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View additional product information for pcDNA™6/TR vector Mammalian Expression Vector - FAQs (V102520)
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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。
这里列举了一些可能的原因与解决方案：
•所用检测法可能不适当或不够灵敏： ◦我们推荐您优化检测方案或寻找更为灵敏的方法。如果使用考马斯亮蓝染色/银染法检测过该蛋白，我们则推荐您使用免疫印迹法来增加检测灵敏度。裂解产物中存在的内源蛋白可能会在考马斯亮蓝染色/银染过程中掩盖目的蛋白。如果可能，我们推荐您在免疫印迹实验中包括一个阳性对照。
•筛选到的克隆数不够：至少筛选出20个克隆。
•在稳转筛选中使用了不适当的抗生素浓度：请确保正确获取了抗生素的杀死曲线。由于某一既定抗生素的效力依赖于细胞类型，血清，培养基和培养技术，因此必须在每次进行稳定筛选的时候确定抗生素的用量。如果采用的培养基或血清条件明显不同，则即使是我们所提供的稳转细胞系对于我们推荐的剂量也可能出现更敏感或更不敏感的情况。
•基因产物（即使低水平）的表达可能与该细胞系的生长不相容（如毒性基因）：使用一个可诱导的表达系统。
•阴性克隆可能由基因表达的关键载体位点处优先发生了线性化所致：在一个不影响表达的位点实施载体线性化，如在细菌抗药性标志物序列中。
这里列举了一些可能的原因与解决方案：
•尝试试剂盒自带的表达对照。
•可能的检测问题：
◦检测瞬转的表达蛋白可能有难度，因为转染效率可能过低，以致用于整个转染群体的评估手段无法成功实现检测。我们推荐您通过稳转筛选或采用能够逐个检测单一细胞的技术手段来优化您的转染操作。您也可尝试通过改变启动子或细胞类型来提高表达水平。
◦细胞中的蛋白表达水平对于所选择的检测方法来说可能过低。我们推荐您优化检测方案或寻找更为灵敏的方法。如果使用考马斯亮蓝染色/银染法检测过该蛋白，我们则推荐您使用免疫印迹法来增加检测灵敏度。裂解产物中存在的内源蛋白可能会在考马斯亮蓝染色/银染过程中掩盖目的蛋白。如果可能，我们推荐您在免疫印迹实验中包括一个阳性对照。
◾蛋白可能降解或截短了：使用Northern杂交进行检测。
◾可能的时程问题：由于蛋白表达随时间延长而发生的变化依赖该蛋白的天然属性，我们一般推荐您先获取一份表达的时程曲线。尝试进行一次时程分析将帮助您确定最优的表达时间窗。
◾可能的克隆问题：通过限制性酶切和/或测序来验证克隆。
不可以；新霉素对哺乳动物细胞有毒性。我们推荐您使用Geneticin（又称 G418硫酸盐)，这一产品的毒性较低，是在哺乳动物细胞中进行有效筛选的新霉素的替代品。
即使缺乏Kozak序列，翻译也还是会在核糖体遇到的第一个ATG处启始，不过启始效率可能相对较低。只要处于最初ATG的阅读框内，任何下游的插入序列都可能表达为融合蛋白，不过如果这里没有Kozak保守序列，则蛋白的表达水平预期会比较低。如果载体中包含一个非Kozak型的保守ATG，我们则推荐您将基因克隆至该ATG上游，再包含一个Kozak序列来优化表达效果。
我们未提供anti-TetR的抗体。即使使用anti-TetR抗体的免疫印迹技术能够筛选出不表达TetR蛋白的克隆，这也不能作为筛选功能性克隆的理想方法。通过瞬转表达LacZ的对照质粒来进行功能测试可以满足这一需求，用户可挑选那些在无四环素条件下β-半乳糖苷酶表达水平极低，而加入四环素后β-半乳糖苷酶表达水平极高的那些克隆。
我们提供pJTI R4 Exp CMV EmGFP pA载体，货号A14146，您可使用这一产品来监控转染和表达情况。
在小鼠细胞系中，人们已知CMV启动子的效率会随时间延长而逐渐下降。因此，我们推荐您使用一款非CMV型的载体，如EF1α或UbC启动子，以在小鼠细胞系中长时间表达蛋白。
保守的Kozak序列为A/G NNATGG，其中的ATG表示起始密码子。ATG周围的核苷酸点突变会影响翻译效率。尽管我们通常情况下都推荐加入一段Kozak保守序列，不过这一操作的必要性还是基于具体的目的基因，一般只需ATG就足以高效地启始翻译过程。最佳的建议是保持cDNA中天然起始位点，除非确定这一位点的功能性不理想。如果从表达的角度来考虑，推荐构建并测试两种载体，一个具有天然的起始位点，另一个具有保守的Kozak序列。通常情况下，所有具有N-融合表达的表达载体都已经包含了一个翻译起始位点。
ATG通常对于高效的翻译启始是足够的，尽管翻译效率要视目的基因而定。最佳的建议应是保持cDNA中天然起始位点，除非确定这一位点的功能性不理想。如果从表达的角度来考虑，推荐构建并测试两种载体，一个具有天然的起始位点，另一个具有保守的Kozak序列。通常情况下，所有N-端融合型表达载体都已包含了一个RBS或翻译起始位点。
原核生物mRNA含有Shine-Dalgarno序列，也称为核糖体结合位点(RBS)，它是由AUG起始密码子5’端的多嘌呤序列AGGAGG组成。该序列与16S rRNA 3’端的互补，有助于mRNA有效结合到核糖体上。同理，真核生物（特别是哺乳动物）mRNA也含有完成有效翻译所需的重要序列信息。然而，Kozak序列不是真正的核糖体结合位点，而是一种翻译起始增强子。Kozak共有序列是ACCAUGG，其中AUG是起始密码子。-3位的嘌呤(A/G)具有重要作用；若-3位是一个嘧啶(C/T)，翻译过程会对-1、-2和+4位的改变更敏感。当-3位从嘌呤变为嘧啶时，可使表达水平降低多达95%。+4位对表达水平的影响相对较小，可以使表达水平降低约50%。
注：果蝇的最佳Kozak序列稍有不同，酵母完全不遵循这些规则。见下列参考文献：
•Foreign Gene Expression in Yeast: a Review. Yeast, vol. 8, p. 423-488 (1992).
•Caveneer, Nucleic Acids Research, vol. 15, no. 4, p. 1353-1361 (1987).
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.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
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.
Here are possible causes and solutions:
Detection method may not be appropriate or sensitive enough:
- We recommend optimizing the detection protocol or finding more sensitive methods. If the protein is being detected by Coomassie/silver staining, we recommend doing a western blot for increased sensitivity. The presence of endogenous proteins in the lysate may obscure the protein of interest in a Coomassie/silver stain. If available, we recommend using a positive control for the western blot.
- Insufficient number of clones screened: Screen at least 20 clones.
- Inappropriate antibiotic concentration used for stable selection: Make sure the antibiotic kill curve was performed correctly. Since the potency of a given antibiotic depends upon cell type, serum, medium, and culture technique, the dose must be determined each time a stable selection is performed. Even the stable cell lines we offer may be more or less sensitive to the dose we recommend if the medium or serum is significantly different.
- Expression of gene product (even low level) may not be compatible with growth of the cell line: Use an inducible expression system.
- Negative clones may result from preferential linearization at a vector site critical for expression of the gene of interest: Linearize the vector at a site that is not critical for expression, such as within the bacterial resistance marker.
Here are possible causes and solutions:
- Try the control expression that is included in the kit
Possible detection problem:
- Detection of expressed protein may not be possible in a transient transfection, since the transfection efficiency may be too low for detection by methods that assess the entire transfected population. We recommend optimizing the transfection efficiency, doing stable selection, or using methods that permit examination of individual cells. You can also increase the level of expression by changing the promoter or cell type.
- Expression within the cell may be too low for the chosen detection method. We recommend optimizing the detection protocol or finding more sensitive methods. If the protein is being detected by Coomassie/silver staining, we recommend doing a western blot for increased sensitivity. The presence of endogenous proteins in the lysate may obscure the protein of interest in a Coomassie/silver stain. If available, we recommend using a positive control for the western blot.
Protein might be degraded or truncated: Check on a Northern.
Possible time-course issue: Since the expression of a protein over time will depend upon the nature of the protein, we always recommend doing a time course for expression. A pilot time-course assay will help to determine the optimal window for expression.
Possible cloning issues: Verify clones by restriction digestion and/or sequencing.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
No; neomycin is toxic to mammalian cells. We recommend using Geneticin (a.k.a. G418 Sulfate), as it is a less toxic and very effective alternative for selection in mammalian cells.
Translation initiation will occur at the first ATG encountered by the ribosome, although in the absence of a Kozak sequence, initiation will be relatively weak. Any insert downstream would express a fusion protein if it is in frame with this initial ATG, but levels of expressed protein are predicted to be low if there is a non-Kozak consensus sequence. If the vector contains a non-Kozak consensus ATG, we recommend that you clone your gene upstream of that ATG and include a Kozak sequence for optimal expression.
We do not offer an anti-TetR antibody. Even though a western using an anti-TetR antibody can be used to screen out clones that do not express any TetR protein, it would not be the optimal way to screen for functional clones. Functional testing by performing a transient transfection with the lacZ expression control plasmid is recommended for this purpose, followed by picking a clone that shows lowest basal levels of expression of beta-galactosidase in the absence of tetracycline, and highest levels of beta-galactosidase expression upon addition of tetracycline.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
We offer pJTI R4 Exp CMV EmGFP pA Vector, Cat. No. A14146, which you can use to monitor your transfection and expression.
The CMV promoter is known to be downregulated over time in mouse cell lines. Hence, we recommend using one of our non-CMV vectors, such as those with the EF1alpha or UbC promoter, for long-term expression in mouse cell lines.
The consensus Kozak sequence is A/G NNATGG, where the ATG indicates the initiation codon. Point mutations in the nucleotides surrounding the ATG have been shown to modulate translation efficiency. Although we make a general recommendation to include a Kozak consensus sequence, the necessity depends on the gene of interest and often, the ATG alone may be sufficient for efficient translation initiation. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a consensus Kozak. In general, all expression vectors that have an N-terminal fusion will already have an initiation site for translation.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
ATG is often sufficient for efficient translation initiation although it depends upon the gene of interest. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a Shine Dalgarno sequence/RBS or consensus Kozak sequence (ACCAUGG), as the case may be. In general, all expression vectors that have an N-terminal fusion will already have a RBS or initiation site for translation.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
No. The two systems are not compatible since they utilize different strategies for promoter regulation. The T-REx system is designed such that native E. coli tet-repressor protein molecules bind to specific tet-operator sequences (2X TO) just downstream of the TATA box in the full length CMV promoter in the expression vector. This binding keeps the promoter silent simply by preventing the normal transcription machinery from productive assembly at the TATA box. Incidentally, it is this full length CMV promoter region that permits higher induced expression levels relative to other systems.
The recombinant 'repressor' proteins utilized in Clontech's system are actually recombinant fusion proteins which also contain a potent transcriptional transactivator. The Clontech system places operator sequences 5' to the TATA box and relies upon the VP16 transactivator to promote transcription. These repressor-transactivator fusion constructs would have unpredictable and unreliable effects at the CMV promoter in our expression constructs. Additionally, the tet-repressor protein produced from the pCDNA6/TR construct in the T-REx system has no transactivation domain and so would exert little regulatory effect at the minimal promoter region (non-full length CMV) found in the Clontech response plasmids.
Prokaryotic mRNAs contain a Shine-Dalgarno sequence, also known as a ribosome binding site (RBS), which is composed of the polypurine sequence AGGAGG located just 5’ of the AUG initiation codon. This sequence allows the message to bind efficiently to the ribosome due to its complementarity with the 3’-end of the 16S rRNA. Similarly, eukaryotic (and specifically mammalian) mRNA also contains sequence information important for efficient translation. However, this sequence, termed a Kozak sequence, is not a true ribosome binding site, but rather a translation initiation enhancer. The Kozak consensus sequence is ACCAUGG, where AUG is the initiation codon. A purine (A/G) in position -3 has a dominant effect; with a pyrimidine (C/T) in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Expression levels can be reduced up to 95% when the -3 position is changed from a purine to pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. See the following references:
- Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.
- Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947-950.
- Kozak, M. (1987) An analysis of 5´-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
- Kozak, M. (1989) The scanning model for translation: An update. J. Cell Biol. 108, 229-241.
- Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 18, 2828.
Note: The optimal Kozak sequence for Drosophila differs slightly, and yeast do not follow this rule at all. See the following references:
- Romanos, M.A., Scorer, C.A., Clare, J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423-488.
- Cavaneer, D.R. (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 15, 1353-1361.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Our vectors have not been completely sequenced. Your sequence data may differ when compared to what is provided. Known mutations that do not affect the function of the vector are annotated in public databases.
No, our vectors are not routinely sequenced. Quality control and release criteria utilize other methods.
Sequences provided for our vectors have been compiled from information in sequence databases, published sequences, and other sources.
Eukaryotic (and specifically mammalian) mRNA contains sequence information that is important for efficient translation. However, this sequence, termed a Kozak sequence, is not a true ribosome binding site, but rather a translation initiation enhancer. The Kozak consensus sequence is ACCAUGG, where AUG is the initiation codon. A purine (A/G) in position -3 has a dominant effect; with a pyrimidine (C/T) in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Expression levels can be reduced up to 95% when the -3 position is changed from a purine to pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. See the following references:
Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.
Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947-950.
Kozak, M. (1987) An analysis of 5´-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
Kozak, M. (1989) The scanning model for translation: An update. J. Cell Biol. 108, 229-241.
Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 18, 2828.
Note: The optimal Kozak sequence for Drosophila differs slightly, and yeast do not follow this rule at all. See the following references:
Romanos, M.A., Scorer, C.A., Clare, J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423-488.
Cavaneer, D.R. (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 15, 1353-1361.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.