我们感谢 Prof. Zappavigna, 摩德纳大学和雷焦艾米利亚, 摩德纳, 意大利为我们提供了 pCCL-PGKrtTA2SM2 质粒。我们也承认 z 伊维西 (保罗 Ehlrich 研究所, 兰, 德国) 和 Prof. z Izvak (马克斯汗分子医学中心, 德国柏林) pCMVSB100X 和 pT2/BH 质粒。这项工作得到了黛布拉国际和意大利大学和研究部的支持。

1. 质粒受雇
注: 质粒 pCCL-PGKrtTA2S-M2 提供了 Prof. Zappavigna (摩德纳大学和雷焦艾米利亚, 摩德纳, 意大利)。pCMVSB100X 质粒携带的多动转和 pT2/BH 转质粒的编码序列由 z 伊维西 (保罗, 兰, 德国) 和 Prof. z Izvak (最大汗分子医学中心, 柏林,德国)。
<ol><li>对于大肠杆菌(大肠杆菌) 中的所有克隆, 请遵循选择和限制酶程序的克隆策略或克隆片段的 PCR 扩增. 注:表 1总结了本协议中使用的所有质粒。提供了每个质粒的描述和参考。</li>
</ol>2. 病毒载体生产
注意: 所有的病毒载体都是在生物安全防护罩的 BSL2 中产生的, 根据机构的规章制度。
<ol><li>Dulbecco 氏改良鹰培养基中的培养附着 HEK293T 细胞, 辅以 10% HyClone 血清、100 U/毫升青霉素-链霉素和2毫米谷氨酰胺。</li>
	<li>0天: 准备五15厘米的盘子/病毒制剂和种子5.5x10 在30毫升的培养基中6细胞。</li>
	<li>1天: 转染<ol>
<li>在开始转染前, 准备100毫升的 2x HEPES 缓冲盐水 (HBS): 氯化钠 (5 米) 5.6 毫升 HEPES (1 米) 10.0 毫升 Na2HPO4 (0.5 M) 0.3 mL 无菌水84.1 毫升 调整到 pH 值7.1 与 37% HCl。 注: 2x HBS 可存储在4° c。准确的 pH 值对于高效转染非常重要。最佳 pH 值范围为7.10 到7.12。</li>
		<li>在转染前取出培养基, 加入22.5 毫升/新培养基4小时。</li>
		<li>染 HEK293T 细胞与转移质粒, pMD. Lg/pRRE D64VInt 包装质粒, pMD2. G 信封编码质粒, 和 pRSV-转速 (表 1) 根据以下数量/板块: 转移质粒25.00 µg Lg/pRRE D64VInt 16.25 µg pMD2 8.75 µg pRSV-启示录6.25 µg 注: 质粒 dna 是根据 Maniatis22描述的中海协议编写的, 或使用无内毒素质粒纯化试剂盒。<ol>
<li>重56.25 µg 的 DNA (混合4质粒) 在1.125 毫升的无菌水和添加125µL 2.5 M CaCl2 (解决方案 A)。</li>
			<li>增加1.250 毫升的 2x HBS 到一个无菌15毫升圆锥离心管 (解决方案 B)。</li>
			<li>添加解决方案 a (1.250 毫升), 以 B (1.250 毫升) 滴, 而起泡与1或2毫升吸管 (转染解决方案, 总容量2.500 毫升)。</li>
			<li>室温孵育20分钟 (RT)。</li>
			<li>使用 P1000 微, 将所有的转染溶液 (2.500 毫升) 分布到细胞单层滴。</li>
		</ol>
</li>
		<li>在37° c、5% CO2的细胞培养箱中过夜。</li>
	</ol>
</li>
	<li>2天: 取出培养基, 加入15毫升的新鲜培养基 (见步骤 2.1)。</li>
	<li>3天: 收集并浓缩含有慢颗粒的上清液。<ol>
<li>从所有 (n=5) 转染的细胞板中收集上清液 (70 毫升), 过滤通过0.45 µm-过滤器和填充 2 polyallomer 管 (1 英寸 x 3.5 英寸)/病毒制剂。</li>
		<li>将矢量粒子离心在 10.6万 x g 为2.5 小时, 在15° c 与制动器。</li>
		<li>在停止离心 (为了避免悬浮的颗粒在试管中), 轻轻地将上清液倒入含有10% 漂白剂的容器中, 并在100µL 1x PBS (+ 1% BSA) 中悬浮2勉强可见的小球。这是正常的, 因为颗粒是明确和非常小的。使用在-80 ° c 的新制备的浓缩载体, 或分和存储病毒制剂。 警告: 上清液中含有慢的微粒, 在生物危害废物中丢弃之前必须彻底漂白。</li>
	</ol>
</li>
	<li>为了滴定病毒载体的准备, 根据制造商的协议, 使用 HIV-1 堵嘴 p24 免疫套件。 注: 为规范病毒载体的生产, 可采用 liposomes-based 试剂。然而, 病毒载体的效价高度依赖于质粒 DNA 的转染效率和纯度。</li>
</ol>3. 人类细胞系的转导 (如 HeLa 细胞)
注意: HeLa 细胞在 Dulbecco 氏改良的鹰培养基中进行培养, 并辅以10% 胎小牛血清、100 U/毫升青霉素-链霉素和2毫米谷氨酰胺。表 1总结了此协议中使用的所有向量。提供了每个向量的描述。HeLa 细胞的转导使转在两个 IDLV 载体的最佳剂量组合中对转录调控进行验证。IDLV 剂量的变化可能与载体颗粒滴定和靶细胞类型有关。
<ol><li>0天: 种子 2x105细胞在3毫升培养基中为6井板的每一个井。准备4口井。</li>
	<li>1天: HeLa 细胞的转导。<ol>
<li>对于每个条件, 稀释慢向量到最后的体积1毫升培养基 (见上文注) 在10μ l 的凝聚 (最终浓度8微克/毫升): 稀释为井 #1: 模拟转细胞 (阴性控制)。 稀释为井 #2: IDLVTKSB 载体 (2600 ng p24)。 稀释为井 #3, #4: IDLVTKSB 向量 (2600 ng p24) + IDLVrtTA2s-M2 向量 (9600 ng p24)。</li>
		<li>在0天将 HeLa 细胞镀在每一个井中, 并在相应的井中添加慢矢量稀释。</li>
		<li>Spinoculate 6 井板在 754 x g 为45分钟在20-25 ° c, 然后转移 6-井板到细胞培养孵化器在37° c 和 5% CO2。</li>
		<li>6小时后, 在所有井中用新鲜培养基取代培养基, 并在井 #4 中加入强力霉素至1μ m。将板材放入37° c 48 小时的细胞培养箱中。</li>
	</ol>
</li>
	<li>3天: RNA 提取用细胞颗粒的制备。<ol>
<li>在分离细胞之前, 准备一个胰蛋白酶工作溶液 (1x): 2.5% 胰蛋白酶 (10x) 5.0 毫升 500毫米 EDTA (最终浓度 = 5 mm) 0.5 毫升 1x PBS 44.5 毫升</li>
		<li>热胰蛋白酶工作溶液在37° c 使用前。储存胰蛋白酶工作溶液在4° c。</li>
		<li>用 1x PBS 去除培养基和洗涤细胞。添加0.5 毫升的 5ml 37 ° c 胰蛋白酶工作溶液, 并在37° c 孵育7分钟 (在细胞培养孵化器)。</li>
		<li>在孵化后, 在每个井中加入0.5 毫升的含血清培养基, 使胰蛋白酶失效, 并在离心管中收集细胞。用3毫升 1x PBS 冲洗一次, 并将细胞悬浮液离心5分钟, 在 240 x g。</li>
		<li>用5毫升 1x PBS 洗涤细胞, 离心5分钟, 在 240 x g 和丢弃上清液。使用新鲜收集的细胞小球或储存在-80 ° c。从小球中提取总 RNA, 进行半定量 rt-pcr (见步骤 6)。</li>
	</ol>
</li>
</ol>4. 人原发性细胞的转导 (如角质形成)
注意: 人类原发性角质形成细胞被播种到致命辐照的 3T3-J2 电池 (饲养层)23, 一种来自燕 Barrandon (洛桑, 洛桑, 瑞士) 的礼物。
<ol><li>在 Dulbecco 的改良鹰培养基中种植瑞士老鼠 3T3 J2 细胞, 辅以10% 供体牛血清、50种 U/毫升青霉素-链霉素和4毫米谷氨酰胺 (3T3 培养基)。</li>
	<li>在 cFAD 培养基上致命辐照的 3T3 J2 细胞生长角质形成, Dulbecco 的改良鹰培养基和火腿的 F12 培养基混合物 (3:1) 含有胎儿牛血清 (10%), 青霉素-链霉素 (1%), 谷氨酰胺 (2%), 胰岛素 (5 µg/毫升), 腺嘌呤 (0.18 毫米), 氢化可的松 (0.4 µg/毫升), 霍乱毒素 (0.1 nm), 和甲状腺 (2 nm)。</li>
	<li>0天: 种子 3x10^-75致命辐照的 3T3 J2 细胞, 以前稀释为3T3 培养基, 最终浓度为 1X105细胞/毫升, 每井 6-井板。准备9口井。</li>
	<li>1天: 原发性角质形成细胞的转导 注意: 角质形成细胞的转导允许定量的转录调节 SB 转的最佳剂量组合的两个 IDLV 载体 (通过 qRT PCR) 和验证的融合, 印度 (GFP) 的目标单元 (由 cytofluorimetric分析)。<ol>
<li>分离细胞前, 准备胰蛋白酶工作溶液: 0.5% 胰蛋白酶-EDTA (10x) 5.0 毫升 500mM EDTA (最终浓度 = 5 mM) 0.5 毫升 1x PBS 44.5 毫升</li>
		<li>热胰蛋白酶工作溶液在37° c 使用前。储存胰蛋白酶工作溶液在4° c。</li>
		<li>分离 subconfluent 角质形成细胞的培养, 去除培养基, 用 1x PBS 清洗电池, 并添加1毫升的5ml 胰蛋白酶工作液, 每一个井。孵育在37° c 为15分钟在细胞培养孵化器。</li>
		<li>在孵化后, 重彻底的细胞, 并将细胞悬液转移到含有2毫升含血清培养基的离心管中 (如果仍有许多细胞附着, 则在37° c 下孵育一分钟)。用3毫升的 1x PBS 或新鲜培养基冲洗每一次井, 并在离心管上添加冲洗液, 使其与细胞悬浮液一起使用。</li>
		<li>离心机5分钟, 在 580 x 克, 并丢弃上清。</li>
		<li>用5毫升的 1x PBS 洗涤细胞, 离心5分钟, 在 580 x g 和丢弃上清液。</li>
		<li>在15毫升聚丙烯管, 稀释 1.6x105角质形成1毫升的 cFAD 培养基, 一个稀释为每个条件。</li>
		<li>在1毫升的 cFAD 培养基中, 在20μ l 凝聚 (最终浓度为8µg/毫升, 在最后的2毫升) 中稀释慢载体: 稀释为井 #1, #2, #3: 模拟转细胞 (阴性控制没有传染媒介)。 稀释为井 #4, #5: IDLVTKSB 向量 (1.3万 ng p24) + IDLVrtTA2s-M2 向量 (4.8万 ng p24)。 稀释为井 #6, #7, #8, #9: IDLVTKSB 向量 (1.3万 ng p24) + IDLVrtTA2s-M2 向量 (4.8万 ng p24) + IDLVT2 向量 (9160 ng p24)。</li>
		<li>传感器细胞在悬浮通过增加每个慢向量稀释 (1 毫升) 到相应的角质形成悬浮 (1.6x105角质形成1毫升)。</li>
		<li>从致命照射的 3T3-J2 细胞中去除培养基, 在0天和平板转角质形成 (1.6x105角质形成2毫升) 到他们身上。在换以后一个井继续与下一个。</li>
		<li>孵育在25° c 为30分钟, 然后转移细胞到37° c 在细胞培养孵化器为 6 h。 注意: 不要 spinoculate 原发性角质形成细胞, 因为存活和增殖会受到强烈的影响。</li>
		<li>6小时后, 每井加1毫升的 cFAD 培养基。在水井 #5、#8 和 #9 中加入1μ m 强力霉素 (最终浓度)。返回细胞到37° c。</li>
	</ol>
</li>
	<li>2天: 用3毫升的 KC 培养基替换 cFAD 培养基, 10 ng/毫升 EGF。</li>
	<li>3天: 处理和洗涤细胞从井 #1, #4 和 #5 前面描述 (参见4.4.1 到 4.4.6)。在-80 ° c 下冷冻细胞颗粒提取总 RNA qRT-PCR 分析 (见步骤 7)。</li>
	<li>处理细胞从井 #2, #6 和 #8 的步骤 4.4.1 4.4.5 和执行 cytofluorimetric 分析 GFP 表达 (见步骤 5)。</li>
	<li>保持从水井 #3, #7 和 #9 至少 3-4 doublings 的文化, 足以稀释七零八落 IDLVT2 载体 (5 天的人原代角质形成在饲养层)。</li>
	<li>6天: 大约5天后转导, 处理细胞 (见步骤 4.4.1 4.4.5) 从水井 #3, #7, 并 #9 执行 cytofluorimetric 分析 GFP 表达 (见步骤 5)。 注意: 实验时间和转导效率受原细胞类型和采集样本变异性的影响很大。</li>
</ol>5. 转原代角质形成细胞的 Cytofluorimetric 分析
注: 使用蓝色激光 (488 nm)、红色激光 (633 nm) 和用于检测 GFP 和 APC 荧光的过滤器配置的流式细胞仪 (参见材料表)。
<ol><li>要区分角质形成细胞从 3T3 J2 饲养层, 标签转角质形成细胞分离在3天和一天 6 (见步骤4.7 和 4.9) 与鼠标单克隆 APC 共轭抗馈线抗体。<ol>
<li>为每个样品准备4毫升的染色缓冲液: 5% FBS 200 μ l 500毫米 EDTA (最终浓度 = 2.5 毫米) 20 μ l PBS 1x 3780 μ l</li>
		<li>用1毫升的染色缓冲液冲洗分离细胞。离心机5分钟, 在 580 x 克, 并丢弃上清。</li>
		<li>在流式细胞仪采集管中, 重 5x104细胞在100μ l 的染色缓冲液中, 并添加2μ l 抗馈线抗体 (1:50)。在黑暗中混合好, 在冰上孵育30分钟。</li>
		<li>30分钟后, 用2毫升的染色缓冲液冲洗。离心机5分钟, 在 580 x 克, 并丢弃上清。重在200μ l 的染色缓冲液中。</li>
		<li>通过 cytofluorimetric 分析18获取 APC 和 GFP 信号。APC 单元格中的 GFP+单元格分数表示转角质形成细胞. 注: 如果需要, 在流式细胞仪前固定染色样品, 2% 甲醛在 PBS 1x 和存储在4° c 在黑暗中, 直到运行。</li>
	</ol>
</li>
	<li>分析流式细胞仪数据通过绘制端点 (5 天) 的 gfp+单元格百分比和 gfp+单元格的百分比 (2 天后转导), 归入 IDLVT2-transduced 的剩余水平。细胞.</li>
</ol>6. 半定量 rt-pcr
注: 使用 PCR 热循环。
<ol><li>根据制造商的协议, 使用 rna 纯化试剂盒从转或控制细胞提取总 rna。 注: 总 RNA 可以被存放在-80 ° c。</li>
	<li>根据制造商的协议, 用 100 ng 总 RNA 和逆转录酶试剂盒合成20µL 反应中的 cDNA。 注: cDNA 可贮存于-20 ° c。</li>
	<li>设计底漆专门为 SB100X, rtTA2s-M2 和 GAPDH (一个管家基因)。 建议的设计: SB 前锋底漆: 5-GCCACTCAGCAAGGAAGAAG-3 " SB 反转底漆: 5-GTGTTGGAAGACCCATTTGC-3 " rtTAM2 前引物: 5"-GACGACAAGGAAACTCGCTC-3" rtTAM2 反向底漆: 5-TTACCCGGGAGCATGTCAA-3 " GAPDH 前引物: 5 "-GACCACAGTCCATGCCATCAC-3" GAPDH 反向引物: 5 "-CCACCACCCTGTTGCTGTAG-3"</li>
	<li>在50µL 的最终反应量中进行 PCR。特别是, 在每个 PCR 管集合以下反应: cDNA (1:5 稀释) (从步骤 6.2) 1.00 µL 前引底漆 (10 µM 库存) 1.00 µL 反向底漆 (10 µM 库存) 1.00 µL dNTPs (10 µM 库存) 1.00 µL 缓冲区 (+ 氯化镁2) (10x 库存) 5.00 µL Taq 聚合酶 (5 U/µL 库存) 0.25 µL 无菌水40.75 µL</li>
	<li>使用热循环的以下节目: 1 周期在95° c 为5分钟然后继续与95° c 为三十年代, 58 ° c 为三十年代和72° c 为三十年代为34个周期为 SB100X, 32 周期为 rtTA2s-M2, 25 周期为 GAPDH。</li>
	<li>准备1% 琼脂糖凝胶。将1克琼脂糖溶于100毫升的 1x (10x 库存稀释在无菌水中), 在烧杯或烧瓶中。融化在微波炉, 每分钟旋转, 直到琼脂糖完全溶解。冷却融化的琼脂糖, 直到它达到约50° c, 然后添加5µL 溴化溴 (10 毫克/毫升的股票)。倒入融化的琼脂糖在一个组装的凝胶托盘与梳子, 为凝胶铸造。</li>
	<li>在琼脂糖凝胶上装载样品 (10 µL), 并进行电泳。</li>
	<li>如果需要, 获取凝胶图片和执行密度分析的 PCR 带。</li>
</ol>7. 定量 rt-pcr (qRT pcr)
注意: 采用了商业序列检测系统。PCR 引物和 6-羧基 (家族) 探针为甘油 3-磷酸脱氢酶 (GAPDH) 是购买商业。
<ol><li>对于复古转录, 见步骤6.2。</li>
	<li>使用软件为 SB100X 设计底漆和探头。 建议设计: SB 2 前引物: 5 "-GAAGAAGCCACTGCTCCAAAA-3", SB 2 反向底漆: 5 "-CCCCATGTGCAGTTGCAA-3" 探索 SB: 5-CATAAGAAAGCCAGACTACGG-3 "</li>
	<li>在25µL 的最终反应量中, 用 pcr 主混合和引物 + 探针混合, 在96井板上进行实时 pcr。特别是, 考虑以下反应为每井: cDNA (1:10 稀释在无菌水中) 1.00 µL 2x 主混合12.50 µL 底漆 + 20x 探头混合1.25 µL 无菌水10.25 µL 注: 执行所有反应三份。为了防止移的错误, 准备一个混合至少 n+3 反应 (没有 cDNA)。分使用多通道吸管。</li>
	<li>使用以下程序运行 real-time PCR: 1 周期在95° c 为 10 min, 然后40个周期95° c 为十五年代和60° c 为 1 min 和举行在4° c。</li>
	<li>采用典型的数据分析软件, 通过 2-ΔΔCT量化, 将 SB100X 的相对表达式 (RQ 值) 正常化到同一 cDNA 样品的 GAPDH 水平, 对 qRT PCR 数据进行分析。</li>
</ol>

<ol>
	<li>Mates, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+evolution+of+a+novel+hyperactive+Sleeping+Beauty+transposase+enables+robust+stable+gene+transfer+in+vertebrates.">Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates.</a> Nat Genet. 41 (6), 753-761 (2009).</li><li>Maiti, S. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleeping+beauty+system+to+redirect+T-cell+specificity+for+human+applications.">Sleeping beauty system to redirect T-cell specificity for human applications.</a> J Immunother. 36 (2), 112-123 (2013).</li><li>Johnen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleeping+Beauty+transposon-mediated+transfection+of+retinal+and+iris+pigment+epithelial+cells.">Sleeping Beauty transposon-mediated transfection of retinal and iris pigment epithelial cells.</a> Invest Ophthalmol Vis Sci. 53 (8), 4787-4796 (2012).</li><li>Koso, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposon+mutagenesis+identifies+genes+that+transform+neural+stem+cells+into+glioma-initiating+cells.">Transposon mutagenesis identifies genes that transform neural stem cells into glioma-initiating cells.</a> Proc Natl Acad Sci U S A. 109 (44), E2998-E3007 (2012).</li><li>Belay, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+hyperactive+transposons+for+genetic+modification+of+induced+pluripotent+and+adult+stem+cells:+a+nonviral+paradigm+for+coaxed+differentiation.">Novel hyperactive transposons for genetic modification of induced pluripotent and adult stem cells: a nonviral paradigm for coaxed differentiation.</a> Stem Cells. 28 (10), 1760-1771 (2010).</li><li>Grabundzija, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleeping+Beauty+transposon-based+system+for+cellular+reprogramming+and+targeted+gene+insertion+in+induced+pluripotent+stem+cells.">Sleeping Beauty transposon-based system for cellular reprogramming and targeted gene insertion in induced pluripotent stem cells.</a> Nucleic Acids Res. 41 (3), 1829-1847 (2013).</li><li>Kues, W. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+and+characterization+of+sleeping+beauty+transposon-mediated+porcine+induced+pluripotent+stem+cells.">Derivation and characterization of sleeping beauty transposon-mediated porcine induced pluripotent stem cells.</a> Stem Cells Dev. 22 (1), 124-135 (2013).</li><li>Turchiano, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+analysis+of+Sleeping+Beauty+transposon+integration+in+human+somatic+cells.">Genomic analysis of Sleeping Beauty transposon integration in human somatic cells.</a> PLoS One. 9 (11), e112712 (2014).</li><li>Jin, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hyperactive+Sleeping+Beauty+transposase+SB100X+improves+the+genetic+modification+of+T+cells+to+express+a+chimeric+antigen+receptor.">The hyperactive Sleeping Beauty transposase SB100X improves the genetic modification of T cells to express a chimeric antigen receptor.</a> Gene Ther. 18 (9), 849-856 (2011).</li><li>Latella, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correction+of+Recessive+Dystrophic+Epidermolysis+Bullosa+by+Transposon-Mediated+Integration+of+COL7A1+in+Transplantable+Patient-Derived+Primary+Keratinocytes.">Correction of Recessive Dystrophic Epidermolysis Bullosa by Transposon-Mediated Integration of COL7A1 in Transplantable Patient-Derived Primary Keratinocytes.</a> J Invest Dermatol. 137 (4), 836-844 (2017).</li><li>Zhang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+profile+and+safety+of+an+adenovirus+hybrid-vector+utilizing+hyperactive+sleeping+beauty+transposase+for+somatic+integration.">Integration profile and safety of an adenovirus hybrid-vector utilizing hyperactive sleeping beauty transposase for somatic integration.</a> PLoS One. 8 (10), e75344 (2013).</li><li>Zhang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+adeno-associated+viral+vectors+utilizing+transposase-mediated+somatic+integration+for+stable+transgene+expression+in+human+cells.">Hybrid adeno-associated viral vectors utilizing transposase-mediated somatic integration for stable transgene expression in human cells.</a> PLoS One. 8 (10), e76771 (2013).</li><li>Turunen, T. A., Laakkonen, J. P., Alasaarela, L., Airenne, K. J., Yla-Herttuala, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sleeping+Beauty-baculovirus+hybrid+vectors+for+long-term+gene+expression+in+the+eye.">Sleeping Beauty-baculovirus hybrid vectors for long-term gene expression in the eye.</a> J Gene Med. 16 (1-2), 40-53 (2014).</li><li>Galla, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avoiding+cytotoxicity+of+transposases+by+dose-controlled+mRNA+delivery.">Avoiding cytotoxicity of transposases by dose-controlled mRNA delivery.</a> Nucleic Acids Res. 39 (16), 7147-7160 (2011).</li><li>Moldt, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+genomic+integration+profiling+of+Sleeping+Beauty+transposons+mobilized+with+high+efficacy+from+integrase-defective+lentiviral+vectors+in+primary+human+cells.">Comparative genomic integration profiling of Sleeping Beauty transposons mobilized with high efficacy from integrase-defective lentiviral vectors in primary human cells.</a> Mol Ther. 19 (8), 1499-1510 (2011).</li><li>Recchia, A., Perani, L., Sartori, D., Olgiati, C., Mavilio, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+integration+of+functional+transgenes+into+the+human+genome+by+adeno/AAV+hybrid+vectors.">Site-specific integration of functional transgenes into the human genome by adeno/AAV hybrid vectors.</a> Mol. Ther. 10, 660-670 (2004).</li><li>Urlinger, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+sequence+space+for+tetracycline-dependent+transcriptional+activators:+novel+mutations+yield+expanded+range+and+sensitivity.">Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity.</a> Proc Natl Acad Sci U S A. 97 (14), 7963-7968 (2000).</li><li>Cocchiarella, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptionally+regulated+and+nontoxic+delivery+of+the+hyperactive+Sleeping+Beauty+Transposase.">Transcriptionally regulated and nontoxic delivery of the hyperactive Sleeping Beauty Transposase.</a> Mol Ther Methods Clin Dev. 3, 16038 (2016).</li><li>Yanez-Munoz, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+gene+therapy+with+nonintegrating+lentiviral+vectors.">Effective gene therapy with nonintegrating lentiviral vectors.</a> Nat Med. 12 (3), 348-353 (2006).</li><li>Dull, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+third-generation+lentivirus+vector+with+a+conditional+packaging+system.">A third-generation lentivirus vector with a conditional packaging system.</a> J Virol. 72 (11), 8463-8471 (1998).</li><li>Bak, R. O., Mikkelsen, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobilization+of+DNA+transposable+elements+from+lentiviral+vectors.">Mobilization of DNA transposable elements from lentiviral vectors.</a> Mob Genet Elements. 1 (2), 139-144 (2011).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A laboratory Manual. , (2012).</li><li>Dellambra, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corrective+transduction+of+human+epidermal+stem+cells+in+laminin-5-dependent+junctional+epidermolysis+bullosa.">Corrective transduction of human epidermal stem cells in laminin-5-dependent junctional epidermolysis bullosa.</a> Hum Gene Ther. 9, 1359-1370 (1998).</li></ol>

作者没有什么可透露的。

在这里, 我们描述一个广泛的方法, 以稳定地整合到目标细胞的基因组通过睡美人介导的转位。尽管 SB 系统的开发是为了提供一种病毒的基因组编辑方法, 但有效地交付集成机械 (转和 T2 转) 是强制性的。因此, 使用 sb 系统在几乎不 transfectable 的主要细胞, 病毒交付 sb 组分在最近几年被追求10,12,15。然而, 目前使用的病毒?...

多动 SB100X 转耦合到 T2-based 转已被用于临床前基因治疗应用1,2,3, 基因组修改4,5, 以及诱导多能干细胞 (iPSC) 重新编程6,7。典型地, SB 组分由质粒转染瞬时地提供, 并且一个强的本构子推动转的表示。然而, 尽管改进了新的转染方法, 但两组分系统的交付仍然是许多应用的挑战。因此, 新的交付议定书的发展引起了越来越多的兴趣。除转染方法为质粒8和 mRNA9, 病毒传递的基础上腺病毒10,11, 腺相关病毒 (AAV)12, 病毒性13, gammaretroviral14和慢向量15已在过去提出。值得注意的是, 选择系统必须保证在不集成病毒载体的情况下暂时交付 SB 部件。然而, 由于转的不可控 rehopping 的风险, 转的本构表达引起了安全问题。
因此, SB100X 的转录调控是第一个挑战。通过将原发育的最小巨细胞病毒 (CMV) 启动子替换为单纯疱疹 Virus-1 (HSV-1)16的 Timidine 激酶 (TK) 基因的最小启动子 (-81), 在SB100X cDNA (pTetOTKSB100X)。突变的反转四环素-反 rtTA2s-M217是在强本构子 (甘油促进剂, PGK) 的控制下, 在不同质粒 (pCCL-PGKrtTA2s-M2) 中克隆的。当添加到培养基中时, dox 绑定 rtTA2s-M2 调制器和系的新的启动子复合体或, 从而导致严格调节的 SB100X 表达式的归纳18。
第二个主要的挑战是由几种物理和化学方法对人类原细胞的低效转染。为了有效地将转在人类细胞中抗转染, SB100X 和 rtTA2s-M2 表达式磁带被打包成两个集成缺陷慢向量 (IDLVs)19: IDLVTKSB 和 IDLVrtTA2sM2病毒载体瞬时产生作为第三代载体在 HEK293T 细胞20和型的糖蛋白 g 的 Vescicular 口炎病毒 (VSV-g), 它提供了广泛的感染范围。矢量粒子由离心和滴定 HIV-1 塞 p24 免疫。对于传感器细胞系和原细胞, 载体颗粒与凝聚复合, 在 dox 存在或缺失时与靶细胞孵育。用定量 rt-pcr (qRT-pcr)18测定了转细胞中 SB100X 表达的药物依赖性活化。
一旦春节调控的 SB100X 表达被证明, 将印度 (如绿色荧光蛋白, GFP) 转到靶细胞基因组中的分子事件, 由 Bak 和泰尔杰·密盖尔森21清楚地化。在 T2-transposon 国税局 (IDLVT2) 之间复制的一个表达式卡带的第三 IDLV 载体必须在上面提到的构造和包装。最后, 三 IDLV 载体可用于有效地传感器人体细胞 (如, 原发角质形成细胞)在体外, 并在强力霉素18的存在中整合印度。

<table><tbody><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium with 4.5g/L glucose w/o L-Glutamine</td><td>Lonza</td><td>BE12-614F</td><td>Cell culture medium.</td></tr><tr><td>HyClone Fetal Bovine Serum (U.S.), Characterized</td><td>GE Healthcare Life Sciences</td><td>SH30071.03</td><td>Serum used HEK293T cell culture for lentiviral particle production.</td></tr><tr><td>Penicillin 10.000 UI/ml Streptomycin 10.000 UI/ml</td><td>Lonza</td><td>DE17-602E</td><td>Reagents for cell culture medium.</td></tr><tr><td>L-Glutamine 200mM</td><td>Lonza</td><td>BE17-605E</td><td>Reagent for cell culture medium.</td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Sigma-Aldrich</td><td>S7653</td><td>Reagent for HBS 2X preparation.</td></tr><tr><td>HEPES Buffer, 1M Stock in normal saline</td><td>Lonza</td><td>BE17-737E</td><td>Reagent for HBS 2X preparation.</td></tr><tr><td>Disodium hydrogen phosphate dihydrate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O), 99.5%</td><td>Merck</td><td>106580</td><td>Reagent for HBS 2X preparation.</td></tr><tr><td>Hydrochloric acid - ACS reagent 37% (HCl)</td><td>Sigma-Aldrich</td><td>320331</td><td>Reagent for HBS 2X preparation.</td></tr><tr><td>Sterile water for injection</td><td>Fresenius Kabi</td><td>-</td><td>Reagent for HBS 2X preparation.</td></tr><tr><td>0.45 &amp;micro;m PESS filter</td><td>Whatman</td><td>10462100</td><td>Filters used to remove cell debris from viral supernatant.</td></tr><tr><td>Polyallomer Beckman tubes</td><td>Beckman Coulter</td><td>326823</td><td>Tubes for concentrating IDLV vectors by ultracentrifugation.</td></tr><tr><td>PBS-1X w/o Ca, Mg</td><td>Lonza</td><td>BE17-516F</td><td>Buffer for cell wash and lentiviral particle resuspension.</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A2153</td><td>(Optional) Buffer for lentiviral particle resuspension.</td></tr><tr><td>HIV-1 Gag p24 immunocapture kit</td><td>Perkin Elmer</td><td>NEK50001KT</td><td>Kit for IDLV particle titration.</td></tr><tr><td>Polybrene</td><td>Sigma-Aldrich</td><td>107689</td><td>Reagent to enhance transduction efficiency.</td></tr><tr><td>Trypsin (10X) 2.5% in HBSS w/o Ca, Mg</td><td>GIBCO</td><td>BE17-160E</td><td>Reagent to detach HeLa cells (stock solution, to be diluted).</td></tr><tr><td>Ethylenediaminetetraacetic acid disodium salt (EDTA)</td><td>100938B</td><td>AnalaR BDH</td><td>Reagent for trypsin working solution preparation.</td></tr><tr><td>0.5% Trypsin-EDTA, no phenol red (10X)</td><td>GIBCO</td><td>15400-054</td><td>Reagent to detach keratinocytes (stock solution, to be diluted).</td></tr><tr><td>Donor Bovine Serum, New Zealand Origin</td><td>GIBCO</td><td>16030-074</td><td>Serum for Swiss mouse 3T3-J2 cell culture (feeder layer).</td></tr><tr><td>Ham&amp;rsquo;s F12 media</td><td>GIBCO</td><td>21765</td><td>Medium for keratinocytes.</td></tr><tr><td>Fetal bovine serum</td><td>Lonza</td><td>DE14-801F</td><td>Serum for HeLa cell culture medium.</td></tr><tr><td>Fetal Bovine Serum, qualified, Australia Origin</td><td>GIBCO</td><td>10099-141</td><td>Serum for human primary keratinocyte culture medium.</td></tr><tr><td>Insulin from bovine pancreas</td><td>Sigma-Aldrich</td><td>I5500</td><td>Reagents for keratinocyte growth.</td></tr><tr><td>Adenine</td><td>VWR</td><td>1152-25</td><td>Reagents for keratinocyte growth.</td></tr><tr><td>Hydrocortisone</td><td>VWR</td><td>3867-1</td><td>Reagents for keratinocyte growth.</td></tr><tr><td>Cholera Toxin from Vibrio cholerae</td><td>Sigma-Aldrich</td><td>C8052</td><td>Reagents for keratinocyte growth.</td></tr><tr><td>Triiodothyronine</td><td>Sigma-Aldrich</td><td>T5516</td><td>Reagents for keratinocyte growth.</td></tr><tr><td>Human EGF</td><td>Austral Biological</td><td>GF-010-9</td><td>Reagents for keratinocyte growth.</td></tr><tr><td>Mouse monoclonal APC-conjugated Anti-Feeder antibody</td><td>Miltenyi Biotech</td><td>130-096-099</td><td>To label feeder layer.</td></tr><tr><td>RNeasy Plus Mini Kit</td><td>Qiagen</td><td>74134</td><td>RNA purification kit.</td></tr><tr><td>SuperScript III Reverse Transcriptase</td><td>Life Technologies</td><td>18080051</td><td>Reverse transcriptase kit.</td></tr><tr><td>Deoxynucleotide Mix, 10 mM (dNTP)</td><td>Sigma-Aldrich</td><td>D7295</td><td>Reagent for semi-quantitative PCR.</td></tr><tr><td>UltraPure DNase/RNase-Free Distilled Water</td><td>(any)</td><td>-</td><td>Reagent for semi-quantitative PCR and for qRT-PCR.</td></tr><tr><td>GoTaq G2 Hot Start Polymerase</td><td>Promega</td><td>M740B</td><td>Taq polymerase.</td></tr><tr><td>Agarose, for molecular biology</td><td>Sigma-Aldrich</td><td>A9539</td><td>Reagent for agarose gel electrophoresis.</td></tr><tr><td>UltraPure TBE Buffer, 10X</td><td>Invitrogen</td><td>15581028</td><td>Reagent for agarose gel electrophoresis, to be dilute (1X) in ddH2O.</td></tr><tr><td>Ethidium bromide</td><td>Sigma-Aldrich</td><td>E1510</td><td>Reagent for agarose gel electrophoresis.</td></tr><tr><td>Doxycycline</td><td>Sigma-Aldrich</td><td>D-9891</td><td>drug to induce transposase expression in HeLa cells and in keratinocytes</td></tr><tr><td>TaqMan Universal PCR Master Mix</td><td>Applied Biosystem</td><td>4304437</td><td>TaqMan Universal PCR Master Mix including primers and probe.</td></tr><tr><td>15mL High Clarity polypropylene Centrifuge Tube, Conical Bottom, with Dome Seal Screw Cap, Sterile</td><td>Falcon Corning</td><td>352096</td><td>Tubes for cell collection and vector dilution preparation.</td></tr><tr><td>150 mm TC-Treated Cell Culture Dish with 20 mm Grid, Sterile</td><td>Falcon Corning</td><td>353025</td><td>Cell culture dish for viral production.</td></tr><tr><td>6 Well Clear Flat Bottom TC-Treated Multiwell Cell Culture Plate, with Lid, Individually Wrapped, Sterile</td><td>Falcon Corning</td><td>353046</td><td>Cell culture plate for HeLa and human primary keratinocyte transduction.</td></tr><tr><td>Safe-Lock microcentrifuge tubes, volume 1.5 mL</td><td>Eppendorf</td><td>0030 120.086</td><td>Tubes for dilution preparation.</td></tr><tr><td>Safe-Lock microcentrifuge tubes, volume 2 mL</td><td>Eppendorf</td><td>0030 120.094</td><td>Tubes for dilution preparation.</td></tr><tr><td>0.2ml PCR Tubes with flat caps, RNase, DNase, DNA and PCR Inhibitor free</td><td>(any)</td><td>-</td><td>Tubes for semi-quantitative PCR.</td></tr><tr><td>MicroAmp Fast Optical 96-Well Reaction Plate with Barcode, 0.1 mL</td><td>Applied Biosystem</td><td>4346906</td><td>96-well for qRT-PCR.</td></tr><tr><td>5mL Round Bottom Polystyrene Tube, without Cap</td><td>BD Falcon</td><td>352052</td><td>Tubes for flow cytometry acquisition.</td></tr><tr><td>Disposable Serological Pipets, Polystyrene, Individually Wrapped, Sterile (different volumes)</td><td>(any)</td><td>-</td><td>Pipettes for sterile tissue culture applications.</td></tr><tr><td>Filter Tips, Sterile, and RNase, DNase, DNA and Pyrogen free (different volumes)</td><td>(any)</td><td>-</td><td>Filter tips for sterile tissue colture and molecular biology applications.</td></tr><tr><td>pH meter</td><td>(any)</td><td>-</td><td>Equipment for measurement of HBS 2X pH.</td></tr><tr><td>PCR Thermal cycler</td><td>(any)</td><td>-</td><td>Equipment for semi-quantitative PCR.</td></tr><tr><td>Agarose gel electrophoresis equipment</td><td>(any)</td><td>-</td><td>Equipment for agarose gel electrophoresis of semi-quantitative PCR.</td></tr><tr><td>BD FACS Canto II 2LSR 4/2</td><td>BD</td><td>-</td><td>Flow cytometer.</td></tr><tr><td>7900HT Fast Real-Time PCR system</td><td>Applied Biosystem</td><td>7900HT</td><td>Equipment for qRT-PCR.</td></tr><tr><td>Benchtop centrifuges for efficient sample processing in cell culture applications, refrigerated, with microplate buckets.</td><td>(any)</td><td>-</td><td>Centrifuge for cell culture processing and spinoculation.</td></tr><tr><td>Optima L-90K Ultracentrifuge equipped with SW32TI rotor</td><td>Beckman Coulter</td><td>-</td><td>Ultracentrifuge for lentiviral particle production.</td></tr><tr><td>Equipment for cell culture and molecular biology laboratory (e.g., Class II laminar flow hood designed for work involving BSL2 materials, cell culture incubator,refrigerators and freezers (+4 &amp;deg;C, -20 &amp;deg;C, -80 &amp;deg;C), micropipettes and pipet-aid).</td><td></td><td></td><td></td></tr><tr><td>Protective laboratory coats, gloves, face protection in accordance with Institute rules and regulations, in particular for lentiviral particle production and transduction.</td><td></td><td></td><td></td></tr></tbody></table>

an efficient in vitro transposition method by a transcriptionally regulated sleeping beauty system packaged into an integration defective lentiviral vector

睡美人(SB) 转是一种病毒的集成系统, 具有有效的基因转移和功能基因组学的功效。为优化 SB 转机械, 采用转录调节过动转 (SB100X) 和 T2-based 转。通常, 转和转通过质粒转染而瞬时提供, SB100X 表达由本构子驱动。在这里, 我们描述了一个有效的方法, 将 sb 组件传递给那些对几种物理和化学转染方法有抵抗力的人体细胞, 控制 SB100X 表达, 并通过 "剪切和粘贴", 稳定地整合一个感兴趣的基因 (印度)。机制.自1992年以来, 多动转的表达受到了系统的严格控制, 广泛用于控制基因表达。该基因的兴趣是两侧的反向重复 (IR) 的 T2 转。在 HEK293T 细胞中, 两个部件都被封装在集成缺陷的慢载体上。人类细胞, 无论是细胞系或人类组织的主要细胞, 都是在体外瞬时转与病毒载体。在添加强力霉素 (dox, 四环素模拟) 到培养基中, fine-tuning 的转表达的测量, 并导致长期的基因的兴趣在处理细胞基因组的结合。这种方法是有效的和适用于细胞线 (如, HeLa 细胞) 和原细胞 (如, 人原发角质形成), 从而代表了一个有价值的工具, 基因工程和治疗基因转移。

使用这里介绍的程序, 三 IDLV 载体运载被调控的 sb 组分 (SB100X, rtTA2S-M2 和 T2-GFP 转) 被包装并且用于高效率地运载和严密地调控 sb 系统在人的细胞。图 1A显示了一个用于 HeLa 细胞的体外转导的方案, 它是用两个 IDLV 向量的最佳剂量组合 (在表 2中报告) 来评估 SB 转的转录调节。用 qRT PCR (图 1B) 测量了 SB10...

Watch this Scientific Journal Video about An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector at

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

一种有效的体外转位方法由转录调节的睡眠美容系统封装到一个集成缺陷的慢向量

该协议描述了一种方法, 以实现稳定的基因的兴趣进入人类基因组由转录调控的睡美人系统。本文报道了有缺陷的慢载体的合成、人体细胞的体外转导和转细胞的分子检测。

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...

an-efficient-vitro-transposition-method-transcriptionally-regulated

Research

JoVE Journal

Biology

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

8.8K 次观看.  University of Modena and Reggio Emilia. 该协议描述了一种方法, 以实现稳定的基因的兴趣进入人类基因组由转录调控的睡美人系统。本文报道了有缺陷的慢载体的合成、人体细胞的体外转导和转细胞的分子检测。

视频: An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells and mediating high levels of gene expression. However, their ability to integrate into the host cell genome enhances the risk of insertional mutagenicity and thus raises safety concerns and limits their usage in clinical settings. Further, the persistent expression of gene-editing components delivered by these integration-competent lentiviral vectors (ICLVs) increases the probability of promiscuous gene targeting. As an alternative, a new generation of integrase-deficient lentiviral vectors (IDLVs) has been developed that addresses many of these concerns. Here the production protocol of a new and improved IDLV platform for CRISPR-mediated gene editing and list the steps involved in the purification and concentration of such vectors is described and their transduction and gene-editing efficiency using HEK-293T cells was demonstrated. This protocol is easily scalable and can be used to generate high titer IDLVs that are capable of transducing cells in vitro and in vivo. Moreover, this protocol can be easily adapted for the production of ICLVs.

We describe the production strategy of integrase-deficient lentiviral vectors (IDLVs) as vehicles for delivering CRISPR/Cas9 to cells. With an ability to mediate quick and robust gene editing in cells, IDLVs present a safer and equally effective vector platform for gene delivery compared to integrase-competent vectors.

整合酶缺陷慢病毒载体载体的制备及其在细胞 CRISPR/Cas9-mediated 基因敲除中的表达

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells ...

科研

遗传学

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a routine procedure is widely utilized throughout the world and its main limitation resides in the poor efficacy of transgene integration, resulting in a low yield of founder animals. Only few percent of animals born after implantation of injected fertilized oocytes have integrated the transgene. In contrast, lentiviral vectors are powerful tools for integrative gene transfer and their use to transduce fertilized oocytes allows highly efficient production of founder transgenic mice with an average yield above 70%. Furthermore, any mouse strain can be used to produce transgenic animal and the penetrance of transgene expression is extremely high, above 80% with lentiviral mediated transgenesis compared to DNA microinjection. The size of the DNA fragment that can be cargo by the lentiviral vector is restricted to 10 kb and represents the major limitation of this method. Using a simple and easy to perform injection procedure beneath the zona pellucida of fertilized oocytes, more than 50 founder animals can be produced in a single session of microinjection. Such a method is highly adapted to perform, directly in founder animals, rapid gain and loss of function studies or to screen genomic DNA regions for their ability to control and regulate gene expression in vivo.

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.

慢病毒载体介导的转基因小鼠生产: 一种简单高效的创始人直接研究方法

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

慢病毒载体平台, 用于将表观基因编辑工具高效地传递到人类诱导的多能干细胞衍生疾病模型中

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

Production of Lentiviral Vectors for Transducing Cells from the Central Nervous System

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing therapeutic approaches. Lentiviral vectors are attractive tools in transduction of neurons and other cell types in CNS as they transduce both dividing and non-dividing cells, support sustained expression of transgenes, and have relatively large packaging capacity and low toxicity 1-3. Lentiviral vectors have been successfully used in transducing many neural cell types in vitro 4-6 and in animals 7-10.
Great efforts have been made to develop lentiviral vectors with improved biosafety and efficiency for gene delivery. The current third generation replication-defective and self-inactivating (SIN) lentiviral vectors are depicted in Figure 1. The required elements for vector packaging are split into four plasmids. In the lentiviral transfer plasmid, the U3 region in the 5' long terminal repeat (LTR) is replaced with a strong promoter from another virus. This modification allows the transcription of the vector sequence independent of HIV-1 Tat protein that is normally required for HIV gene expression 11. The packaging signal (&Psi;) is essential for encapsidation and the Rev-responsive element (RRE) is required for producing high titer vectors. The central polypurine tract (cPPT) is important for nuclear import of the vector DNA, a feature required for transducing non-dividing cells 12. In the 3' LTR, the cis-regulatory sequences are completely removed from the U3 region. This deletion is copied to 5' LTR after reverse transcription, resulting in transcriptional inactivation of both LTRs. Plasmid pMDLg/pRRE contains HIV-1 gag/pol genes, which provide structural proteins and reverse transcriptase. pRSV-Rev encodes Rev which binds to the RRE for efficient RNA export from the nucleus. pCMV-G encodes the vesicular stomatitis virus glycoprotein (VSV-G) that replaces HIV-1 Env. VSV-G expands the tropism of the vectors and allows concentration via ultracentrifugation 13. All the genes encoding the accessory proteins, including Vif, Vpr, Vpu, and Nef are excluded in the packaging system. The production and manipulation of lentiviral vectors should be carried out according to NIH guidelines for research involving recombinant DNA (<a href="http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf" target="_blank">http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf</a>). An approval from individual Institutional Biological and Chemical Safety Committee may be required before using lentiviral vectors. Lentiviral vectors are commonly produced by cotransfection of 293T cells with lentiviral transfer plasmid and the helper plasmids encoding the proteins required for vector packaging. Many lentiviral transfer plasmids and helper plasmids can be obtained from Addgene, a non-profit plasmid repository (<a href="http://www.addgene.org/" target="_blank">http://www.addgene.org/</a>). Some stable packaging cell lines have been developed, but these systems provide less flexibility and their packaging efficiency generally declines over time 14, 15. Commercially available transfection kits may support high efficiency of transfection 16, but they can be very expensive for large scale vector preparations. Calcium phosphate precipitation methods provide highly efficient transfection of 293T cells and thus provide a reliable and cost effective approach for lentiviral vector production.
In this protocol, we produce lentiviral vectors by cotransfection of 293T cells with four plasmids based on the calcium phosphate precipitation principle, followed by purification and concentration with ultracentrifugation through a 20% sucrose cushion. The vector titers are determined by fluorescence- activated cell sorting (FACS) analysis or by real time qPCR. The production and titration of lentiviral vectors in this protocol can be finished with 9 days. We provide an example of transducing these vectors into murine neocortical cultures containing both neurons and astrocytes. We demonstrate that lentiviral vectors support high efficiency of transduction and cell type-specific gene expression in primary cultured cells from CNS.

In this protocol we describe production, purification and titration of lentiviral vectors. We provide an example of lentiviral vector-mediated gene delivery in primary cultured neurons and astrocytes. Our methods may also apply to other cell types in vitro and in vivo.

从中枢神经系统转导细胞的慢病毒载体生产

Efficient gene delivery in the central nervous system (CNS) is important in studying gene functions, modeling neurological diseases and developing ...

神经科学

Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary brain tumor in adults with dismal prognosis. New potential therapies are rapidly emerging from the bench and require systematic testing in experimental models which closely reproduce the salient features of the human disease. Herein we describe in detail a method to induce new models of GBM with transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mice. We present a simple way to clone new transposons amenable for genomic integration using the Sleeping Beauty transposon system and illustrate how to monitor plasmid uptake and disease progression using bioluminescence, histology and immuno-histochemistry. We also describe a method to create new primary GBM cell lines. Ideally, this report will allow further dissemination of the Sleeping Beauty transposon system among brain tumor researchers, leading to an in depth understanding of GBM pathogenesis and progression and to the timely design and testing of effective therapies for patients.

Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.

质粒DNA转座子介导整合到新生小鼠的脑室下区，以生成胶质母细胞瘤的新模型

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary ...

医学

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique has been widely used to encode fluorescent proteins, opto- or chemo-genetic controllers of cell activity, or heterologous expression of human genes in model organisms. These viruses have also successfully been used to deliver recombinases to relevant target sites in transgenic animals, or even deliver small hairpin or micro RNAs in order to manipulate gene expression. While these techniques have been fruitful, they rely on transgenic animals (recombinases) or frequently lack high efficacy and specificity (shRNA/miRNA). In contrast, the CRISPR/Cas system uses an exogenous Cas nuclease which targets specific sites in an organism's genome via an exogenous guide RNA in order to induce double stranded breaks in DNA. These breaks are then repaired by non-homologous end joining (NHEJ), producing insertion and deletion (indel) mutations that can result in deleterious missense or nonsense mutations. This manuscript provides detailed methods for the design, production, injection, and validation of single lenti/retro virus particles that can stably transduce neurons to express a fluorescent reporter, Cas9, and sgRNAs to knockout genes in a model organism.

The CRISPR/Cas9 system offers the potential to make targeted genome editing accessible and affordable to the scientific community. This protocol is intended to demonstrate how to create viruses that will knockout a gene of interest using the CRISPR/Cas9 system, and then inject them stereotaxically into the adult mouse brain.

设计，包装，并通过立体定向注射高效价CRISPR复古和慢交付

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

慢病毒介导的击倒在体外</em人造血干细胞>红细胞

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

生物学

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications.

Lentiviral CRISPR/Cas9介导基因组编辑，用于疾病模型中造血细胞的研究

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting ...

免疫与感染

<meta charset="utf8"></head><body>用于体内基因调节的慢病毒载体制备

Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue&#45;Resident Macrophages In Vivo in Mice

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring tissues. Their functions are dictated by microenvironmental cues; thus, it is essential to investigate their behavior in an in vivo physiological niche. Currently, specific peritoneal macrophage-targeting methodologies employ whole-mouse transgenic models. Here, a protocol for effective in vivo modulation of mRNA and small RNA species (e.g., microRNA) expression in peritoneal macrophages using lentivirus particles is described. Lentivirus preparations were made in HEK293T cells and purified on a single sucrose layer. In vivo validation of lentivirus effectivity following intraperitoneal injection revealed predominant infection of macrophages restricted to local tissue. Targeting of peritoneal macrophages was successful during homeostasis and thioglycolate-induced peritonitis. The limitations of the protocol, including low-level inflammation induced by intraperitoneal delivery of lentivirus and time restrictions for potential experiments, are discussed. Overall, this study presents a quick and accessible protocol for the rapid assessment of gene function in peritoneal macrophages in vivo.

<meta charset="utf8"></head><body>作者聚焦:通过慢病毒载体介导的基因操作探索巨噬细胞免疫代谢

We demonstrate a step-by-step protocol for the investigation of gene function in peritoneal tissue-resident macrophages in vivo, using lentiviral vectors.

小鼠 体内 腹膜腔组织驻留巨噬细胞高效基因和 MicroRNA 调控的慢病毒载体制备

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring ...

一种有效的体外转位方法由转录调节的睡眠美容系统封装到一个集成缺陷的慢向量

摘要

探索更多视频

此视频中的章节

整合酶缺陷慢病毒载体载体的制备及其在细胞 CRISPR/Cas9-mediated 基因敲除中的表达

慢病毒载体介导的转基因小鼠生产: 一种简单高效的创始人直接研究方法

慢病毒载体平台, 用于将表观基因编辑工具高效地传递到人类诱导的多能干细胞衍生疾病模型中

从中枢神经系统转导细胞的慢病毒载体生产

质粒DNA转座子介导整合到新生小鼠的脑室下区，以生成胶质母细胞瘤的新模型

设计，包装，并通过立体定向注射高效价CRISPR复古和慢交付

慢病毒介导的击倒在体外红细胞

Lentiviral CRISPR/Cas9介导基因组编辑，用于疾病模型中造血细胞的研究

小鼠体内腹膜腔组织驻留巨噬细胞高效基因和 MicroRNA 调控的慢病毒载体制备

小鼠体内腹膜腔组织驻留巨噬细胞高效基因和 MicroRNA 调控的慢病毒载体制备

一种有效的体外转位方法由转录调节的睡眠美容系统封装到一个集成缺陷的慢向量

摘要

探索更多视频

此视频中的章节

整合酶缺陷慢病毒载体载体的制备及其在细胞 CRISPR/Cas9-mediated 基因敲除中的表达

慢病毒载体介导的转基因小鼠生产: 一种简单高效的创始人直接研究方法

慢病毒载体平台, 用于将表观基因编辑工具高效地传递到人类诱导的多能干细胞衍生疾病模型中

从中枢神经系统转导细胞的慢病毒载体生产

质粒DNA转座子介导整合到新生小鼠的脑室下区，以生成胶质母细胞瘤的新模型

设计，包装，并通过立体定向注射高效价CRISPR复古和慢交付

慢病毒介导的击倒在体外红细胞

Lentiviral CRISPR/Cas9介导基因组编辑，用于疾病模型中造血细胞的研究

小鼠 体内 腹膜腔组织驻留巨噬细胞高效基因和 MicroRNA 调控的慢病毒载体制备

小鼠 体内 腹膜腔组织驻留巨噬细胞高效基因和 MicroRNA 调控的慢病毒载体制备

小鼠体内腹膜腔组织驻留巨噬细胞高效基因和 MicroRNA 调控的慢病毒载体制备

小鼠体内腹膜腔组织驻留巨噬细胞高效基因和 MicroRNA 调控的慢病毒载体制备