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View additional product information for BLOCK-iT™ Lentiviral RNAi Gateway™ Vector Kit - FAQs (K494300)
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不能，您使用的入门载体应含有可使shRNA发生RNA聚合酶III依赖性表达所需的元件（即，Pol III启动子和终止子）。
在建立稳定细胞系时，量效曲线或杀伤曲线是一种用于确定最佳抗生素使用浓度的简单方法。为确定杀死全部未转染细胞所需的最低抗生素用量，可将未转染细胞置于含有不同浓度抗生素的培养基中生长。做量效曲线或杀伤曲线的基本步骤如下：
•以汇合度25%的细胞密度将未转染细胞接种到培养皿中，并加入含递增浓度抗生素的培养基使细胞生长。对于某些抗生素，您将需要计算活性药物量以控制批次间差异。
•每3-4天补充选择培养基。10-12天后，检测培养皿中的活细胞数。在出现细胞死亡前，细胞可能在选择培养基中分裂过1-2次。
•找到杀死全部细胞所需的最低抗生素浓度，即用于建立稳定细胞系所需的最佳抗生素浓度。
很遗憾，pENTR/U6载体不含筛选标记；因此，只能实现瞬时RNAi分析。如果您想建立稳定细胞系，可通过LR反应将shRNA克隆进入合适的Gateway目的载体中，生成表达克隆。
pENTR/H1/TO载体含有Zeocin抗性基因，方便制作能够诱导表达目的shRNA的细胞系。可通过做一个杀死曲线，确定杀死未转染哺乳细胞所需的Zeocin最低浓度。请注意，Zeocin敏感性细胞不会聚集和脱离培养皿，但可能会出现体积增大、细胞形态异常、细胞质中形成较大的空泡或细胞膜/和膜分解。
您可使用长度在4-11个核苷酸范围内的任何环序列，但是，通常优先选择较短的环（即，4-7个核苷酸）。应避免使用含有胸腺嘧啶核苷酸（T）的环序列，否则有可能会导致过早转录终止，特别是在目标序列本身以1个或多个T核苷酸终止的情况下。以下是一些我们推荐使用的环序列：
•5’ – CGAA – 3’
•5’ – AACG – 3’
•5’ – GAGA – 3’
shRNA的转录从U6启动子序列末端后面的第一个碱基开始。在上游链寡核苷酸中，转录起始位点相当于4 碱基 CACC序列后的第一个核苷酸，加入4 碱基 CACC序列是为了实现定向克隆。我们建议以鸟嘌呤核苷酸（G）作为shRNA序列的起始，因为天然U6snRNA的转录是以G开始的。请注意下列情况：
•如果G是目标序列的一部分，则将G并入上游链寡核苷酸的茎序列，并在上游链寡核苷酸的3’端加一个互补C。
•如果G不是目标序列的第一个碱基，我们建议直接在上游链寡核苷酸5’末端紧随CACC突出序列后加一个G。这种情况下，不要在上游链寡核苷酸的3’末端添加互补C。注意：我们已经发现，在这种情下添加互补C，可导致shRNA活性降低。或者，如果没有特别想以G作为转录起始，则应使用腺嘌呤核苷酸（A），而不要使用C或T。但是，应注意的是，除了G以外，使用其它任何一种核苷酸都会影响起始效率和位置。
请遵循以下步骤：
•访问 RNAi Designer（https://rnaidesigner.thermofisher.com/rnaiexpress/setOption.do?designOption=shrna&pid=1407484891722110832）
•输入检索号或提供核苷酸序列
•确定设计的靶标区域：ORF、5’ UTR或3’ UTR
•选择Blast数据库
•选择最小和最大G/C比例
选择载体和链方向，点击“RNAi Design”开始设计shRNA。
为得到最佳结果，连接时双链寡核苷酸片段与载体的摩尔比应为10:1。
我们建议您另取一份分装的的退火双链寡核苷酸（5 μL的500 nM储液）进行电泳，并与等体积的各起始单链寡核苷酸（将200 μM储液稀释400倍至500 nM；取5μL稀释液进行凝胶电泳分析）进行对比。应确保使用合适的分子量标准品。我们通常使用以下凝胶和分子量标准品：
•琼脂糖凝胶：4% E-Gel（货号G5000-04）
•分子量标准品：10 bp DNA Ladder（货号10821-015）
当使用琼脂糖凝胶电泳对退火双链寡核苷酸反应的小样进行分析时，我们通常可以看到以下结果：
•一条可检测到的高分子量条带代表退火的双链寡核苷酸。
•一条可检测到的低分子量条带代表未退火的单链寡核苷酸。应注意，这个条带应该是能检测到的因为仍有大量的单链寡核苷酸未退火。
您将需要退火等量的上游链和下游链寡核苷酸，从而生成双链寡核苷酸。如果您的单链寡核苷酸是冻干形式的，可在使用前用水或TE缓冲液将其重悬至终浓度200 µM。我们通常在单链寡核苷酸终浓度为50 μM时进行退火。在浓度低于50 μM时退火，会显著降低效率。请注意，退火步骤效率不是100%的，即使在浓度为50 μM时，也会有约一半的单链寡核苷酸仍未退火。请参见以下步骤：
1. 使用0.5 mL无菌微量离心管，在室温下设置以下退火反应：
“上游链”DNA寡核苷酸（200 μM） - 5 μL，“下游链”DNA寡核苷酸（200 μM）- 5 μL，10X寡核苷酸退火缓冲液 - 2 μL，无DNase/RNase水 - 8 µL，至总体积 20 μL。
2. 如果对lacZ双链对照寡核苷酸进行退火，则将离心管短暂离心（约5秒），然后将离心管的内容物转移至一个单独的0.5 mL无菌微量离心管中。
3. 在95°C孵育反应4分钟。
4. 将含有退火反应的离心管从水浴或加热模块中取出，放置在实验桌上。
5. 放置5-10分钟，等待反应混合物冷却到室温。在这段时间内，单链寡核苷酸会退火。
6. 将样品置于一个微量离心机中并短暂离心（约5秒）。轻轻混合。
7. 取出1 μL退火混合物，按照说明稀释双链寡核苷酸。
8. 将剩余的50 μM双链寡核苷酸混合物保存于-20°C。
如有需要，您可通过琼脂糖凝胶电泳来验证退火双链寡核苷酸的完整性。
您将需要双链寡核苷酸，用于编码待克隆入上述任一载体的目标shRNA。使用我们的 RNAi Designer ，设计和合成2个互补的单链DNA寡核苷酸，其中一个用于编码目标shRNA。
TO代表四环素操纵子，因为该入门载体含有shRNA在哺乳细胞中发生四环素诱导型表达所需的元件。四环素操纵子序列使目标shRNA能够以四环素依赖性方式进行表达，因此，这是一个诱导型系统。
BLOCK-iT诱导型H1和U6入门载体试剂盒分别使用Pol III依赖的H1或U6启动子。经过修饰的H1启动子含有2个侧翼四环素操纵子（TetO2）位点。因此，在表达四环素阻遏蛋白（TR）的细胞中，可对从该启动子开始表达的shRNA进行调控。H1和U6都是Pol III型启动子；但是，所使用的细胞系不同，它们的有效性可能存在轻微差异。
我们可提供pENTR/U6（货号K492000）和pENTR/H1/TO（货号K494500）载体用于shRNA传递。两种载体都是Gateway兼容的，分别利用U6或H1/TO启动子驱动shRNA表达。pENTR/H1/TO载体可用于shRNA诱导型表达，而pENTR/U6载体可用于组成型表达。如果您想设计可同时兼容这两种载体的shRNA寡核苷酸，应选择pENTER/U6载体。
外源性短发夹RNA可在RNA聚合酶III的作用下发生转录（Paule&White，2000），它通常具有以下结构特性：
来源于目标基因的19–29个核苷酸的短序列，紧随其后的是4-5个核苷酸的短分隔序列（即，茎环）以及19-29个核苷酸的和起始靶序列反向互补的序列。所得RNA分子会形成分子内茎环结构，随后在Dicer酶的作用下形成siRNA双链。
短发夹RNA（shRNA）是一种人工设计的RNA分子，可通过与RNAi和miRNA通路中常见的细胞成分相互作用而诱导基因沉默。尽管shRNA在结构上是miRNA的一种简化形式，但shRNA分子诱导RNAi效应的方式与siRNA相似，即诱导目标转录本发生断裂和降解（Brummelkamp et al，2002；Paddison et al，2002；Paul et al，2002；Sui et al，2002；Yu et al，2002）。RNA聚合酶III（Pol III），如U6和H1，可驱动shRNA转录本的转录。发夹结构的shRNA离开细胞核并被Dicer酶加工后输送到细胞质，形成siRNA。
为实现有效的shRNA表达，应使用Pol III型启动子。这些Pol III型启动子包含表达RNA所有必需的上游启动子元件，并以一个较短的多聚胸腺嘧啶束终止。一旦shRNA开始表达，它们便被运输出细胞核并在胞质中被Dicer酶加工成siRNA。Dicer酶优先识别Pol III型启动子生成的shRNA，因为它们不带有5’或3’侧翼序列。siRNA进入RISC复合物并在哺乳细胞中产生RNAi效应。
在BP/LR Clonase反应的一步法实验方案中，不建议用BP Clonase酶和LR Clonase酶替代BP Clonase II 酶/LR Clonase II酶，因为这样的重组效率非常低。
有的，我们能提供针对BP/LR Clonase反应的一步式实验方案DNA可以在一步反应后被克隆到目的载体中，从而节省了您的时间和金钱。
建议使用一个供体载体进行一次BP反应以获得一个入门克隆。然后将这一入门克隆和目的载体进行一次LR反应以获得新的表达克隆。
5X LR Clonase缓冲液或5X BP Clonase缓冲液不作为单独产品出售。它们作为酶试剂盒的一部分进行销售。
我们不提供任何用于在植物内表达的Gateway载体。
理论上，pDONR载体在BP反应时对插入片段没有大小的限制。我们自己测试过的最大片段是12 kb。TOPO载体对插入片段大小更敏感一些，要获得较高的克隆效率其插入片段长度的上限是3-5 kb。
在得到attB-PCR产物之后，我们建议对产物进行纯化以去除PCR缓冲液，残留的dNTP，attB引物，以及attB引物二聚体。引物和引物二聚体在BP反应中会高效的与供体载体重组，因而会增加转化E. coli时的背景，而残留的PCR缓冲液可能会抑制BP反应。使用酚/氯仿抽提，加醋酸铵和乙醇或异丙醇沉淀的标准PCR产物纯化方案不适合对attB-PCR产物进行纯化，因为这些实验方案通常仅能去除小于100 bp的杂质，而在去除较大的引物二聚体时效果不佳。我们推荐一种PEG纯化方案（请参见使用Clonase II的Gateway技术手册第17页）。如果使用上述实验方案您的attB-PCR产物仍然不够纯，您可以进一步对其进行凝胶纯化。我们推荐使用Purelink Quick 凝胶纯化试剂盒。
请检查您所用的菌株的基因型。我们的Gateway目的载体通常含有一个ccdB基因元件，该元件如果不被破坏，则E. Coli生长将受到抑制。因此，未进行克隆的载体应该在ccdB survival菌株如我们的ccdB Survival 2 T1R感受态细胞中扩增。
目的基因必须两端带有合适的att位点，或者是入门克隆中的attL (100 bp)位点，或者是PCR产物中的 attB (25 bp)位点。对于入门克隆而言，所有位于attL位点之间的部分都将被转移到含有attR位点的Gateway目的载体中，而两端带有attB位点的PCR产物需被转移到一个含有attP位点的供体载体，例如pDONR221。
翻译起始位点的位置，终止子，或者用于表达的融合标签必须在最开始的克隆设计中考虑到。例如，如果您的目的载体包含一个N末端标记而非C末端标记，则该载体应当已经带有合适的翻译起始位点，但是终止子应当被包含在插入片段当中。
小抽（碱裂解）纯化的DNA即适用在Gateway克隆反应中。重要的一点是要将RNA污染去除干净以便得到精确的定量。推荐使用通过我们的S.N.A.P. 核酸纯化试剂盒，ChargeSwitch试剂盒，或PureLink试剂盒纯化的质粒DNA。
理论上没有片段大小限制。长度在100 bp到11 kb之间的PCR产物可以被直接克隆到pDONR Gateway载体中。其它DNA片段如带有att位点的150 kb DNA片段可以成功和一个Gateway兼容载体发生重组。对于大的插入片段，推荐进行过夜孵育反应。
请使用推荐的滤波装置对所用荧光进行检测。使用倒置荧光显微镜进行分析。如有需要，可使蛋白表达持续1-3天，再进行荧光检测。
所用目标序列可能与其他基因具有较高的同源性；请选择一个不同的目标区域。
做一个杀死曲线，确定细胞株对抗生素的敏感性。应确保将病毒储液正确保存于-80°C，并且冻融次数不超过3次。最后，使用Polybrene试剂，将重组慢病毒转导至细胞。
应确保所用的感受态细胞被正确保存于-80°C，在冰上融化并立即使用。加入DNA时，轻轻混合感受态细胞：不要使用移液管反复吹打混合。同时，转化所用DNA不要超过最大推荐用量（100 ng），或者DNA加入体积不要超过感受态细胞体积的10%，否则会抑制转化。
请确保您使用的含胎牛血清（FBS）的培养基已减少了四环素含量。许多FBS都含有四环素，因为FBS往往是从饮食中含四环素的牛体内分离出来的，这导致出现低水平的shRNA本底表达。应确保使用可表达Tet阻遏蛋白的细胞系，并以合适的MOI进行转导。如果您自行建立了可表达Tet阻遏蛋白的细胞系，则应在使用shRNA重组体转导细胞前至少等待24小时。
有多种因素可导致敲低效果较差。请参见以下建议：
•低转染效率：应确保转染所用培养基不含抗生素，并且细胞的汇合度合适；通过改变转染试剂用量而优化转染条件。
•做一个时间梯度检测，确定达到最高基因敲低水平的时间点。
•重组子中存在突变：对转化子中双链寡核苷酸插入片段进行测序验证。
•目标区域不是最佳的：选择一个不同的目标区域。
•应根据相应使用手册中的指南，设计siRNA。
你可尝试减少转染试剂的用量，或使用其他转染试剂。此外，应确保使用的质粒是纯净的，并为转染实验准备的。
难以测序可能是因为发夹序列是一种反向重复序列，在测序期间可形成二级结构，从而导致在测序进行到发夹区域时出现信号跌落。如果您遇到测序困难的情况，请尝试以下建议：
•使用高质量的纯化质粒DNA进行测序。我们建议使用Invitrogen PureLink HQ小量质粒纯化试剂盒（货号K2100-01）或S.N.A.P.质粒DNA中量提取试剂盒（货号K1910-01）来制备DNA。
•在测序反应中加入DMSO至终浓度为5%。
•增加反应中的模板用量（高达正常浓度的2倍）。
•标准测序试剂盒通常使用dITP代替dGTP，以减少G:Ccompression。其他含dGTP的试剂盒可用于对富含G和富含GT的模板进行测序。如果您在使用含有dITP的标准商业化测序试剂盒，再买一个含dGTP的测序试剂盒（如，dGTPBigDye Terminator v3.0 Ready Reaction Cycle Sequencing试剂盒，货号4390229），并在测序反应中使用摩尔比为7:1的dITP:dGTP。
我们强烈建议对阳性转化子进行测序，确认双链寡核苷酸插入片段的序列。在筛选转化子时，我们发现多达20%的克隆可能包含突变的插入片段（通常在双链寡核苷酸中有1或2 bp缺失）。其原因尚不清楚，但可能是由于双链寡核苷酸插入片段中的反向重复序列触发了E. coli的修复机制引起的。注意：双链寡核苷酸插入片段有突变的入门克隆，在哺乳细胞中RNAi效果通常较差。应确认入门克隆具有正确的双链寡核苷酸序列，并将这种克隆用于您的RNAi分析。
使用劣质的单链寡核苷酸也会导致出现突变的插入片段。为避免出现这类问题，可使用质谱分析法来检验质量错误的峰，或订购HPLC或PAGE纯化的寡核苷酸。
•应确认下游寡核苷酸链的序列与上游寡核苷酸链的序列是互补的。
•使用shRNA载体时，应将互补序列的单链寡核苷酸混合。上游寡核苷酸链的5’末端应含有CACC，而下游寡核苷酸链的5’末端应含有AAAA。
•使用miRNA载体时，应确保上游寡核苷酸链的5’末端含有TGCT，而下游寡核苷酸链的5’末端含有CCTG
请查看以下可能原因：
•单链寡核苷酸的设计错误；应确认下游链寡核苷酸的序列与上游链寡核苷酸的序列是互补的。
•在寡核苷酸加热至95°C后，确保在室温下退火5-10分钟。
•应检查退火所用的上游链和下游链寡核苷酸的摩尔比，用量应相同。
使用pLenti4/BLOCK-iT-DEST构建的重组慢病毒进行RNAi研究时，我们通常可在转导后48-120小时内观察到对基因表达的显著抑制。基因敲低的程度取决于检测时间、目标蛋白的稳定性以及其他因素。请注意，通常无法实现100%的基因敲低，但是，经过优化条件可能会达到80%以上。
BLOCK-iT慢病毒RNAi表达系统中的慢病毒和包装载体于1998年由Dull等人开发，它们是以慢病毒载体为基础的第三代载体。第三代慢病毒系统带有许多安全特性，可提高生物安全性，并将该系统与野生型人类HIV-1病毒的关联性降至最低。BLOCK-iT慢病毒RNAi表达系统的主要安全特性如下：
•pLenti6/BLOCK-iT-DEST表达载体删除了3’ LTR（ΔU3），这不会影响生产细胞中病毒基因组的生成，但会导致慢病毒在转导至靶细胞后发生“自我失活”（Yee et al.，1987；Yu et al，1986；Zufferey et al，1998）。一旦整合到转导的靶细胞中，慢病毒基因组将不能再生产可包装的病毒基因组。
•用于该系统的HIV-1基因已被减少至3个（即，gag、pol和rev）。
•水泡性口炎病毒的VSV-G基因被用于代替HIV-1的包膜（Burns et al，1993；Emi et al，1991；Yee et al，1994）。
•编码结构和病毒基因组包装元件的基因被分散在4个质粒上。这4个质粒经过加工改造，彼此之间不含有任何同源性区域，可防止发生不必要的重组事件而生成可复制病毒（Dull et al，1998）。
•虽然3个包装质粒均可实现在293FT生产细胞中生产子代病毒（如gal、pol、rev、env）所需的反式蛋白表达，但它们3个都不含有LTRs或Ψ包装序列。这表示，包装病毒基因组中实际上不含任何HIV-1结构基因，因此，转导的靶细胞中也永远不会表达这些基因。不会产生新的可复制病毒。
•该系统生产的慢病毒颗粒是复制缺陷型病毒，只负责携带目的基因。不会产生其他病毒种类。
•pLP1的gag和pol基因表达具有Rev依赖性，因为gag/pol mRNA转录本中含有HIV-1 RRE。在无Rev的情况下，加入RRE可阻止gag和pol的表达（Dull et al，1998）。
•在pLenti6/BLOCK-iT-DEST表达载体的5’ LTR上游已加入了一个组成型启动子（RSV启动子），从而抵消有效生成病毒RNA时对Tat的需求（Dull et al，1998）。
尽管前文讨论了许多安全特性内容，但是该系统生产的慢病毒仍具有一些生物危害风险，因为它能够转导至原代人细胞。因此，我们强烈建议您将该系统生成的慢病毒储液作为生物安全2级（BL-2）生物体进行处理，严格遵守所有已发布的BL-2指南并采取适当的废物处理措施。此外，在建立携带潜在有害或有毒基因的慢病毒（如活化的致癌基因）时，应格外小心。关于BL-2指南和慢病毒处理的更多信息，请参考由疾病控制和预防中心（CDC）发布的第五版《微生物和生物医学实验室的生物安全性》。
这两种启动子在HEK 293细胞中的组成型敲低能力基本相同。有报道显示，在其他细胞类型中，H1或U6将有一个更活跃，但是它们之间的差异通常非常小。
使用BLOCK-iT慢病毒RNAi表达系统行使使用慢病毒导入shRNA至哺乳细胞，可带来以下优势：
•pENTR/U6入门载体可快速、有效地将编码所需shRNA目标序列的双链寡核苷酸克隆到含RNA Pol III依赖性表达盒（即，U6 RNAi表达盒）的载体中，用于RNAi分析。
•该系统中的载体是Gateway兼容的，可轻松将pENTR/U6载体的U6 RNAi表达盒重组到pLenti6/BLOCK-iT-DEST载体中。
•生成的复制缺陷型慢病毒可有效转导至分裂和非分裂哺乳细胞，从而拓宽该系统的RNAi应用潜能，超越了其他传统逆转录病毒系统的应用范围（Naldini，1998）。
•可将目标shRNA高效导入至培养的或体内的哺乳细胞中。
•目标shRNA的表达比传统腺病毒系统更稳定、更长久。
•产生的假病毒适用的宿主范围更广（Yee，1999）。
•具有多种可增强生物安全性的设计特点。
腺病毒不是一种活跃的裂解病毒，一般成熟病毒颗粒经过2-3天可在细胞中积累。随着细胞内病毒的积累和绝对数量增加，生产细胞发生聚合并最终破裂。一旦发生这种情况，邻近细胞也会被感染，并开始重复为期3天的循环。“细胞病变效应”或CPE，可用于描述上述情况，该效应通常出现在感染后7天内，表现为经过2个周期感染、复制和细胞破裂形成的“彗星状”空斑。
•感染7天后，CPE会扩大；感染后约10天，CPE将最终遍及整个培养皿。
•培养皿中的细胞被感染后，需要约10天的时间才能收获病毒（如上所述）。
•一旦获得原代病毒储液，即可使用感染复数（MOI）为3的病毒储液感染新鲜的293A细胞而直接进行扩增。
请参见以下定义：
•感染：适用于发生病毒复制并生成传染性子代病毒的情况。只有稳定表达E1的细胞系能够被感染。
•转导：适用于无病毒复制且不生成传染性子代病毒的情况。不表达E1的哺乳细胞可被转导。在这种情况下，您可使用腺病毒作为shRNA的传递载体。
请参见以下步骤：
1.将编码shRNA或miR RNAi的双链DNA寡核苷酸克隆进入一种BLOCK-iT入门（shRNA）或表达（miR RNAi）载体中。
2.通过Gateway重组反应将RNAi表达盒转移到腺病毒（仅shRNA）或慢病毒目的载体。
3.将RNAi载体转染到病毒生产细胞中，获得病毒储液，该储液可立即使用或保存于–80°C。
4.收集病毒上清液并测定滴度（根据需要对腺病毒储液进行扩增）。
5.将慢病毒或腺病毒储液转导至任意细胞类型。
No, you should use an entry vector that contains the elements necessary for RNA Polymerase III-dependent expression of your shRNA (i.e., Pol III promoter and terminator).
A dose response curve or kill curve is a simple method for determining the optimal antibiotic concentration to use when establishing a stable cell line. Untransfected cells are grown in a medium containing antibiotic at varying concentrations in order to determine the lowest amount of antibiotic needed to achieve complete cell death. The basic steps for performing a dose response curve or kill curve are as follows:
- Plate untransfected cells at 25% confluence, and grow them in a medium containing increasing concentrations of the antibiotic. For some antibiotics, you will need to calculate the amount of active drug to control for lot variation.
- Replenish the selective medium every 3-4 days. After 10-12 days, examine the dishes for viable cells. The cells may divide once or twice in the selective medium before cell death begins to occur.
- Look for the minimum concentration of antibiotic that resulted in complete cell death. This is the optimal antibiotic concentration to use for stable selection.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Unfortunately, the pENTR/U6 vector does not contain a selection marker; therefore, only transient RNAi analysis may be performed. If you wish to generate stable cell lines, perform an LR reaction into an appropriate Gateway destination vector to generate expression clones.
The pENTR/H1/TO vector contains the Zeocin resistance gene to facilitate generation of cell lines that inducbily express the shRNA of interest. Perform a kill curve to determine the minimum concentration of Zeocin that is required to kill your untransfected mammalian cell line. Please note that Zeocin-sensitive cells do not round up and detach from the plate, but rather may increase in size, show abnormal cell shape, display presence of large empty vesicles in the cytoplasm, or show breakdown of plasma/nuclear membranes.
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You can use a loop sequence of any length ranging from 4 to 11 nucleotides, although short loops (i.e., 4-7 nucleotides) are generally preferred. Avoid using a loop sequence containing thymidines (Ts), as they may cause early termination. This is particularly true if the target sequence itself ends in one or more T nucleotides. Here are some loop sequences we recommend:
- 5' - CGAA - 3'
- 5' - AACG - 3'
- 5' - GAGA - 3'
Transcription of the shRNA initiates at the first base following the end of the U6 promoter sequence. In the top-strand oligo, the transcription initiation site corresponds to the first nucleotide following the 4 bp CACC sequence added to permit directional cloning. We recommend initiating the shRNA sequence at a guanosine (G) because transcription of the native U6 snRNA initiates at a G. Note the following:
- If G is part of the target sequence, then incorporate the G into the stem sequence in the top-strand oligo and add a complementary C to the 3' end of the top-strand oligo.
- If G is not the first base of the target sequence, we recommend adding a G to the 5' end of the top-strand oligo directly following the CACC overhang sequence. In this case, do not add the complementary C to the 3' end of the top-strand oligo. Note: We have found that adding the complementary C in this situation can result in reduced activity of the shRNA. Alternative, if use of a G to initiate transcription is not desired, use an adenosine (A) rather than C or T. Note, however, that use of any nucleotide other than G may affect initiation efficiency and position.
Please follow the steps outlined below:
- Visit RNAi Designer
- Enter an accession number or provide a nucleotide sequence
- Determine the region for target design: ORF, 5' UTR, or 3' UTR
- Choose database for Blast
- Choose minimum and maximum G/C percentage
Select vector and strand orientation and click “RNAi Design” to design shRNA.
For optimal results, use a 10:1 molar ratio of ds oligo insert:vector for ligation.
We suggest running an aliquot of the annealed ds oligo (5 µL of the 500 nM stock) and comparing it to an aliquot of each starting single-stranded oligo (dilute the 200 µM stock 400-fold to 500 nM; use 5 µL for gel analysis). Be sure to include an appropriate molecular weight standard. We generally use the following gel and molecular weight standard:
- Agarose gel: 4% E-Gel (Cat. No. G5000-04)
- Molecular weight standard: 10 bp DNA Ladder (Cat. No. 10821-015)
When analyzing an aliquot of the annealed ds oligo reaction by agarose gel electrophoresis, we generally see the following:
- A detectable higher molecular weight band representing annealed ds oligo.
- A detectable lower molecular weight band representing unannealed single-stranded oligos. Note that this band is detected since a significant amount of the single-stranded oligo remains unannealed.
You will want to anneal equal amounts of the top- and bottom-strand oligos to generate the ds oligos. If your single-stranded oligos are supplied lyophilized, resuspend them in water or TE buffer to a final concentration of 200 µM before use. We generally perform the annealing reaction at a final single-stranded oligo concentration of 50 µM. Annealing at concentrations lower than 50 µM can significantly reduce the efficiency. Note that the annealing step is not 100% efficient; approximately half of the single-stranded oligos remain unannealed even at a concentration of 50 µM. Please see the steps below:
1. In a 0.5 mL sterile microcentrifuge tube, set up the following annealing reaction at room temperature.
“Top-strand” DNA oligo (200 µM) - 5 µL, “Bottom-strand” DNA oligo (200 µM)- 5 µL, 10X Oligo Annealing Buffer - 2 µL, DNase/RNase-Free Water - 8 µL which should make a total volume of 20 µL.
2. If reannealing the lacZ ds control oligo, centrifuge its tube briefly (approximately 5 seconds), then transfer the contents to a separate 0.5 mL sterile microcentrifuge tube.
3. Incubate the reaction at 95 degrees C for 4 minutes.
4. Remove the tube containing the annealing reaction from the water bath or the heat block, and set it on your laboratory bench.
5. Allow the reaction mixture to cool to room temperature for 5-10 minutes. The single-stranded oligos will anneal during this time.
6. Place the sample in a microcentrifuge and centrifuge briefly (approximately 5 seconds). Mix gently.
7. Remove 1 µL of the annealing mixture and dilute the ds oligo as directed.
8. Store the remainder of the 50 µM ds oligo mixture at -20 degrees C.
You can verify the integrity of your annealed ds oligo by agarose gel electrophoresis, if desired.
You will need a double-stranded oligo that encodes the shRNA of interest to be cloned into one of the above-mentioned vectors. Use our RNAi Designer to design and synthesize two complementary single-stranded DNA oligonucleotides, with one encoding the shRNA of interest.
TO stands for tetracycline operator, as this entry vector contains elements required for tetracycline-inducible expression of the shRNA in mammalian cells. The presence of the Tet operator sequences enables the shRNA of interest to be expressed in a tetracycline-dependent manner, thereby making this an inducible system.
The BLOCK-iT Inducible H1 and U6 Entry Vector Kits use either the Pol III-dependent H1 or the U6 promoter, respectively. The H1 promoter is modified to contain two flanking tetracycline operator (TetO2) sites within the H1 promoter. This allows the shRNA expressed from this promoter to be regulated in cells that express the tetracycline repressor (TR) protein. Both the H1 and the U6 are Pol III type promoters; however, there may be some minor differences in their effectiveness, depending on the cell line used.
We offer our pENTR/U6 (Cat. No. K494500) and pENTR/H1/TO (Cat. No. K492000) vectors for shRNA delivery. Both vectors are Gateway compatible and drive expression through either the U6 or H1/TO promoter, respectively. The pENTR/H1/TO vector is for inducible shRNA expression, while the pENTR/U6 can be used for constitutive expression. If you want to design shRNA oligos compatible with both vectors, select the pENTER/U6 vector.
Exogenous short hairpin RNAs can be transcribed by RNA Polymerase III (Paule and White, 2000) and generally contain the following structural features: A short nucleotide sequence ranging from 19-29 nucleotides derived from the target gene, followed by a short spacer of 4-15 nucleotides (i.e., loop) and a 19-29 nucleotide sequence that is the reverse complement of the initial target sequence. The resulting RNA molecule forms an intramolecular stem-loop structure that is then processed to an siRNA duplex by the Dicer enzyme.
Short hairpin RNA (shRNA) is an artificially designed class of RNA molecules that can trigger gene silencing through interaction with cellular components common to the RNAi and miRNA pathways. Although shRNA is a structurally simplified form of miRNA, these RNA molecules behave similarly to siRNA in that they trigger the RNAi response by inducing cleavage and degradation of target transcripts (Brummelkamp et al., 2002; Paddison et al., 2002; Paul et al., 2002; Sui et al., 2002; Yu et al., 2002). An RNA Polymerase III (Pol III), such as U6 and H1, drives transcription of shRNA transcripts. shRNA hairpins are exported from the nucleus and processed by Dicer into the cytosol, resulting in siRNA.
For efficient shRNA expression, a Pol III type promoter is used. These Pol III promoters contain all of their essential elements upstream of the expressed RNA and terminate with a short polythymidine tract. Once the shRNA is expressed, it is transported from the nucleus and processed into siRNA in the cytoplasm by the enzyme Dicer. Dicer preferentially recognizes shRNAs generated from a Pol III promoter because they carry no 5' or 3' flanking sequences. The siRNAs enter into RISC complexes and generate an RNAi response in mammalian cells.
In the single-step protocol for the BP/LR Clonase reaction, we would not recommend substituting the BP Clonase II/LR Clonase II enzymes with BP Clonase /LR Clonase enzymes as this would result in very low recombination efficiency.
Yes, we have come up with a single-step protocol for BP/LR Clonase reaction (http://www.thermofisher.com/us/en/home/life-science/cloning/gateway-cloning.html#1), where DNA fragments can be cloned into Destination vectors in a single step reaction, allowing you to save time and money.
We would recommend performing a BP reaction with a Donor vector in order to obtain an entry clone. This entry clone can then be used in an LR reaction with the Destination vector to obtain the new expression clone.
We do not offer the 5X LR Clonase buffer and 5X BP Clonase buffer as standalone products. They are available as part of the enzyme kits.
We do not offer any Gateway vectors for expression in plants.
There is no theoretical limit to insert size for a BP reaction with a pDONR vector. Maximum size tested in-house is 12 kb. TOPO vectors are more sensitive to insert size and 3-5 kb is the upper limit for decent cloning efficiency.
After generating your attB-PCR product, we recommend purifying it to remove PCR buffer, unincorporated dNTPs, attB primers, and any attB primer-dimers. Primers and primer-dimers can recombine efficiently with the Donor vector in the BP reaction and may increase background after transformation into E. coli, whereas leftover PCR buffer may inhibit the BP reaction. Standard PCR product purification protocols using phenol/chloroform extraction followed by ammonium acetate and ethanol or isopropanol precipitation are not recommended for purification of the attB-PCR product as these protocols generally have exclusion limits of less than 100 bp and do not efficiently remove large primer-dimer products. We recommend a PEG purification protocol (see page 17 of the Gateway Technology with Clonase II manual). If you use the above protocol and your attB-PCR product is still not suitably purified, you may further gel-purify the product. We recommend using the PureLink Quick Gel Extraction kit.
Check the genotype of the cell strain you are using. Our Gateway destination vectors typically contain a ccdB cassette, which, if uninterrupted, will inhibit E. coli growth. Therefore, un-cloned vectors should be propagated in a ccdB survival cell strain, such as our ccdB Survival 2 T1R competent cells.
LR Clonase II Plus contains an optimized formulation of recombination enzymes for use in MultiSite Gateway LR reactions. LR Clonase and LR Clonase II enzyme mixes are not recommended for MultiSite Gateway LR recombination reactions, but LR Clonase II Plus is compatible with both multi-site and single-site LR recombination reactions.
Both systems are used for gene targeting or gene knockdown but each has distinctive features. The shRNA expression vectors like pENTR/U6 or pENTR/H1-TO use Pol III promoters, whereas the miRNA expression vectors are flexible to use more common and more processive Pol II promoters like CMV, EF1 or other mammalian expression promoters. You can only clone a single shRNA sequence into an shRNA vector to target a single gene, whereas multiple miRNA sequences can be cloned together into an miRNA vector to target one or more genes, or multiple locations in a gene. An additional feature of the miRNA expression vectors is that, due to use of Pol II promoters, the miRNA can be expressed directly in fusion with a reporter gene like EmGFP to monitor transfection and transcription.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
When the LR reaction is complete, the reaction is stopped with Proteinase K and transformed into E. coli resulting in an expression clone containing a gene of interest. A typical LR reaction followed by Proteinase K treatment yields about 35,000 to 150,000 colonies per 20ul reaction. Without the Proteinase K treatment, up to a 10 fold reduction in the number of colonies can be observed. Despite this reduction, there are often still enough colonies containing the gene of interest to proceed with your experiment, so the Proteinase K step can be left out after the LR reaction is complete if necessary.
The lentiviruses produced in this system will not replicate under any conditions. You must perform a fresh transfection each time you need more virus.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Yes, it will work as an expression vector by itself and can be stably selected with blasticidin. Please note that the vector will be about twice the size of most regular vectors. Therefore you may need to increase the amount of transfected vector to approximate molar equivalents.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Lentiviruses produced with this system do not carry or express ANY viral genes and therefore have no associated toxicity issues. Only the protein expressed from the coding region between the LTR sites is incorporated into the mammalian cell chromosome and expressed. The lentivirus itself cannot replicate because of the built-in safety features.
For routine maintenance of 293FT cells, you need to add Geneticin (G418) antibiotic at a concentration of 500 µg/mL to maintain the Large T antigen plasmid/phenotype.
The F stands for the high transfection efficiency of this particular 293 cell clone (called 293F) and the T stands for the SV40 large T antigen. If you want to use regular 293 cells or another 293T cell line, you will be able to produce virus, but the titers will be lower. The large T antigen expression plasmid is stably integrated in the 293FT cell and confers resistance to Geneticin antibiotic in these cells.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
For HT1080 cells we typically use 10 µg/mL, but we strongly recommend that you generate a kill-curve for each antibiotic and cell line before proceeding. Most cell types respond to between 1 µg/mL and 10 µg/mL of blasticidin. For HT1080 cells, we typically use 100 µg/mL of Zeocin for Zeocin-containing lentiviral vectors. But again, generation of a kill-curve is strongly suggested.
We strongly recommend titering on HT1080 cells to determine the absolute titer of infectious virus in your supernatant. The primary reason is that it's a way to standardize titers obtained in different labs. Transduction efficiency is high in these cells, and titering results are very accurate and reproducible, making HT1080 cells the gold standard for titering. You can then try different MOIs in other cell types based on HT1080 titers. For instance, you may require an MOI of 50 in one cell type or MOI of 10 in another cell type based on titers obtained in HT1080.Accurate titer, however, can be obtained in essentially any mammalian cell line, but 3T3 and HeLa cells have a lower transduction efficiency than HT1080 cells (for reasons unknown). Do not use 293FT cells for titering.
Yes, you can use restriction enzymes Cla I (cutting at 1796) and BamH I (cutting at 2401) to remove the CMV promoter from the pLent6/V5-D-TOPO vector. Use Cla I and Spe I for the pLenti6/V5-DEST vector. Alternatively, we offer promoter-less lentiviral vector, pLenti6.4/R4R2/V5-DEST (Cat. No. A11145).
Ultracentrifugation is the most commonly used approach and is typically very successful (see Burns et al. (1993) Proc Natl Acad Sci USA 90:8033-8037; Reiser (2000) Gene Ther 7:910-913). Others have used PEG precipitation. Some purification methods are covered by patents issued to the University of California and Chiron.
Adenovirus is concentrated using CsCl density gradient centrifugation (there is a reference for this procedure in our adenovirus manual) or commercially available columns.
Titers between 1 x 10e5 and 3 x 10e5 cfu/mL (unconcentrated) are typical. If the titer is lower than 1x 10e5 cfu/mL, virus production was not optimal (arising for various reasons). Titers for the LacZ virus are typically in this low to mid 10e5 range. The sample lentiviral titer experiment shown in the ViraPower instruction manual shows lacZ lentivirus with a titer of 4.8 x 10e6 cfu/mL.
We strongly suggest that you titer your lentivirus on HT1080 cells, which allows you to compare titers from day-to-day within your lab and also with external labs. Transduction efficiency is high in these cells, and titering results are very accurate and reproducible--making HT1080 cells the gold standard for titering. You can then try different MOIs in other cell types based on HT1080 titers. For instance, you may require an MOI of 50 in one cell type or MOI of 10 in another cell type based on titers obtained in HT1080.
The ViraPower Lentiviral System:
(1) effectively transduces both dividing and non-dividing cells
(2) efficiently delivers the gene of interest to mammalian cells in culture or in vivo
(3) produces a pseudotyped virus with a broadened host range
(4) includes multiple features designed to enhance the biosafety of the system
Clone your gene of interest into one of our lentiviral expression vectors. We have a Directional TOPO version (pLenti6/V5/D-TOPO) and a Gateway version (pLenti6/V5-DEST vector). Co-transfect your recombinant vector along with the optimized ViraPower packaging mix into the 293FT producer cell line using Lipofectamine 2000 reagent (if using a different transfection reagent, follow the manufacturer's recommendations). Harvest the viral supernatant and determine the titer of the virus. Add the viral supernatant to your mammalian cell line of interest at the appropriate MOI. Assay for "transient" expression of your recombinant protein or select for stably transduced cells using the appropriate selection antibiotic, if desired, then examine expression of your protein of interest.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
This depends entirely on the target cell. Adenovirus requires the coxsackie-adenovirus receptor (CAR) and an integrin for efficient transduction. Lentivirus (with VSV-G) binds to a lipid in the plasma membrane (present on all cell types). With two totally different mechanisms of entry into the cell, there will always be differences in transduction efficiencies. However, the efficiency of transduction for both viral systems is easily modulated by the multiplicity of infection (MOI) used.
We use mycoplasma-tested Gibco FBS (Cat. No. 16000-044) without any modifications. We have observed that when 293FT cells are cultured in the presence of this FBS following the instructions in the manual, virus production is better than that obtained with many other serum sources.
We use the following plasticware for 293A and 293FT cells:
T175--Fisher Cat. No. 10-126-13; this is a Falcon flask with 0.2 µm vented plug seal cap.
T75--Fisher Cat. No. 07-200-68; this is a Costar flask with 0.2 µm vented seal cap.
100 mm plate--Fisher Cat. No. 08-772E; this is a Falcon tissue culture-treated polystyrene plate
We get excellent adherence on these plates under routine cell culture/maintenance conditions (expect cell lysis in 293A cells when making adenovirus).
Viral vectors:
Store lentiviral and adenoviral expression vectors (plasmid DNA) at -20 degrees C. Due to their relatively large sizes, we do not recommend storing these vectors at -80 degrees C, as the vector solutions will completely freeze and too many freeze thaws from -80 degrees C will affect the cloning efficiency. At -20 degrees C, the vectors will be stable but will not freeze completely. Glycerol stocks of vectors transformed into bacteria should always be stored at -80 degrees C.
Virus:
Both adenovirus and lentivirus particles should be aliquoted immediately after production and stored at -80 degrees C.
Lentivirus is more sensitive to storage temperature and to freeze/thaw than adenovirus and should be handled with care. Adenovirus can typically be frozen/thawed up to 3 times without loss of titer, while lentivirus can lose up to 5% or more activity with each freeze/thaw. It is recommended to aliquot your virus into small working volumes immediately after production, freeze at -80 degrees C, and then thaw just one aliquot for titering. This way, every time you thaw a new aliquot it should be the same titer as your first tube.
Adenovirus particles can be kept overnight at 4 degrees C if necessary, but it is best to avoid this. Viruses will be most stable at -80 degrees C.
When stored properly, viral stocks should maintain consistent titer and be suitable for use for up to one year. After long-term storage, we recommend re-titering your viral stocks before use.
Both the lentiviral and adenoviral systems should be used following Biosafety Level 2 (BSL-2). We recommend strict adherence to all CDC guidelines for BSL-2 (as well as institutional guidelines). Thermo Fisher Scientific has also engineered specific safety features into the lentiviral system.
Consult the "Biosafety in Microbiological and Biomedical Laboratories" publication (www.cdc.gov, published by the CDC in the USA, describes BSL-2 handling) and the "Laboratory Biosafety Guidelines" publication (www.phac-aspc.gc.ca, published by the Centre for Emergency Preparedness and Response in Canada) for more information on safe handling of various organisms and the physical requirements for facilities that work with them.
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If you're interested in stable integration and selection, choose the lentiviral system. We offer both a Directional TOPO (D-TOPO) and Gateway version of the kit to provide flexibility in the cloning of the gene of interest.
If you're looking for transient gene expression, choose the adenoviral system. We offer the Gateway cloning method for this product. It should be noted, however, that gene expression from both systems is typically detected within 24-48 hours of transduction, so both systems can be used for experiments of a transient nature. The main difference is that lentivirus integrates into the host genome and adenovirus does not. Higher viral titers are achieved with the adenovirus.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
No, neither lentivirus nor adenovirus can take an insert as large as 9 Kb. Lentiviral packaging limits are around 6 kb and adenoviral packaging limits are around 7-7.5 kb. Above that, no virus is made.
For lentivirus, titers will generally decrease as the size of the insert increases. We have effectively packaged inserts of 5.2 kb with good titer (approx. 0.5 x 10^5 cfu/mL). The size of the wild-type HIV-1 genome is approximately 10 kb. Since the size of the elements required for expression from pLenti vectors add up to approximately 4-4.4 kb, the size of your gene of interest should theoretically not exceed 5.6-6 kb for efficient packaging (see below for packaging limits for individual vectors).
pLenti4/V5-DEST vector: 6 kb
pLenti6/V5-DEST vector: 6 kb
pLenti6/V5/D-TOPO vector: 6 kb
pLenti6/UbC/V5-DEST vector: 5.6 kb
For adenovirus, the maximum packagable size is approximately 7-7.5 Kb (see below for packaging limits for individual vectors).
pAd/CMV/V5-DEST vector: 6 kb
pAd/PL-DEST vector: 7.5 kb
In most cases, there will not be enough pENTR vector DNA present to go directly from TOPO cloning into an LR reaction. You need between 100-300 ng of pENTR vector for an efficient LR reaction, and miniprep of a colony from the TOPO transformation is necessary to obtain that much DNA. However, if you want to try it, here are some recommendations for attempting to go straight into LR reactions from the TOPO reaction using pENTR/D, or SD TOPO, or pCR8/GW/TOPO vectors:
1. Heat inactivate the topoisomerase after the TOPO cloning reaction by incubating the reaction at 85 degrees C for 15 minutes.
2. Use the entire reaction (6 µL) in the LR clonase reaction. No purification steps are necessary.
3. Divide the completed LR reaction into 4 tubes and carry out transformations with each tube. You cannot transform entire 20 µL reaction in one transformation, and we have not tried ethanol precipitation and then a single transformation.
When attempting this protocol, we observed very low efficiencies (~10 colonies/plate). So just be aware that while technically possible, going directly into an LR reaction from a TOPO reaction is very inefficient and will result in a very low colony number, if any at all.
To have an N-terminal tag, the gene of interest must be in the correct reading frame when using non-TOPO adapted Gateway entry vectors. All TOPO adapted Gateway Entry vectors will automatically put the insert into the correct reading frame, and to add the N-terminal tag you simply recombine with a destination vector that has N-terminal tag.
To attach a C-terminal tag to your gene of interest, the insert must lack its stop codon, and be in the correct reading frame for compatibility with our C-terminal tagged destination vectors. Again, TOPO adapted Gateway Entry vectors will automatically put the insert into the correct reading frame. If you do not want the C-terminal tag to be expressed, simply include a stop codon at the end of the insert that is in frame with the initial ATG.
Generally, you need to choose a destination vector before you design and clone your insert into the Entry vector. This will determine whether you need to include an initiating ATG or stop codon with your insert.
No, not directly. The attB-PCR product must first be cloned, via a BP Clonase reaction, into a pDONR vector which creates an "Entry Clone" with attL sites. This clone can then be recombined, via an LR Clonase reaction, with a Destination vector containing attR sites. However, It is possible to perform both of these reactions in one step using the "One-Tube Protocol" described in the manual entitled "Gateway Technology with Clonase II".
Yes, this can be done using the Multisite Gateway Technology. MultiSite Gateway Pro Technology enables you to efficiently and conveniently assemble multiple DNA fragments - including genes of interest, promoters, and IRES sequences - in the desired order and orientation into a Gateway Expression vector. Using specifically designed att sites for recombinational cloning, you can clone two, three, or four DNA fragments into any Gateway Destination vector containing attR1 and attR2 sites. The resulting expression clone is ready for downstream expression and analysis applications.
For the BP reaction, approximately 5-10% of the starting material is converted into product. For the LR reaction, approximately 30% of the starting material is converted into product.
The core region of the att sites contains the recognition sequence for the restriction enzyme BsrGI. Provided there are no BsrGI sites in the insert, this enzyme can be used to excise the full gene from most Gateway plasmids. The BsrGI recognition site is 5'-TGTACA and is found in both att sites flanking the insertion site.
If a different restriction site is desired, the appropriate sequence should be incorporated into your insert by PCR.
We do have an alternative method called the "attB Adapter PCR" Protocol in which you make your gene specific primer with only 12 additional attB bases and use attB universal adapter primers. This protocol allows for shorter primers to amplify attB-PCR products by utilizing four primers instead of the usual two in a PCR reaction. You can find the sequence of these primers in the protocol on page 45 of the "Gateway Technology with Clonase II" manual.
There is a protocol in which all 4 primers mentioned above are in a single PCR reaction. You can find this protocol at in the following article: Quest vol. 1, Issue 2, 2004. https://www.thermofisher.com/us/en/home/references/newsletters-and-journals/quest-archive.reg.in.html. The best ratio of the first gene-specific and the second attB primers was 1:10.
We do not offer pre-made primers, but we can recommend the following sequences that can be ordered as custom primers for sequencing of pDONR201:
Forward primer, proximal to attL1: 5'- TCGCGTTAACGCTAGCATGGATCTC
Reverse primer, proximal to attL2: 5'-GTAACATCAGAGATTTTGAGACAC
1. Yeast two-hybrid protein-protein interaction studies Walhout AJ, Sordella R, Lu X, Hartley JL, Temple GF, Brasch MA, Thierry-Mieg N, Vidal M.
2. Protein Interaction Mapping in C. elegans Using Proteins Involved in Vulval Development. Science Jan 7th 2000; 287(5450), 116-122 Davy, A. et al.
3. A protein-protein interaction map of the Caenorhabditis elegans 26S proteosome. EMBO Reports (2001) 2 (9), p. 821-828. Walhout, A.J.M. and Vidal, M. (2001).
4. High-throughput Yeast Two-Hybrid Assays for Large-Scale Protein Interaction mapping. Methods: A Companion to Methods in Enzymology 24(3), pp.297-306
5. Large Scale Analysis of Protein Complexes Gavin, AC et al. Functional Organization of the Yeast Proteome by Systematic Analysis of Protein Complexes. Nature Jan 10th 2002, 415, p. 141-147.
6. Systematic subcellular localisation of proteins Simpson, J.C., Wellenreuther, R., Poustka, A., Pepperkok, R. and Wiemann, S.
7. Systematic subcellular localization of novel proteins identified by large-scale cDNA sequencing. EMBO Reports (2000) 1(3), pp. 287-292.
8. Protein-over expression and crystallography Evdokimov, A.G., Anderson, D.E., Routzahn, K.M. & Waugh, D.S.
9. Overproduction, purification, crystallization and preliminary X-ray diffraction analysis of YopM, an essential virulence factor extruded by the plague bacterium Yersinia pestis. Acta Crystallography (2000) D56, 1676-1679.
10. Evdokimov, et al. Structure of the N-terminal domain of Yersinia pestis YopH at 2.0 A resolution. Acta Crystallographica D57, 793-799 (2001).
11. Lao, G. et al. Overexpression of Trehalose Synthase and Accumulation of Intracellular Trehalose in 293H and 293FTetR:Hyg Cells. Cryobiology 43(2):106-113 (2001).
12. High-throughput cloning and expression Albertha J. M. Walhout, Gary F. Temple, Michael A. Brasch, James L. Hartley, Monique A. Lorson, Sander Van Den Huevel, and Marc Vidal.
13. Gateway Recombinational Cloning: Application to the Cloning of Large Numbers of Open Reading Frames or ORFeomes. Methods in Enzymology, Vol. 328, 575-592.
14. Wiemann, S. et.al., Toward a Catalog of Human Genes and Proteins: Sequencing and Analysis of 500 Novel Complete Protein Coding Human cDNAs, Genome Research (March 2001) Vol. 11, Issue 3, pp.422-435
15. Reviewed in NATURE: Free Access to cDNA provides impetus to gene function work. 15 march 2001, p. 289. Generating directional cDNA libraries using recombination
16. Osamu Ohara and Gary F. Temple. Directional cDNA library construction assisted by the in vitro recombination reaction. Nucleic Acids Research 2001, Vol. 29, no. 4. RNA interference (RNAi)
17. Varsha Wesley, S. et al. Construct design for efficient, effective and highthroughput gene silencing in plants. The Plant Journal 27(6), 581-590 (2001). Generation of retroviral constructs
18. Loftus S K et al. Generation of RCAS vectors useful for functional genomic analyses. DNA Res 31;8(5):221 (2001).
19. James L. Hartley, Gary F. Temple and Michael A. Brasch. DNA Cloning Using In Vitro Site-Specific Recombination. Genome Research (2000) 10(11), pp. 1788-1795.
20. Reboul et al. Open-reading frame sequence tags (OSTs) support the existence of at least 17,300 genes in C. elegans. Nature Genetics 27(3):332-226 (2001).
21. Kneidinger, B. et al. Identification of two GDP-6-deoxy-D-lyxo-4-hexulose reductase synthesizing GDP-D-rhamnose in Aneurinibacillus thermoaerophilus L420-91T*. JBC 276(8) (2001).
The attP1 sequence (pDONR) is:
AATAATGATT TTATTTTGAC TGATAGTGAC CTGTTCGTTG CAACAAATTG ATGAGCAATGCTTTTTTAT AATGCCAACT TTGTACAAAA AAGC[TGAACG AGAAACGTAA AATGATATAA ATATCAATAT ATTAAATTAG ATTTTGCATA AAAAACAGACTA CATAATACTG TAAAACACAA CATATCCAGT CACTATGAAT CAACTACTTA GATGGTATTA GTGACCTGTA]
The region within brackets is where the site is "cut" and replaced by the attB1-fragment sequence to make an attL1 site. The sequence GTACAAA is the overlap sequence present in all att1 sites and is always "cut" right before the first G.
The overlap sequence in attP2 sites is CTTGTAC and cut before C. This is attP2:
ACAGGTCACT AATACCATCT AAGTAGTTGA TTCATAGTGA CTGGATATGT TGTGTTTTAC AGTATTATGT AGTCTGTTTT TTATGCAAAA TCTAATTTAA TATATTGATA TTTATATCAT TTTACGTTTC TCGTTCAGCT TTCTTGTACA AAGTTGGCAT TATAAGAAAG CATTGCTTAT AATTTGTTG CAACGAACAG GTCACTATCA GTCAAAATAA AATCATTATT
So, attL1 (Entry Clone) should be:
A ATAATGATTT TATTTTGACT GATAGTGACC TGTTCGTTGC AACAAATTGA TGAGCAATGC TTTTTTATAA TGCCAACT TT G TAC AAA AAA GC[A GGC T]NN NNN
attL2 (Entry Clone) should be:
NNN N[AC C]CA GCT TT CTTGTACA AAGTTGGCAT TATAAGAAAG CATTGCTTAT CAATTTGTTG CAACGAACAG GTCACTATCA GTCAAAATAA AATCATTATT
The sequence in brackets comes from attB, and N is your gene-specific sequence.
Note: When creating an Entry Clone through the BP reaction and a PCR product, the vector backbone is not the same as Gateway Entry vectors. The backbone in the case of PCR BP cloning is pDONR201.
There is no size restriction on the PCR fragments if they are cloned into a pDONR vector. The upper limit for efficient cloning into a TOPO adapted Gateway Entry vector is approximately 5 kb. A Gateway recombination reaction can occur between DNA fragments that are as large as 150 kb.
Destination vectors that contain N-terminal fusion partners will express proteins that contain amino acids contributed from the attB1 site, which is 25 bases long. This means that in addition to any tag (6x His and/or antibody epitope tag), the N-terminus of an expressed protein will contain an additional 9 amino acids from the attB1 sequence - the typical amino acid sequence is Thr-Ser-Leu-Tyr-Lys-Lys-Ala-Gly-nnn, where nnn will depend on the codon sequence of the insert.
Effects on protein function: A researcher (Simpson et al. EMBO Reports 11(31):287-292, 2000) demonstrated that GFP fusions (N- terminal and C-terminal) localized to the proper intracellular compartment. The expression constructs were generated using Gateway cloning, so the recombinant protein contained the attB1 or attB2 amino acid sequence. The localization function of the cloned recombinant proteins was preserved.
Effects on expression: We have seen no effect of the attB sites on expression levels in E. coli, insect and mammalian cells. The gus gene was cloned into bacterial expression vectors (for native and N-terminal fusion protein expression) using standard cloning techniques and expressed in bacteria. Gus was also cloned into Gateway Destination vectors (for native and N-terminal fusion expression) and expressed. When protein expression is compared, there was no difference in the amount of protein produced. This demonstrates that for this particular case, the attB sites do not interfere with transcription or translation.
Effects on solubility: A researcher at the NCI has shown that Maltose Binding Protein fusions constructed with Gateway Cloning were soluble. The fusion proteins expressed had the attB amino acid sequence between the Maltose Binding Protein and the cloned protein. It is possible that some proteins containing the attB sequence could remain insoluble when expressed in E.coli.
Effects on folding: Two Hybrids screens show the same interacters identified with and without the attB sequence. Presumably correct protein folding would be required for protein-protein interactions to take place. It is possible that some proteins containing the attB sequence may not fold correctly.
Since the attB sequences are on the 5' end of oligos, they will not anneal to the target template in the first round of PCR. Sometimes the PCR product is more specific with the attB primers, probably due to the longer annealing sequence (all of attB plus gene specific sequence) after the first round of amplification. Generally there is no need to change PCR reaction conditions when primers have the additional attB sequence
No, this is not really feasible due to the fact that the attL sequence is approximately 100 bp, which is too long for efficient oligo synthesis. Our own maximum sequence length for ordering custom primers is 100 nucleotides. In contrast, the attB sequences are only 25 bp long, which is a very reasonable length for adding onto the 5' end of gene-specific PCR primers.
Vector information can be found in the product manuals or directly on our web site by entering the catalog number of the product in the search box. The vector map, cloning site diagram, and sequence information will be linked to the product page.
The Gateway nomenclature is consistent with lambda nomenclature, but we use numbers to differentiate between modified versions of the att sites (attB1, attB2, attP1, attP2, and so on). We have introduced mutations in the att sites to provide specificity and directionality to the recombination reaction. For example, attB1 will only recombine with attP1 and not with attP2.
The first step is to create an Entry clone for your gene of interest. We have 3 options to do this: The first is by BP recombination reaction using the PCR Cloning System with Gateway Technology. This is recommended for cloning large (>5 kb) PCR products. We also have Gateway compatible TOPO Cloning vectors such as pCR8/GW/TOPO and pENTR/D-TOPO. The final option is to use restriction enzymes to clone into a pENTR Dual Selection vector.
The gene of interest must be flanked by the appropriate att sites, either attL (100 bp) in an Entry clone or attB (25 bp) in a PCR product. For Entry clones, everything between the attL sites will be shuttled into the Gateway destination vector containing attR sites, and a PCR product flanked by attB sites must be shuttled into an attP-containing donor vector such as pDONR221.
The location of translation initiation sites, stop codons, or fusion tags for expression must be considered in your initial cloning design. For example, if your destination vector contains an N-terminal tag but does not have a C-terminal tag, the vector should already contain the appropriate translation start site but the stop codon should be included in your insert.
Yes, increasing the incubation time from 1 hour to 4 hours will generally increase colony numbers 2-3 fold. An overnight incubation at room temperature will typically increase colony yield by 5-10 fold.
BP Clonase II and LR Clonase II can be freeze/thawed at least 10 times without significant loss of activity. However, you may still want to aliquot the enzymes to keep freeze/thaw variability to a minimum.
These enzymes are more stable than the original BP and LR Clonase and can be stored at -20 degrees C for 6 months.
Mini-prep (alkaline lysis) DNA preparations work well in Gateway cloning reactions. It is important that the procedure remove contaminating RNA for accurate quantification. Plasmid DNA purified with our S.N.A.P. nucleic acid purification kits, ChargeSwitch kits, or PureLink kits are recommended.
A simple way to express a protein with a leader sequence is to have the leader sequence encoded in the destination vector. The other option is to have the leader sequence subcloned into the entry vector using restriction enzymes, or incorporate the leader sequence into the forward PCR primer when cloning a PCR product into the entry vector. Please see Esposito et al. (2005), Prot. Exp. & Purif. 40, 424-428 for an example of how a partial leader sequence for secretion was incorporated into an entry vector.
This depends on whether you are expressing a fusion or a native protein in the Gateway destination vector. For an N-terminal fusion protein the ATG will be given by the destination vector and it will be upstream of the attB1 site. For a C-terminal fusion protein or a native protein, the ATG should be provided by your gene of interest, and it will be downstream of the attB1 site.
The Gateway attB sites are derived from the bacteriophage lambda site-specific recombination, but are modified to remove stop codons and reduce secondary structure. The core regions have also been modified for specificity (i.e., attB1 will recombine with attP1 but not with attP2).
Expression experiments have shown that the extra amino acids contributed by the attB site to a fusion protein will most likely have no effect on protein expression levels or stability. In addition, they do not appear to have any effect on two-hybrid interactions in yeast. However, as is true with the addition of any extra sequences that result from tags, the possible effects will be protein-dependent.
No, attB primers are highly specific under standard PCR conditions. We have amplified from RNA (RT-PCR), cDNA libraries, genomic DNA, and plasmid templates without any specificity problems.
The smallest size we have recombined is a 70 bp piece of DNA located between the att sites. Very small pieces are difficult to clone since they negatively influence the topology of the recombination reaction.
There is no theoretical size limitation. PCR products between 100 bp and 11 Kb have been readily cloned into a pDONR Gateway vector. Other DNA pieces as large as 150 kb with att sites will successfully recombine with a Gateway-compatible vector. Overnight incubation is recommended for large inserts.
Standard desalted purity is generally sufficient for creating attB primers. We examined HPLC-purified oligos for Gateway cloning (about 50 bp long) and found only about a 2-fold increase in colony number over standard desalted primers. If too few colonies are obtained, you may try to increase the amount of PCR product used and/or incubate the BP reaction overnight.
Please ensure that the recommended filter sets for detection of fluorescence are used. Use an inverted fluorescence microscope for analysis. If desired, allow the protein expression to continue for 1-3 days before assaying for fluorescence.
The target sequence used may contain strong homology to other genes; please select a different target region.
Perform a kill curve to determine the antibiotic sensitivity of your cell line. Ensure that viral stocks are stored properly at -80 degrees C, and do not undergo freeze/thaw more than 3 times. Lastly, transducer the lentiviral contruct into cells in the presence of Polybrene reagent.
Ensure that the competent cells used were stored properly at -80 degrees C, and thawed on ice for immediate use. When adding DNA, mix competent cells gently: do not mix by pipetting up and down. Also do not exceed the maximum recommended amount of DNA for transformation (100 ng) or allow the volume of DNA added to exceed 10% of the volume of the competent cells, as these may inhibit the transformation.
Please check to ensure that your medium containing fetal bovine serum (FBS) is reduced in tetracycline. Many lots of FBS contain tetracycline, as FBS is often isolated from cows that have been fed a diet containing tetracycline, leading to low basal expression of shRNA. Ensure that a cell line expressing the Tet repressor is being used, and that the cells used are transduced at a suitable MOI. If creating your own Tet repressor-expressing cell line, wait at least 24 hours before transducing cells with your shRNA construct.
Low expression levels can be due to several factors. Please see the suggestions below:
- Low transfection efficiency: ensure that antibiotics are not added to the media during transfection, and that cells are at the proper cell confluency; optimize transfection conditions by varying the amount of transfection reagent used.
- Try a time course assay to determine the point at which the highest degree of gene knockdown occurs.
- Mutations are present in your construct: analyze the transformants by sequencing the ds oligo insert to verify its sequence.
- Target region is not optimal: select a different target region.
- Ensure siRNA is designed according to guidelines listed in the respective manual.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
You can try to scale back the amount of transfection reagent used, or use a different reagent for the transfection. Additionally, ensure that the plasmid used is pure and properly prepared for transfection.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
Difficulties sequencing could occur because the hairpin sequence is an inverted repeat that can form secondary structure during sequencing, resulting in a drop in the sequencing signal when entering the hairpin. If you encounter difficulties while sequencing, please try the following:
- Use high-quality, purified plasmid DNA for sequencing. We recommend preparing DNA using the Invitrogen PureLink HQ Mini Plasmid Purification Kit (Cat. No. K2100-01) or S.N.A.P. Plasmid DNA MidiPrep Kit (Cat. No. K1910-01).
- Add DMSO to the sequencing reaction to a final concentration of 5%.
- Increase the amount of template used in the reaction (up to twice the normal concentration).
- Standard sequencing kits typically use dITP in place of dGTP to reduce G:C compression. Other kits containing dGTP are available for sequencing G-rich and GT-rich templates. If you are using a standard commercial sequencing kit containing dITP, obtain a sequencing kit containing dGTP (e.g., dGTP BigDye Terminator v3.0 Ready Reaction Cycle Sequencing Kit, Cat. No. 4390229) and use a 7:1 molar ratio of dITP:dGTP in your sequencing reaction.
We highly recommend sequencing positive transformants to confirm the sequence of the ds oligo insert. When screening transformants, we find that up to 20% of the clones may contain mutated inserts (generally 1 or 2 bp deletions within the ds oligo). The reason for this is not known, but may be due to triggering of repair mechanisms within E. coli as a result of the inverted repeat sequence within the ds oligo insert. Note: Entry clones containing mutated ds oligo inserts generally elicit a poor RNAi response in mammalian cells. Identify entry clones with the correct ds oligo sequence and use these clones for your RNAi analysis.
Mutated inserts could also be caused by using poor-quality single-stranded oligos. Use mass spectrometry to check for peaks of the wrong mass, or order HPLC- or PAGE-purified oligos to avoid this problem.
- Verify that the sequence of the bottom-strand oligo is complementary to the sequence of the top-strand oligo.
- For the shRNA vectors, make sure that you mix single-stranded oligos with complementary sequences. The top-strand oligo should include CACC on the 5' end, while the bottom-strand oligo should include AAAA on the 5' end.
- For the miRNA vectors, make sure that the top-strand oligo includes TGCT at the 5' end and that the bottom-strand oligo includes CCTG at the 5' end.
Please review the possibilities below:
- Single-stranded oligos designed incorrectly; verify that the sequence of the bottom-strand oligo is complementary to the sequence of the top strand oligo.
- Ensure that oligos are annealed at room temp for 5-10 minutes after heating to 95 degrees C.
- Check the molar ratio you are using for annealing top and bottom-strand oligo; equal amounts should be used.
When performing RNAi studies using pLenti4/BLOCK-iT-DEST lentiviral constructs, we generally observe significant inhibition of gene expression within 48-120 hours after transduction. The degree of gene knockdown depends on the time of assay, stability of the protein of interest, and on the other factors. Note that 100% gene knockdown is generally not observed, but >80% is possible with optimized conditions.
The lentiviral and packaging vectors supplied in the BLOCK-iT Lentiviral RNAi Expression System are third-generation vectors based on lentiviral vectors developed by Dull et al., 1998. This third-generation lentiviral system includes a significant number of safety features designed to enhance its biosafety and to minimize its relation to the wild-type human HIV-1 virus. The BLOCK-iT Lentiviral RNAi Expression System includes the following key safety features:
- The pLenti6/BLOCK-iT-DEST expression vector contains a deletion in the 3' LTR (?U3) that does not affect generation of the viral genome in the producer cell line, but results in “self-inactivation” of the lentivirus after transduction of the target cell (Yee et al., 1987; Yu et al., 1986; Zufferey et al., 1998). Once integrated into the transduced target cell, the lentiviral genome is no longer capable of producing packageable viral genome.
- The number of genes from HIV-1 that are used in the system has been reduced to three (i.e., gag, pol, and rev).
- The VSV-G gene from Vesicular Stomatitis Virus is used in place of the HIV-1 envelope (Burns et al., 1993; Emi et al., 1991; Yee et al., 1994).
- Genes encoding the structural and viral genome packaging components are separated onto four plasmids. All four plasmids have been engineered not to contain any regions of homology with each other to prevent undesirable recombination events that could lead to the generation of a replication competent virus (Dull et al., 1998).
- Although the three packaging plasmids allow expression in trans of proteins required to produce viral progeny (e.g., gal, pol, rev, env) in the 293FT producer cell line, none of them contain LTRs or the ? packaging sequence. This means that none of the HIV-1 structural genes are actually present in the packaged viral genome, and thus, are never expressed in the transduced target cell. No new replication-competent virus can be produced.
- The lentiviral particles produced in this system are replication-incompetent and only carry the gene of interest. No other viral species are produced.
- Expression of the gag and pol genes from pLP1 has been rendered Rev dependent by virtue of the HIV-1 RRE in the gag/pol mRNA transcript. Addition of the RRE prevents gag and pol expression in the absence of Rev (Dull et al., 1998).
- A constitutive promoter (RSV promoter) has been placed upstream of the 5' LTR in the pLenti6/BLOCK-iT-DEST expression vector to offset the requirement for Tat in the efficient production of viral RNA (Dull et al., 1998).
Despite the inclusion of the safety features discussed on the previous page, the lentivirus produced with this system can still pose some biohazard risks since it can transduce primary human cells. For this reason, we highly recommend that you treat lentiviral stocks generated using this system as Biosafety Level 2 (BL-2) organisms and strictly follow all published BL-2 guidelines with proper waste decontamination. Furthermore, exercise extra caution when creating lentiviruses carrying potential harmful or toxic genes (e.g., activated oncogenes). For more information about the BL-2 guidelines and lentivirus handling, refer to the document “Biosafety in Microbiological and Biomedical Laboratories,”.
Constitutive knockdown is virtually identical for these two promoters in HEK 293 cells. In other cell types there are reports that either H1 or U6 may be more active, though in general, differences are minimal.
Use of the BLOCK-iT Lentiviral RNAi Expression System to facilitate lentiviral based delivery of shRNA to mammalian cells provides the following advantages:
- The pENTR/U6 entry vector provides a rapid and efficient way to clone ds oligo duplexes encoding a desired shRNA target sequence into a vector containing an RNA Pol III-dependent expression cassette (i.e., U6 RNAi cassette) for use in RNAi analysis.
- The vectors in the System are Gateway-adapted for easy recombination of the U6 RNAi cassette from the pENTR/U6 vector into the pLenti6/BLOCK-iT-DEST vector.
- Generates a replication-incompetent lentivirus that effectively transduces both dividing and nondividing mammalian cells, thus broadening the potential RNAi applications beyond those of other traditional retroviral systems (Naldini, 1998).
- Efficiently delivers the shRNA of interest to mammalian cells in culture or in vivo.
- Provides stable, long-term expression of the shRNA of interest beyond that offered by traditional adenovirus-based systems.
- Produces a pseudotyped virus with a broadened host range (Yee, 1999).
- Includes multiple features designed to enhance the biosafety of the system.
Adenovirus is not an actively lytic virus, meaning that mature viral particles accumulate in the cell over the course of two to three days. As virus accumulates, the producer cell rounds up and eventually bursts due to the sheer number of virus particles inside. Once this occurs, neighboring cells become infected and the three-day cycle begins again. The term “cytopathic effect”, or CPE, is used to describe this and is typically visible within approximately 7 days posttransfection in the form of “comet-shaped” plaques resulting from two rounds of infection, replication and cell burst.
After 7 days, CPE will expand and eventually take over the plate by approximately 10 days posttransfection.
10 days are required to produce virus from a transfected dish of cells (as just described).
Once an initial viral stock is produced, it can be amplified directly by infection of fresh 293A cells at a multiplicity of infection (MOI) of 3.
Please see the definitions below:
Infection: Applies to situations where viral replication occurs and infectious viral progeny are generated. Only cell lines that stably express E1 can be infected.
Transduction: Applies to situations where no viral replication occurs and no infectious viral progeny are generated. Mammalian cell lines that do not express E1 are transduced. In this case, you are using adenovirus as a vehicle to deliver shRNA.
Please see the steps below:
Clone the double-stranded DNA oligo encoding an shRNA or miR RNAi into one of the BLOCK-iT entry (shRNA) or expression (miR RNAi) vectors.
Transfer the RNAi cassette into the adenoviral (shRNA only) or lentiviral destination vector by Gateway recombination.
Transfect RNAi vectors into the viral producer cells to produce viral stocks, which can be used immediately or stored at -80 degrees C.
Harvest viral supernatants and determine the titer (amplify adenoviral stocks if desired).
Transduce lentiviral or adenoviral stocks to any cell type.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.