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查看更多产品信息 T-REx™-293 Cell Line - FAQs (R71007)
19 个常见问题解答
可行，您可使用我们的一株T-REx细胞系作为pLenti6.3/TO/V5-DEST慢病毒载体的表达宿主。不过，请注意pLenti6.3/TO/V5-DEST载体是一款包含WPRE和cPPT元件的HiPerform慢病毒载体，能够提升病毒滴度和表达水平；而我们所提供的T-REx细胞系则不含这些遗传元件。此外，您使用这些T-REx细胞系仅能用于瞬时表达，因为 Lenti6.3/TO/V5-DEST慢病毒表达质粒和Tet抑制子质粒（pcDNA6/TR）均能够稳定整合进T-REx细胞中，而这两者又都含有杀稻瘟素筛选标志物，因此建立稳转细胞系的操作就无法实现。
几乎所有批次的FBS中都含有四环素，因为FBS通常从奶牛中获得，而它们的饮食中有四环素。如果细胞培养于含有未去除四环素的FBS的培养基中时，目的基因就会出现低水平的基底表达情况，即使用户未主动添加四环素。在这一情况下，我们推荐您购买我们的Gibco细胞培养低四环素含量的FBS。为了确保四环素的清除效果，这些批次产品中四环素的含量应低于19.7 ng/mL（这一数值应为分析检测阈值）。
注意：Tet-抑制子蛋白与四环素的结合常数为3 nM。假设培养基中含有10%的血清，而血清中四环素的浓度为19.7 ng/mL，则相当于4 nM的四环素。因此请记得，即便使用了低四环素的FBS，仍有可能存在本底水平的表达。
当执行共转染时，用户无法在稳转细胞系中同时完成功能性TetR或GeneSwitch蛋白的双重测试。另一方面，如果执行连续转染，用户就可对所生成的T-REx或GeneSwitch细胞系进行功能性测试，他们可将LacZ对照表达质粒瞬转进入细胞，并挑取那些在诱导剂缺乏条件下表达LacZ的本底水平最低，而在含诱导剂条件下LacZ表达水平最高的克隆。之后可对这一克隆进行扩增，并按需用于T-REx或GeneSwitch表达载体的转染实验。
使用GeneSwitch系统能够确保目的基因处于极低的基础表达水平，而T-REx系统可能会有少量渗漏表达，因为FBS中不可避免的存在着一些四环素。GeneSwitch系统的诱导表达水平可能甚至高于CMV启动子。GeneSwitch系统的劣势在于，尽管该系统能够在转基因条件下以优异的性能工作，但在培养系统中关闭表达的操作不是很容易实现。而另一方面，T-REx系统可通过加入和去除诱导剂来切换开关状态。
Flp-In T-REx系统将Flp-In系统的靶向整合与T-REx系统的强大诱导表达能力整合在一起。该系统能够生成同基因，可诱导的稳定表达细胞系，并能够针对这些细胞系实行多克隆筛选。一旦建立了包含整合FRT位点的Flp-In T-REx宿主细胞系，后续建立表达目的基因的Flp-In T-REx细胞系就变得迅速高效了。
强力霉素可作为T-REx系统中诱导剂的代替品。它的作用机理与四环素相近，并在T-REx系统中表现出与四环素相似的剂量效应和诱导性质。强力霉素显示出比四环素更长的半衰期（分别为48小时和24小时）。我们未提供强力霉素，但用户可从Sigma（货号D9891）进行购买。
我们提供T-REx-293，-HeLa，-CHO和-Jurkat细胞系。这些细胞系都源自细胞转染了pcDNA6/TR，，之后通过杀稻瘟素的稳转筛选来获得这些阳性细胞。它们持续和稳定地表达TetR基因，因此在使用T-REx系统的过程中能够帮助用户显著地节省时间和精力。这些细胞系通过瞬转阳性对照载体pcDNA4/TO/LacZ，可功能性地检测表达。在抑制状态下它们表达出极低的bGal基底表达水平，而在四环素诱导条件下可实现高表达。
我们提供三类独特的哺乳动物表达系统来帮助用户实现目的基因可诱导/调控性的表达。
•T-REx系统
•Flp-In T-REx系统
•GeneSwitch系统
请参见下表中的比较结果：
系统 --基础表达水平--诱导表达水平--表达最大化的响应时间--转基因应用
T-REx系统--低--最高--高--合适
Flp-In T-REx系统--较低--高--24-48小时--合适
GeneSwitch系统--最低--高--24-48小时--合适
是的，我们提供4种T-REx细胞系：293、HeLa、CHO或Jurkat细胞。此外，您也可使用pcDNA6/TR自行建立可稳定表达四环素阻遏蛋白的T-REx细胞系。
Yes, you can use one of our T-REx cell lines as the host for your pLenti6.3/TO/V5-DEST lentiviral construct. However, please note that while the pLenti6.3/TO/V5-DEST vector is a HiPerform lentiviral vector containing the genetic elements WPRE and cPPT for enhancing viral titer and expression; the T-REx cell lines we offer do not contain these elements. Further, you can use these T-REx cell lines only for transient expression, because the Lenti6.3/TO/V5-DEST lentiviral expression construct, and the Tet repressor plasmid (pcDNA6/TR) that is stably integrated within the T-REx cells, both contain the blasticidin selection marker, making stable cell line development not possible.
Almost all lots of FBS contain tetracycline, because FBS is generally obtained from cows that have been fed a diet containing tetracycline. If cells are cultured in medium containing FBS that is not reduced in tetracycline, there may be low basal expression of the gene of interest in the absence of added tetracycline. In such cases, we recommend purchasing tetracycline-reduced FBS from our Gibco Cell Culture Division. To be qualified as tetracycline-reduced, these lots must contain below 19.7 ng/mL tetracycline (which happens to be the assay detection limit).
Note: The binding constant for Tet-repressor protein with tetracycline is 3 nM. Assuming that the medium contains 10% serum, a serum tetracycline concentration of 19.7 ng/mL is equivalent to 4 nM tetracycline. Thus, keep in mind that it is possible to get basal level expression even from tetracycline-reduced FBS.
When a co-transfection is performed, there is no way of testing the double stable cell line for functional TetR or GeneSwitch protein, respectively. On the other hand, when sequential transfection is performed, one can functionally test the generated T-REx or GeneSwitch cell line by transiently transfecting the lacZ expression control plasmid and then picking a clone that shows the lowest basal level of expression of lacZ in the absence of the inducer, and the highest level of lacZ in the presence of the inducer. This clone can then be expanded and used to transfect the T-REx or GeneSwitch expression construct, as the case may be.
With the GeneSwitch system, it is possible to have the absolute lowest basal levels of expression of the gene of interest, whereas the T-REx system may be a little leaky due to the inevitable presence of tetracycline in FBS. The induced level of expression in the GeneSwitch system can be even higher than that seen with the CMV promoter. The disadvantage of the GeneSwitch system is that the expression does not appear to switch off very easily in culture, although it has been demonstrated to function beautifully in transgenics. The T-REx system, on the other hand, can be switched on and off by the addition and removal of the inducer.
The Flp-In T-REx system combines the targeted integration offered by the Flp-In system with the powerful inducible expression offered by the T-REx system. It allows generation of isogenic, inducible, stable cell lines and permits polyclonal selection of these cell lines. Once the Flp-In T-REx host cell line containing an integrated FRT site has been created, subsequent generation of Flp-In T-REx cell lines expressing the gene(s) of interest is rapid and efficient.
Doxycycline may be used as an alternative inducing agent in the T-REx system. It is similar to tetracycline in its mechanism of action, and exhibits similar dose-response and induction characteristics as tetracycline in the T-REx system. Doxycycline has been shown to have a longer half-life than tetracycline (48 hours vs. 24 hours, respectively). We do not offer doxycycline, but it may be obtained from Sigma (Cat. No. D9891).
We offer T-REx-293, -HeLa, -CHO, and -Jurkat cell lines. These cell lines are derived by transfection of parental cells with pcDNA6/TR followed by stable selection with blasticidin. They constitutively and stably express the TetR gene, allowing significant time and effort saving when using the T-REx system. These cell lines are functionally tested for expression by transient transfection with the positive control vector, pcDNA4/TO/lacZ. They exhibit extremely low basal expression levels of bGal in the repressed state and high expression upon induction with tetracycline.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
We offer three unique mammalian expression systems for inducible/regulated expression of the gene of interest:
- T-REx system
- Flp-In T-REx system
- GeneSwitch system
Please see below to see how they compare with one another:
System -- Basal Expression Level -- Induced Expression Level -- Response time to Maximal Expression -- Transgenic Appliation
T-Rex system -- Low -- Highest -- High -- Suitable
Flp-In T-REx system -- Lower -- High -- 24-48 hrs -- Suitable
GeneSwitch system -- Lowest -- High -- 24-48 hrs -- Suitable
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.
Yes, we offer 4 T-REx cell lines: 293, HeLa, CHO, or Jurkat cells. Alternatively, you can create your own T-REx cell line that stably expresses the Tet repressor form the pcDNA6/TR.