U T4 连接在14° c 下进行标准结扎, 100 ng 100–1,000, 16 h. 热钝化在65° c 为10分钟。</li>
	<li>使用标准的热休克协议16转换合格的细菌。</li>
	<li>在琼脂板上含有15µg/毫升庆大霉素。孵育在37° c 为16小时。</li>
	<li>选择单一菌落, 纯化质粒 DNA, 并通过测序17使用 pCEP 前引物验证插入: 5 "AGAGCTCGTTTAGTGAACCG 3"。 注: PCR 在单一殖民地可以执行, 无需预先纯化与引物 pCEP 向前和 pCEP 反向 (5 ' AAG CTG GGT CTA ATC TCG 3 ')。这一步可以用来预先选择积极的殖民地。</li>
	<li>继续执行步骤4。</li>
</ol>3. shRNA 用于诱导基因敲击的克隆
<ol><li>根据 pSLIK 克隆协议19设计 shRNA 寡核苷酸。退火和纯化寡核苷酸和稀释 1:20 在 ddH2O。</li>
	<li>文摘3µg 的入口向量与 5 U BfuAI 在50° c 为 3 h, 然后钝化反应在65° c 为 20 min。 注意: 如果需要绿色荧光蛋白 (GFP) 的 co 表达式, 请使用 pEN_TTGmiRc19作为输入向量, 否则使用 pEN_TTmiRc219。</li>
	<li>凝胶纯化消化载体, 如步骤2.5。执行标准结扎 100 ng 载体和1µL 纯化和稀释 shRNA 寡核苷酸 (步骤 3.1) 使用 400 U T4 连接在室温下 1 h. 热钝化在65° c 为10分钟。</li>
	<li>使用标准的热休克协议16转换合格的细菌。</li>
	<li>在琼脂板上含有15µg/毫升庆大霉素。孵育在37° c 为16小时。</li>
	<li>选取单个菌落, 纯化质粒 DNA, 并通过桑格测序17 (测序底漆: 5 "TAGTCGACTAGGGATAACAG 3")。</li>
	<li>继续执行步骤4。</li>
</ol>4. 重组克隆以生成现成的注射克隆
<ol><li>混合 150 ng 输入向量 (从步骤2或步骤 3) 和 150 ng 的 pTC 春节向量20。</li>
	<li>将 TE 缓冲区添加到总容量为8µL (pH=8)。</li>
	<li>将 LR-clonase 酶混合 II (见材料表) 转化为冰, 孵育2分钟涡旋两次。</li>
	<li>添加2µL 的 LR-clonase 酶混合 II 的反应和孵化25° c 为1小时。</li>
	<li>通过添加1µL 蛋白酶 K 溶液来停止反应。孵育在37° c 为10分钟。</li>
	<li>利用标准的热休克协议16将 Stbl3 2 µL 的重组克隆混合菌转化为合格细菌。</li>
	<li>琼脂板上含有100µg/毫升氨苄西林。孵育在30° c 为24小时。</li>
	<li>选择单个菌落, 用无内毒素质粒制备试剂盒纯化质粒 DNA 根据制造商的说明,18 , 并通过桑格测序17 (测序底漆 5 ' 确认向量完整性AGGGACAGCAGAGATCCAGTTTGG 3 ')。</li>
	<li>构造已准备好注入, 请继续执行步骤5。</li>
</ol>5. 水动力尾静脉注射液的制备
注: 在步骤1、2、3和4中描述了本构和诱导基因表达结构的制备。
<ol><li>准备无菌0.9% 盐水注射 (不要使用 PBS). 使用体积相当于10% 左右的鼠标体重。示例: 对于重20克的鼠标, 准备2毫升的溶液。</li>
	<li>准备使用无内毒素质粒纯化试剂盒纯化的注射载体18 (分别参见步骤1.8 或 4.8)。</li>
	<li>添加10µg 或15µg 内毒素-免费的睡美人向量结构 (从步骤1使用10µg, 从步骤4使用15µg, 分别) 和1µg 内毒素-免费 pc-HSB521每毫升无菌盐水。</li>
	<li>存储解决方案可达4小时, 在4° c。不要冻结。</li>
</ol>6. 执行水力尾静脉注射
<ol><li>使用抑制剂的尾巴静脉注射 (商业或准备从50毫升锥管与孔呼吸和尾巴)。用纸巾把管子的底部填满。</li>
	<li>对于注射, 使用大约8-10 周龄的小鼠与重量 20-25 g。</li>
	<li>在注射前称小鼠, 并根据体重 (相当于10% 的体重) 准备注射量 (见步骤 5.1)。准备一个无菌3毫升注射器与27克针注射和填写所需的数量。</li>
	<li>将鼠标放入抑制剂。调整卫生纸的数量 (见步骤 6.1), 只留下最小的运动空间, 但有足够的空间进行呼吸。</li>
	<li>确保鼠标定期呼吸。</li>
	<li>用红外线灯加热尾部30-60 秒小心注意过热的迹象。</li>
	<li>用酒精擦拭尾巴。</li>
	<li>将针几乎水平地插入两个侧向尾脉中的一个, 靠近尾部的底部。 注: 如果成功放置, 少量的血液可能会回流到针头的锥体。不建议积极吸气, 因为任何额外的针头运动都可能导致其移位和/或静脉损伤。</li>
	<li>在8-10 秒内将总容积注入尾静脉。</li>
	<li>立即从抑制剂中取出鼠标。压缩注射伤口至少三十年代或直到任何出血消退。</li>
	<li>将鼠标置于单独的笼子中。一旦鼠标从过程中恢复 (约30-60 分钟), 将鼠标移回原来的笼子。在接下来的24小时中定期检查鼠标。 注: 在注射后2小时内常规观察小鼠的轻度镇静。</li>
	<li>在继续进行进一步的实验之前 (即, 步骤 7), 等待10-15 天来清除非集成向量。</li>
</ol>7. 转染 CreER 与他莫昔芬的诱导
注意: 他莫昔芬是有害的, 可能是致癌或损害生育力。请参阅安全数据表。
<ol><li>连续三天计划腹腔他莫昔芬注射。</li>
	<li>在1天, 溶解10毫克他莫昔芬在40µL 乙醇。在55° c 下孵育10分钟涡流几次, 直到他莫昔芬溶解。</li>
	<li>加960µL 玉米油。在55° c 孵育5分钟。涡数倍, 以得到一个明确的解决方案。</li>
	<li>准备一个1毫升的胰岛素注射器与27克针的解决方案。</li>
	<li>用拇指和第二个手指小心翼翼地抓住鼠标的颈部, 在手的底座和第四和第五手指之间固定尾巴。</li>
	<li>注射0.1 毫升 (= 1 毫克的他莫昔芬) 的溶液腹腔到左下腹腹。</li>
	<li>在2和三天重复注射</li>
</ol>8. 诱导四环素相关基因或 shRNA 表达
注意: 强力霉素可能有害。请参阅安全数据表。
注: 根据试验的类型和持续时间, 强力霉素可在饮用水 (步骤 8.1) 或 chow (步骤 8.2) 中提供。
<ol><li>
短期试验 (< 10 天) 使用以下协议通过饮用水管理强力霉素。
	<ol>
<li>在100毫升的自来水中溶解5克蔗糖。釜.</li>
		<li>将100毫克的强力霉素-盐酸在5毫升的蔗糖溶液 (步 8.1.1) 中溶解于15毫升的锥形管中。</li>
		<li>使用10毫升注射器, 无菌过滤器的解决方案通过0.2 µm 过滤器。添加到步骤8.1.1 中的蔗糖溶液中。</li>
		<li>供应强力霉素-蔗糖溶液作为饮用的水对老鼠。每天检查并更换当溶液变为多云时显示细菌过度生长 (最迟在三天后更换清除溶液)。</li>
	</ol>
</li>
	<li>对于长期的实验 (> 10 天) 或对新陈代谢变化敏感的实验, 使用商业强力霉素周 (例如, 强力霉盐酸周0.625 克/千克), 以避免脱水和/或蔗糖引起的动物肝脏的变化。</li>
</ol>9. 小鼠肝免疫的制备
警告: 甲醛可能有害。请参阅安全数据表。
注意: timepoint 时, 老鼠会被分析, 这取决于实验。建议在不少于三天的强力霉素治疗后分析肝组织, 以确保充分诱导转基因或 shRNA 表达。
<ol><li>准备一个1毫升注射器与27克针1毫升的4% 甲醛溶液 (粉煤灰)。</li>
	<li>根据批准的动物协议, 用适当的方法安乐鼠标。 注: 安乐死的适当方法的指导方针可能会因机构而异。</li>
	<li>使用解剖剪刀和解剖钳, 小心打开腹腔与中间剖腹手术暴露肝脏。将小肠移到右侧以暴露门静脉和下腔静脉。</li>
	<li>插入准备好的注射器的针 (参见步骤 9.1) 入下腔和切开门静脉。注入1毫升的粉煤灰慢慢进入下腔静脉灌注肝组织和删除自动荧光红细胞 (可取的, 如果荧光染色将执行)。</li>
	<li>取出肝脏在水中冲洗, 并转移到5-10 毫升的4% 粉煤灰溶液。</li>
	<li>石蜡切片, 修复组织 36-48 h. 组织准备好脱水和石蜡嵌入。</li>
	<li>
对于冰冻切片, 在4% 粉煤灰中固定1小时组织。
	<ol>
<li>冻, 转移到10% 蔗糖溶液。孵育60分钟。</li>
		<li>转移到20% 蔗糖溶液。孵育60分钟。</li>
		<li>转移到30% 蔗糖溶液。在-20 ° c 的冷冻切片中, 孵育12-16 嵌入化合物中。</li>
	</ol>
</li>
</ol>

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水动力尾静脉注入肝细胞的转染已成为15年前6以来建立的一种方法。注入的体积超过心脏输出, 从下腔静脉流向肝脏的窦7, 导致转染约 10-20%, 在某些情况下多达40% 的肝细胞25,26。成功转染的预测因子是每次注入时间的注入量22,23。因此, 低转染效率 (图 1A, 左面板...

慢性肝病是一个主要的健康负担全球1。动物研究模型是肝脏疾病研究的重要工具, 有助于解答肝脏再生、肝脏炎症、脂肪以及肝癌2等复杂问题。大量的这些动物模型依赖于肝细胞的基因修饰。因此, 有效的工具, 操纵基因表达在肝细胞是有益的3。建立的方法, 如育种的基因工程小鼠菌株或病毒载体的生成肝细胞感染要么费时, 港口安全关注, 或屈服于肝细胞的不良转基因表达在体内4,5. 水动力尾静脉注射 (HTVI) 是一种替代方法的体内转染肝细胞允许方便, 快速, 和经济高效的基因功能的审讯。对于 HTVI, 携带所需 DNA 序列的载体被溶解在相当于注射动物体重10% 的盐水量中。然后在5-10 秒6内将该解决方案注入尾部静脉。超过心脏输出, 盐水从下腔静脉流入肝静脉, 导致肝脏扩张和肝细胞的水动力转染7。为了实现稳定的基因组集成, 该方法已与基于转的矢量 (如休眠美人-转系统) 相结合。该系统介导的重组的目标载体与基因组重组网站的催化睡美人-转8,9。对于肝纤维化或癌变的模型, 在疾病模型的某些时间点过度或沉默基因往往是可取的。为此, 可诱导基因表达的工具, 如 LoxP 系统或四环素诱导基因表达系统 (春节) 可以使用10。
在这里, 我们描述了一个协议的在体内转染小鼠肝细胞使用 HTVI 的睡美人转的系统。除了一个在肝脏特异启动子控制下的转基因的稳定、本构表达的协议外, 我们还描述了一种更先进的向量系统, 它将本构型他莫昔芬重组 (CreER) 表达与可诱导表达的转基因或 rna 适应的 shRNA (和平 shRNA), 称为 pTC 的春节系统11。在这个向量系统中, 可诱导的转基因或和平 shRNAs 为四环素相关的表达被克隆到骨干载体与重组克隆系统, 允许快速和容易生成新的载体12。本视频指南包括制备合适的载体, 注射和治疗小鼠, 以实现诱导转基因/和平 shRNA 表达, 最后准备肝脏组织进行分析。本协议中描述的方法旨在使任何选择的基因 loxP 介导的小鼠系统的表达或击倒, 使得它成为一种广泛适用的肝病研究系统。

<table><tbody><tr><td>&lt;strong&gt;General Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>GeneRuler 1 kb Plus DNA Ladder</td><td>Thermo Fisher</td><td>#SM1331</td><td>DNA ladder for electrophoresis</td></tr><tr><td>Tissue-Tek O.C.T.</td><td>Sakura</td><td>4583</td><td>embedding of cryo-sections</td></tr><tr><td>Biozym LE Agarose</td><td>Biozym</td><td>840004</td><td></td></tr><tr><td>Ethidium bromide</td><td>Sigma-Aldrich</td><td>E7637-1G</td><td></td></tr><tr><td>D(+)-Saccharose</td><td>Carl Roth</td><td>4621.1</td><td>For sweetening of the doxycyline solution</td></tr><tr><td>Ampicillin Sodium Salt</td><td>AppliChem</td><td>A0839,0010</td><td>For selection of Amp-resistant clones</td></tr><tr><td>LB Agar (Luria/Miller)</td><td>Carl Roth</td><td>X969.1</td><td></td></tr><tr><td>LB Broth (Luria/Miller)</td><td>Carl Roth</td><td>X968.1</td><td></td></tr><tr><td>S.O.C. Medium</td><td>Thermo Fischer</td><td>15544034</td><td></td></tr><tr><td>Gentamicin sulfate</td><td>AppliChem</td><td>A1492,0001</td><td>For selection of Gentamicin-resistant clones</td></tr><tr><td>Roti-Histofix 4 %</td><td>Fa. Roth</td><td>P087.6</td><td>para-formaldehyde solution</td></tr><tr><td>T4 DNA Ligase</td><td>New England BioLabs</td><td>M0202S</td><td></td></tr><tr><td>GatewayTM LR ClonaseTM II Enzyme Mix</td><td>invitrogen/ThermoFisher</td><td>11791-020</td><td>contains LR-clonase enzyme mix II and proteinase K</td></tr><tr><td>DB3.1 Competent Cells</td><td>Thermo Fisher</td><td>11782-018</td><td></td></tr><tr><td>Stbl3 Chemically Competent E. coli</td><td>Thermo Fisher</td><td>C737303</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Restriction Enzymes&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PacI</td><td>New England BioLabs</td><td>R0547S</td><td></td></tr><tr><td>AscI</td><td>New England BioLabs</td><td>R0558S</td><td></td></tr><tr><td>FseI</td><td>New England BioLabs</td><td>R0588S</td><td></td></tr><tr><td>SacI</td><td>New England BioLabs</td><td>R0156S</td><td></td></tr><tr><td>SpeI</td><td>New England BioLabs</td><td>R0133S</td><td></td></tr><tr><td>KpnI</td><td>New England BioLabs</td><td>R0142S</td><td></td></tr><tr><td>NotI</td><td>New England BioLabs</td><td>R0189S</td><td></td></tr><tr><td>XhoI</td><td>New England BioLabs</td><td>R0146S</td><td></td></tr><tr><td>BfuAI</td><td>New England BioLabs</td><td>R0701S</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Kits&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>Qiagen</td><td>28704</td><td>For DNA Extraction from gel</td></tr><tr><td>NucleoSpin Gel and PCR Clean Up</td><td>Macherey &amp;amp; Nagel</td><td>740609.10</td><td></td></tr><tr><td>NucleoBond PC20</td><td>Macherey &amp;amp; Nagel</td><td>740571</td><td>Plasmid extraction (Mini prep)</td></tr><tr><td>NucleoBond PC500</td><td>Macherey &amp;amp; Nagel</td><td>740574</td><td>Plasmid extraction (Maxi prep)</td></tr><tr><td>Phusion High-Fidelity DNA Polymerase</td><td>Thermo Fisher</td><td>F530S</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Materials for Mouse Experiments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Injekt Syringe F 1 ml</td><td>Braun</td><td>9166017V</td><td>For intraperitoneal injection</td></tr><tr><td>Omnifix Luer 3 ml</td><td>Braun</td><td>4616025V</td><td>For intravenous injection</td></tr><tr><td>Sterican Cannula 24G</td><td>Braun</td><td>4657675</td><td></td></tr><tr><td>Sterican Cannula 27G</td><td>Braun</td><td>4657705</td><td></td></tr><tr><td>Tamoxifen</td><td>Sigma-Aldrich</td><td>T5648-1G</td><td>For CreER activation</td></tr><tr><td>Corn oil</td><td>Sigma-Aldrich</td><td>C8267-500ML</td><td>Carrier for tamoxifen injections</td></tr><tr><td>Doxycycline hyclate</td><td>AppliChem</td><td>A2951,0025</td><td>Activation of tetracycline-dependent expression</td></tr><tr><td>Injekt 10 ml Syringe</td><td>Braun</td><td>4606108V</td><td></td></tr><tr><td>Filtropur S 0.2</td><td>Sarstedt</td><td>831,826,001</td><td>For filtration of doxycycline</td></tr><tr><td>NaCl 0,9%</td><td>Braun</td><td>3200905</td><td>Carrier for intravenous injections</td></tr><tr><td>Falcon Conical Tube 50ml</td><td>Corning Life Science</td><td>352095</td><td></td></tr><tr><td>Infrared Lamp</td><td>N/A</td><td>N/A</td><td>For warming of mouse tail</td></tr><tr><td>IVIS</td><td>Perkin Elmer</td><td>124262</td><td>In vivo imaging system</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Plasmids for cloning of sleeping beauty-transposon vectors for HTVI.&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pTC</td><td></td><td>n/a</td><td>Vector for constitutive gene expression, ref. 15</td></tr><tr><td>pEN_TTmcs</td><td></td><td>Addgene #25755</td><td>Entry vector for inducible gene expression, ref. 19</td></tr><tr><td>pEN_TTGmiRc2</td><td></td><td>Addgene #25753</td><td>Entry vector for inducible miR-shRNA expression with co-expression of GFP, ref. 19</td></tr><tr><td>pEN_TTmiRc2</td><td></td><td>Addgene #25752</td><td>Entry vector for inducible miR-shRNA expression without co-expression of GFP, ref. 19</td></tr><tr><td>pTC ApoE-Tet</td><td></td><td>Addgene #85578</td><td>Expression vector for inducible gene or miR-shRNA expression with ApoE.HCR.hAAT promotor, ref. 11</td></tr><tr><td>pTC-CMV-Tet</td><td></td><td>Addgene #85577</td><td>Expression vector for inducible gene or miR-shRNA expression with CMV promotor, ref. 11</td></tr></tbody></table>

constitutive and inducible systems for genetic in vivo modification of mouse hepatocytes using hydrodynamic tail vein injection

在肝癌的研究模式, 再生, 炎症和纤维化, 灵活的系统为体内基因表达和沉默是非常有用的。以转为基础的水动力尾静脉注射是一种有效的成年小鼠肝细胞遗传操作方法。除了本构式转基因表达外, 该系统还可用于更先进的应用, 如 shRNA 介导的基因敲除, CRISPR/Cas9 系统诱导基因突变或诱导系统的暗示。将本构 CreER 表达与转基因或 shRNA 选择的诱导表达结合起来, 作为这一技术的一个例子。我们涵盖了多步骤的过程, 从准备的睡美人-转结构, 注射和治疗小鼠, 并准备肝组织进行分析的免疫。该系统是实现肝细胞复杂遗传操作的可靠而有效的方法。它是特别有用的结合与 loxP 的小鼠品系, 可用于各种模型的研究肝病。

水动力尾静脉注射转染效果:单次注射转染水力的小鼠肝细胞百分比是可变的, 取决于注射量、注射时间、注入 DNA 量和注入结构的大小等多个参数6, 22,23。此外, 转染效率一般较低, 在较大的动物, 较大的血管直径, 以及较大的正弦面积导致整体压力下降。为了直观地显示转染效率, CreER 转结?...

Watch this Scientific Journal Video about Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection at JoVE.com

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

血管动力尾静脉注射对小鼠肝细胞基因的本构和诱导系统的修饰

水动力尾静脉注射转的融合载体, 可以稳定转染小鼠肝细胞在体内.在这里, 我们提出了一个实用的转染系统的协议, 使一个单一的转基因或联合本构和强力霉素诱导表达的转基因或和平 shRNA 在肝脏的长期本构表达。

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly ...

constitutive-inducible-systems-for-genetic-vivo-modification-mouse

Universidad San Sebastian

Research

JoVE Journal

Genetics

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

14.0K 次观看.  Technische Universität München. 水动力尾静脉注射转的融合载体, 可以稳定转染小鼠肝细胞在体内.在这里, 我们提出了一个实用的转染系统的协议, 使一个单一的转基因或联合本构和强力霉素诱导表达的转基因或和平 shRNA 在肝脏的长期本构表达。

视频: Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

CRISPR/Cas9-mediated 目标集成在体内使用基于同源介导的端接连接策略

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

科研

遗传学

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.

Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.

诱导多潜能干细胞源性肝细胞的家族性高胆固醇血症人肝嵌合小鼠模型

Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset ...

发育生物学

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in which the liver has been repopulated with functional human hepatocytes, could serve as a useful tool for investigating human hepatic cell biology, drug metabolism, and other preclinical applications. One of the key factors required for successful transplantation of human hepatocytes into mice is the elimination of the endogenous hepatocytes to prevent competition with the human cells and provide a suitable space and microenvironment for promoting human donor cell expansion and differentiation. To date, two major liver injury mouse models utilizing fumarylacetoacetate hydrolase (Fah) and uroplasminogen activator (uPA) mice have been established. However, Fah mice are used mainly with mature hepatocytes and the application of the uPA model is limited by decreased breeding. To overcome these limitations, Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice were used for in vivo differentiation of immature human hepatocytes and humanized liver generation. Human hepatic stem cells (HpSCs) successfully repopulated the livers of Alb-TRECK/SCID mice that had developed lethal fulminant hepatic failure following diphtheria toxin (DT) treatment. This model of a humanized liver in Alb-TRECK/SCID mice will have functional applications in studies involving drug metabolism and drug-drug interactions and will promote other in vivo and in vitro studies.

Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells.

利用人体肝干细胞人源化小鼠肝脏生成

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in ...

医学

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors are offering new opportunities for regenerative medicine. Such clinical applications of iPS cells have been limited so far, mainly due to the poor efficiency of the existing reprogramming methodologies and the risk of the generated iPS cells to form tumors upon implantation.
We hypothesized that the reprogramming of somatic cells towards pluripotency could be achieved in vivo by gene transfer of reprogramming factors. In order to efficiently reprogram cells in vivo, high levels of the Yamanaka (OKSM) transcription factors need to be expressed at the target tissue. This can be achieved by using different viral or nonviral gene vectors depending on the target tissue. In this particular study, hydrodynamic tail-vein (HTV) injection of plasmid DNA was used to deliver the OKSM factors to mouse hepatocytes. This provided proof-of-evidence of in vivo reprogramming of adult, somatic cells towards a pluripotent state with high efficiency and fast kinetics. Furthermore no tumor or teratoma formation was observed in situ.
It can be concluded that reprogramming somatic cells in vivo may offer a potential approach to induce enhanced pluripotency rapidly, efficiently, and safely compared to in vitro performed protocols and can be applied to different tissue types in the future.

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors ...

生物学

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).

In vivo transfection of naked DNA by hydrodynamic gene delivery introduces genes into the tissue of an animal with minimal inflammatory response. Sufficient amounts of gene product are generated such that gene function and regulation as well as protein structure and function can be analyzed.

蛋白通过流体动力基因传递在小鼠体内瞬时表达

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney

Hydrodynamic injection creates a local, high-pressure environment to transfect various tissues with plasmid DNA and other substances. Hydrodynamic tail vein injection, for example, is a well-established method by which the liver can be transfected. This manuscript describes an application of hydrodynamic principles by injection of the mouse kidney directly with plasmid DNA for kidney-specific gene expression. Mice are anesthetized and the kidney is exposed by a flank incision followed by a fast injection of a plasmid DNA-containing solution directly into the renal pelvis. The needle is kept in place for ten seconds and the incision site is sutured. The following day, live animal imaging, Western blot, or immunohistochemistry may be used to assay gene expression, or other assays suited to the transgene of choice are used for detection of the protein of interest. Published methods to prolong gene expression include transposon-mediated transgene integration and cyclophosphamide treatment to inhibit the immune response to the transgene.

This protocol describes a method to inject plasmid DNA into the mouse kidney via the renal pelvis to produce transgene expression specifically in the kidney.

肾水动力肾盂注射非病毒蛋白在肾脏的表达

Hydrodynamic injection creates a local, high-pressure environment to transfect various tissues with plasmid DNA and other substances. Hydrodynamic tail ...

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.

一种简便、有效的心肌注射小鼠心脏特异基因操作方法--体内

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

Establishment and Genetic Manipulation of Murine Hepatocyte Organoids

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the executor for most of the liver functions, primary hepatocytes have limited proliferating capacity. This requires the establishment of ex vivo hepatocyte expansion models for liver physiological and pathological research. Here, we isolated murine hepatocytes by two steps of collagenase perfusion and established a 3D organoid culture as the 'mini-liver' to recapitulate cell-cell interactions and physical functions. The organoids consist of heterogeneous cell populations including progenitors and mature hepatocytes. We introduce the process in detailed to isolate and culture the murine hepatocytes or fetal hepatocyte to form organoids within 2-3 weeks and show how to passage them by mechanically pipetting up and down. In addition, we will also introduce how to digest the organoids into single cells for lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering. The organoids can be used for drug screens, disease modelling, and basic liver research by modeling liver biology and pathobiology.

A long-term ex vitro 3D organoid culture system was established from mouse hepatocytes. These organoids can be passaged and genetically manipulated by lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering.

小鼠肝细胞类器官的建立与遗传操作

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the ...

Inducible Tet-On Regulatable System-Based Gene Expression In Vivo: A Tetracycline-Induced Gene Expression System for Target Gene Transcription Activation in Mouse Model

Source: Hubner, E. K. et al.&#160; <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/56613">Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection.</a> J. Vis. Exp. (2018)
This video demonstrates the tetracycline, or Tet-On, gene expression system in a mouse model. In the presence of doxycycline, a tetracycline derivative, the Tet-On system consisting of reverse tetracycline-controlled transactivator protein becomes activated and expresses the gene of interest.

诱导型 Tet-On 基于调节系统的体内基因表达:用于小鼠模型中靶基因转录激活的四环素诱导基因表达系统

Source: Hubner, E. K. et al.&#160; Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein ...

JoVE Encyclopedia of Experiments

Biological Techniques

Genome Editing Techniques

Gene regulation

CreER-LoxP System-Based Target Gene Inactivation: A Tamoxifen-Inducible Cre Recombinase System for Target Gene Knockout in a Mouse Model

Source: Hubner, E. K., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/56613">Constitutive and Inducible Systems for Genetic&#160;In Vivo&#160;Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection.</a>&#160;J. Vis. Exp. (2018)
This video describes a procedure for target gene knockout using a tamoxifen-inducible Cre-recombinase system following intraperitoneal injection of tamoxifen in a mouse model.

基于 CreER-LoxP 系统的靶基因灭活:用于小鼠模型中靶基因敲除的他莫昔芬诱导型 Cre 重组酶系统

Source: Hubner, E. K., et al. Constitutive and Inducible Systems for Genetic&#160;In Vivo&#160;Modification of Mouse Hepatocytes Using Hydrodynamic Tail ...

血管动力尾静脉注射对小鼠肝细胞基因的本构和诱导系统的修饰

摘要

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此视频中的章节

CRISPR/Cas9-mediated 目标集成在体内使用基于同源介导的端接连接策略

诱导多潜能干细胞源性肝细胞的家族性高胆固醇血症人肝嵌合小鼠模型

利用人体肝干细胞人源化小鼠肝脏生成

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

蛋白通过流体动力基因传递在小鼠体内瞬时表达

肾水动力肾盂注射非病毒蛋白在肾脏的表达

一种简便、有效的心肌注射小鼠心脏特异基因操作方法--体内

小鼠肝细胞类器官的建立与遗传操作

诱导型 Tet-On 基于调节系统的体内基因表达:用于小鼠模型中靶基因转录激活的四环素诱导基因表达系统

基于 CreER-LoxP 系统的靶基因灭活:用于小鼠模型中靶基因敲除的他莫昔芬诱导型 Cre 重组酶系统