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View additional product information for GeneSwitch™ Mammalian Expression Kit, complete kit - FAQs (K106001)
17 product FAQs found
抱歉，所有的GeneSwitch细胞系均已停产。
GeneSwitch蛋白包含了源自不同转录因子的功能域，可作为配体依赖的转录因子行使功能，来激活目的基因或自身内源基因的表达。GeneSwitch能够表现出如下属性：
•由于GAL4 DBD来源于酵母蛋白，GeneSwitch蛋白不会对内源基因造成影响，而只会激活GAL4 UAS元件所控制那些基因的转录过程（即目的基因和调节性融合基因）。
•GAL4 DBD能够以同源二聚体的形式结合独立的17核苷酸GAL-4结合位点。pGene/V5-His与pSwitch质粒中分别包含了6个和4个拷贝的GAL4结合位点，但尚不清楚是否所有这些GAL4-结合位点在任意时间都处于占用状态。
•截短型的hPR-LBD的C-端截去了19个氨基酸，从而失去了与孕酮，其他内源的甾族荷尔蒙及其他孕酮激动剂的结合能力，但仍能够与米非司酮进行高亲和力的结合。
•p65 AD是一个强力的转录激动剂且来源于人体蛋白，因此能够最大程度的减少可能存在的毒性或病毒转录激活域相关的多效影响。
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。
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
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).
Sorry, all GeneSwitch cell lines have been discontinued.
The GeneSwitch protein contains functional domains from different transcription factors, allowing it to function as a ligand-dependent transcription factor to activate expression of both the gene of interest and its own gene. The GeneSwitch protein exhibits the following characteristics:
- Since the GAL4 DBD is derived from a yeast protein, the GeneSwitch protein has no effect on endogenous genes and can only activate transcription of genes whose expression is controlled by a GAL4 UAS (i.e., the gene of interest and the regulatory fusion gene).
- The GAL4 DBD binds to an individual 17-nucleotide GAL4-binding site as a homodimer. The pGene/V5-His and pSwitch plasmids contain 6 and 4 copies of the GAL4 binding site, respectively, although it is not known if all of the GAL4-binding sites are occupied at any given time.
- The truncated hPR-LBD contains a 19 amino acid deletion from its C-terminal end that abolishes its ability to bind to progesterone, other endogenous steroid hormones, or other progesterone agonists, but still enables it to bind with high affinity to mifepristone.
- The p65 AD is a strong transcriptional activator but is derived from a human protein, to minimize possible toxic or pleiotropic effects associated with viral transactivation domains.
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.
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
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.
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.