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查看更多产品信息 Flp-In™ T-REx™ Core Kit - FAQs (K650001)
24 个常见问题解答
我们在内部实验中发现，当FRT位点整合进入具有高度转录活性的宿主细胞基因组位点中时（在Flp-In CHO与Flp-In 293细胞中更为常见，但也出现于Flp-In 3T3细胞及其他任何Flp-In宿主细胞系中），就会出现一些“读通（read-though）”的转录现象，并在Flp-In反应后翻译出lacZ-Zeocin ORF——即使lacZ-Zeocin ORF未包含真正的启动子和ATG起始密码子。在这一情况发生时，潮霉素抗性克隆就可能同时出现lacZ阳性和Zeocin抗生素耐受性。为了确保FRT实现了位点特异性整合而非随机整合，我们推荐用户在无pOG44存在的条件下开展平行的对照转染实验。这一对照组在潮霉素筛选过程中应不存在存活的克隆，表明在pOG44存在条件下获得的所有潮霉素抗性克隆确实为Flp重组酶依赖克隆，因此确定目的基因确实有效整合进入了FRT位点。另外，针对这些克隆进行Southern杂交分析也能够帮助用户验证目的基因确实发生了准确的FRT整合，尽管lacZ也出现表达（尽管通常情况下这是不必要的）。在Flp-In反应之后，一旦您观察到潮霉素耐受克隆，我们就推荐您将其挑选出来，并分析其中的目的基因表达情况。
Flp-In 3T3细胞系源自于NIH3T3细胞，后者是小鼠的成纤维细胞。CMV启动子在小鼠细胞系中已知会随时间延长而逐渐沉默，因此我们推荐您在这类细胞系中使用那些包含非CMV型启动子的Flp-In表达载体，如pEF5/FRT/V5-D-TOPO载体或pEF5/FRT/V5-DEST载体。
在放弃之前，我们推荐您尝试使用pFRT/lacZeo2载体来建立您的宿主细胞系。这一载体包含了截短型的SV40启动子的，它驱动了lacZ-Zeocin融合蛋白的表达。使用这一载体能够帮助用户分离出那些整合在增强子附近的克隆，从而获得更高的目的基因表达效果。
Jump-In系统是由PhiC31-整合酶所介导的一个不可逆转的哺乳动物稳定定向表达系统。这一系统是由Jump-In Fast系统和Jump-In TI（定向整合）系统所组成，前者包含了单一的整合步骤，后者则需要两个整合步骤，两者都是定向且不可逆的。相比而言，Flp-In系统是一款可逆和稳定的哺乳动物靶向表达系统。第一步整合是随机的（pFRT/lacZeo的整合），而第二步整合（Flp-In表达载体的整合）是定向但可逆的。
在理论上来讲，用户能够实现Flp-In表达载体的多重整合效果——这其中包含了一个FRT-特异性的整合事件和一个随机的第二位点整合事件。不过，随机整合的发生概率相对较低。转染过程中所用DNA的有限数量将减少第二位点的整合概率。我们向293细胞（缺少FRT位点）中转染了pcDNA5/FRT载体，并在筛选了200个以上的克隆后，鉴定到一个潜在的第二整合位点。用户可通过Southern杂交来检测DNA的整合位点。单一整合元件会显示为独立的一个条带；两个整合位点：两个条带；三个位点，三条带，如此延续。我们将一些Flp-In表达细胞系培养了四个月以上，无论是否向培养体系中加入潮霉素，均未发现Flp-In表达载体发生任何形式的丢失。
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
We have observed in-house that in cells where the FRT site has integrated into a very transcriptionally active locus in the host cell genome (seen more commonly in Flp-In CHO and Flp-In 293 cells but can also happen in Flp-In 3T3 cells and any other Flp-In host cell line), there is some "read-through" transcription and translation of the lacZ-Zeocin ORF subsequent to the Flp-In reaction, even though the lacZ-Zeocin ORF does not have a bonafide promoter and ATG. In such cases, the hygromycin-resistant clones would also be lacZ-positive and Zeocin antibiotic-resistant. To make sure that the integration is FRT site-specific and not random, we recommend doing a parallel control transfection with no pOG44 present. This should yield no surviving clones upon hygromycin selection, indicating that all the hygromycin-resistant clones obtained in the presence of pOG44 are indeed Flp recombinase-dependent and hence have the gene of interest integrated at the FRT site. Also, a Southern blot analysis of these clones will help verify that they do indeed have proper FRT integration of the gene of interest despite the expression of lacZ (although this is typically not necessary). After the Flp-In reaction, as long as you see hygromycin-resistant clones, we recommend that you select them and assay them for expression of your gene of interest.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
The Flp-In 3T3 cell line is derived from NIH3T3 cells, which are mouse fibroblast cells. The CMV promoter is known to get silenced over time in murine cell lines and hence we would recommend using a Flp-In expression vector with a non-CMV promoter in these cells, such as the pEF5/FRT/V5-D-TOPO vector or the pEF5/FRT/V5-DEST vector.
Before giving up, we would suggest that you try using the pFRT/lacZeo2 vector to generate your host cell line. This vector contains a truncated version of the SV40 promoter driving the lacZ-Zeocin fusion. Use of this vector facilitates the isolation of clones that have integrated the vector near enhancer elements in the genome, thus resulting in higher levels of expression of the gene of interest.
The Jump-In system is PhiC31-integrase mediated and is a stable, targeted, and irreversible mammalian expression system. It consists of the Jump-In Fast system that involves a single integration step and the Jump-InTI (targeted integration) system that needs two integration steps, both of which are targeted and irreversible. In contrast, the Flp-In system is a stable, targeted mammalian expression system that is reversible. The first integration is random (integration of pFRT/lacZeo), and the second integration (integration of the Flp-In expression vector) is targeted but reversible.
In theory, one can get multiple integrations of the Flp-In expression construct—an FRT-specific integration event and a random, second-site integration. However, random integration is a relatively uncommon event. Limiting the amount of DNA in the transfection will reduce the chance of second-site integration. We have transfected 293 cells (lacking the FRT site) with the pcDNA5/FRT vector and have identified one potential second-site integrant after screening over 200 clones. DNA integrations can be detected by Southern blot. A single integrant will display a single band; double: two; triple: three, etc. We have maintained a number of Flp-In expression cell lines for over four months and have not observed any loss of the Flp-In expression construct, whether hygromycin selection was maintained or not.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
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 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
Use irreversible integration (Jump-In system) if the transgene should be sustained in the mammalian genome for a long time. Use reversible integration such as Flp-In system if the transgene needs to be replaced with another gene of interest after a short period of time.
The Jump-In system is PhiC31-integrase mediated and is a stable, targeted, and irreversible mammalian expression system, involving one integration step. The Jump-In TI (Targeted Integration) system needs two integration steps, both of which are targeted and irreversible. In contrast, the Flp-In system is a stable, targeted mammalian expression system that is reversible. The first integration is random (integration of pFRT/lacZeo) and the second integration (integration of the Flp-In expression vector) is targeted but reversible.
We have observed in-house that in cells where the FRT site has integrated into a very transcriptionally active locus in the host cell genome (seen more commonly in Flp-In CHO and Flp-In 293 cells but can also happen in Flp-In 3T3 cells and any other Flp-In host cell line), there is some "read-through" transcription and translation of the lacZ-Zeocin ORF post Flp-In recombination, even though the lacZ-Zeocin ORF does not have a bona fide promoter and ATG. In such cases, the hygromycin-resistant clones would also be lacZ positive and Zeocinantibiotic-resistant. To make sure that the integration is FRT site-specific and not random, we recommend doing a parallel control transfection with no pOG44 present. This should yield no surviving clones upon hygromycin selection, indicating that all the hygromycin-resistant clones obtained in the presence of pOG44 are indeed Flp recombinase-dependent and hence have the gene of interest integrated at the FRT site. Also, a Southern blot analysis of these clones will help verify that they do indeed have proper FRT integration of the gene of interest despite the expression of lacZ (although this is typically not necessary). Post Flp-Inrecombination, as long as you see hygromycin-resistant clones, we recommend that you select them and assay them for expression of your gene of interest.
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.