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&#8217;s modified Eagle&#8217;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 (Na2HPO4&#183;2H2O), 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 &#181;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&#8217;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 colspan="4">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 &#176;C, -20 &#176;C, -80 &#176;C), micropipettes and pipet-aid).</td>
		</tr>
		<tr>
			<td colspan="4">Protective laboratory coats, gloves, face protection in accordance with Institute rules and regulations, in particular for lentiviral particle production and transduction.</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

Universidad de Cartagena UDEC

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

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFR&#945;, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transfection protocol using 293T cells, a human renal epithelial cell line transformed by the adenovirus E1A gene product. 293 cells have the unusual property of being highly transfectable by calcium phosphate (CaPO4), with up to 50-80% transfection efficiency readily attainable. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80&deg;C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model.

This technique demonstrates an efficient way to prepare replication-defective retroviral stocks encoding a human oncogene, and subsequently used for induction of myeloproliferative disease in the mouse model.

瞬时转染293T细胞复制缺陷型逆转录病毒生产

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, ...

科研

生物学

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

自制的网站定向诱变整个质粒

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

人类体细胞重编程为诱导多能干细胞（iPS细胞）用绿色荧光蛋白逆转录病毒载体

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a particularly attractive option for studying membrane proteins, especially in the context of ligand-receptor interactions. The method pioneered by Sligar and colleagues is based on the amphipathic properties of an engineered highly a-helical scaffold protein derived from the apolipoprotein A1. The hydrophobic faces of the scaffold protein interact with the fatty acyl side-chains of the lipid bilayer whereas the polar regions face the aqueous environment. Analyses of membrane proteins in nanodiscs have significant advantages over liposome because the particles are small, homogeneous and water-soluble. In addition, biochemical and biophysical methods normally reserved to soluble proteins can be applied, and from either side of the membrane. In this visual protocol, we present a step-by-step reconstitution of a well characterized bacterial ABC transporter, the MalE-MalFGK2 complex. The formation of the disc is a self-assembly process that depends on hydrophobic interactions taking place during the progressive removal of the detergent. We describe the essential steps and we highlight the importance of choosing a correct protein-to-lipid ratio in order to limit the formation of aggregates and larger polydisperse liposome-like particles. Simple quality controls such as gel filtration chromatography, native gel electrophoresis and dynamic light scattering spectroscopy ensure that the discs have been properly reconstituted.

Nanodiscs are small discoid particles that incorporate membrane proteins into a small patch of phospholipid bilayer. We provide a visual protocol that shows the step-by-step incorporation of the MalFGK2 transporter into a disc.

一个循序渐进的步骤方法的到Nanodisc脂质颗粒的ABC转运蛋白的重组

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

混合式切割:用于顽拗植物组织样品的改进切法

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

一种用于研究卡波西肉瘤细胞转化的体外模型

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...

An Efficient and Flexible Cell Aggregation Method for 3D Spheroid Production

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. Three-dimensional (3D) cell culture techniques are a recent innovation developed to address the shortcomings of adherent cell culture. While several techniques for generating tissue analogues in vitro have been developed, these methods are frequently complex, expensive to establish, require specialized equipment, and are generally limited by compatibility with only certain cell types. Here, we describe a rapid and flexible protocol for aggregating cells into multicellular 3D spheroids of consistent size that is compatible with growth of a variety of tumor and normal cell lines. We utilize varying concentrations of serum and methyl cellulose (MC) to promote anchorage-independent spheroid generation and prevent the formation of cell monolayers in a highly reproducible manner. Optimal conditions for individual cell lines can be achieved by adjusting MC or serum concentrations in the spheroid formation medium. The 3D spheroids generated can be collected for use in a wide range of applications, including cell signaling or gene expression studies, candidate drug screening, or in the study of cellular processes such as tumor cell invasion and migration. The protocol is also readily adapted to generate clonal spheroids from single cells, and can be adapted to assess anchorage-independent growth and anoikis-resistance. Overall, our protocol provides an easily modifiable method for generating and utilizing 3D cell spheroids in order to recapitulate the 3D microenvironment of tissues and model the in vivo growth of normal and tumor cells.

Here, we describe a rapid and flexible protocol for the formation of 3D cell spheroids through cell aggregation. This is easily adapted to multiple cell types and is suitable for use in a variety of applications including cell migration, invasion, or anoikis assays, and for imaging and quantifying cell-matrix interactions.

三维球体制作一个高效，灵活细胞聚集方法

Monolayer cell culture does not adequately model the in vivo behavior of tissues, which involves complex cell-cell and cell-matrix interactions. ...

IR-TEx: An Open Source Data Integration Tool for Big Data Transcriptomics Designed for the Malaria Vector Anopheles gambiae

IR-TEx is an application written in Shiny (an R package) that allows exploration of the expression of (as well as assigning functions to) transcripts whose expression is associated with insecticide resistance phenotypes in Anopheles gambiae mosquitoes. The application can be used online or downloaded and used locally by anyone. The local application can be modified to add new insecticide resistance datasets generated from multiple -omics platforms. This guide demonstrates how to add new datasets and handle missing data. Furthermore, IR-TEx can be completely and easily recoded to use-omics datasets from any experimental data, making it a valuable resource to many researchers. The protocol illustrates the utility of IR-TEx in identifying new insecticide resistance candidates using the the microsomal glutathione transferase, GSTMS1, as an example. This transcript is upregulated in multiple pyrethroid resistant populations from C&#244;te D&#39;Ivoire and Burkina Faso. The identification of co-correlated transcripts provides further insight into the putative roles of this gene.

IR-TEx: An Open Source Data Integration Tool for Big Data Transcriptomics Designed for the Malaria Vector Anopheles gambiae

IR-TEx explores insecticide resistance-related transcriptional profiles in the species Anopheles gambiae. Provided here are full instructions for using the application, modifications for exploring multiple transcriptomic datasets, and using the framework to build an interactive database for collections of transcriptomic data from any organism, generated in any platform.

IR-TEx:专为疟疾病媒阿诺菲斯甘巴病设计的大数据转录组学的开源数据集成工具

IR-TEx is an application written in Shiny (an R package) that allows exploration of the expression of (as well as assigning functions to) transcripts ...

Imaging Molecular Adhesion in Cell Rolling by Adhesion Footprint Assay

Rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic, passive motility in recruiting leukocytes to the site of inflammation. This phenomenon occurs in postcapillary venules, where blood flow pushes leukocytes in a rolling motion on the endothelial cells. Stable rolling requires a delicate balance between adhesion bond formation and their mechanically-driven dissociation, allowing the cell to remain attached to the surface while rolling in the direction of flow. Unlike other adhesion processes occurring in relatively static environments, rolling adhesion is highly dynamic as the rolling cells travel over thousands of microns at tens of microns per second. Consequently, conventional mechanobiology methods such as traction force microscopy are unsuitable for measuring the individual adhesion events and the associated molecular forces due to the short timescale and high sensitivity required. Here, we describe our latest implementation of the adhesion footprint assay to image the P-selectin: PSGL-1 interactions in rolling adhesion at the molecular level. This method utilizes irreversible DNA-based tension gauge tethers to produce a permanent history of molecular adhesion events in the form of fluorescence tracks. These tracks can be imaged in two ways: (1) stitching together thousands of diffraction-limited images to produce a large field of view, enabling the extraction of adhesion footprint of each rolling cell over thousands of microns in length, (2) performing DNA-PAINT to reconstruct super-resolution images of the fluorescence tracks within a small field of view. In this study, the adhesion footprint assay was used to study HL-60 cells rolling at different shear stresses. In doing so, we were able to image the spatial distribution of the P-selectin: PSGL-1 interaction and gain insight into their molecular forces through fluorescence intensity. Thus, this method provides the groundwork for the quantitative investigation of the various cell-surface interactions involved in rolling adhesion at the molecular level.

This protocol presents the experimental procedures to perform the adhesion footprint assay to image the adhesion events during fast cell rolling adhesion.