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查看更多产品信息 Jump-In™TI™ Gateway® System - FAQs (A10895)
33 个常见问题解答
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。
我们推荐您在用于建立平台细胞系的质粒中加入一个表达标志物/报告基因，之后通过该标志物的表达情况来筛选平台细胞系，以鉴定出一个高表达位点。否则，这一过程会非常繁琐，重定位后需要筛选大量的克隆。
pJTI Phic31 Int载体不含NLS信号。加入NLS信号可能会增加假attP位点的特异性整合效率，但尚未获得支持数据。有一篇文献描述了在PhiC31整合酶载体上NLS信号的应用，但作者未检测假attP位点的整合情况。
靶向整合的第二步骤为R4整合酶所介导的重定位事件，这一事件中目的遗传元件由重定位表达载体（使用MultiSite Gateway Pro模块建立）所携带，位点特异性地整合进入平台细胞系的基因组中。整合事件也会将EF1α启动子放在杀稻瘟素，新霉素或Zeocin抗性基因（即 “无启动子”的筛选标志物）的上游，因此就可通过适当的筛选试剂来筛选出成功实现“重定位”的转化子。尽管您使用了杀稻瘟素，新霉素或Zeocin抗性基因筛选出了成功实现重定位的克隆，您可能还是需要进行一个巢氏PCR来扩增从EF1α启动子至适当抗性基因之间的区域。您还可扩增潮霉素抗性基因，来作为一个阳性对照。与建立平台细胞系类似，您也可针对您的目的基因设计探针来开展Southern杂交分析。
当R4 attP重定位（retargeting）序列通过PhiC31 Int介导的重组反应，位点特异性地插入哺乳动物基因组中时，平台细胞系就建立了。除R4重定位序列之外，整合事件还会基于所用的平台载体（即pJTI/Bsd，pJTI/Neo或pJTI/Zeo），来将潮霉素抗性基因（由HSV TK启动子控制），不含启动子的Bsd，Neo或Zeo抗性标志物导入细胞内。尽管您会按照对潮霉素的耐受性筛选出那些携带了R4重定位序列的转化子，您可能还是需要执行PCR分析，来检查R4 attP重定位序列的整合情况。为了实现此种目的，我们推荐用户使用平台细胞系的基因组DNA来扩增从R4 attP序列至适当耐药标志物（基于所使用的平台细胞系）之间的序列。我们推荐用户使用巢氏PCR来降低初始PCR过程中可能观察到的高背景情况。此外，您也可通过PCR扩增一条1.5 kb左右、覆盖重定位序列的区域来制作一条DNA标记探针，之后执行Southern杂交分析。Southern杂交也可作为附加测试，来验证重定位序列向基因组中的整合是否为单一拷贝。
获得单拷贝所需的DNA用量可能要通过不含整合酶的对照试验来确定。在不含整合酶的条件下，生成低于5个克隆时的DNA用量可用于后续含整合酶的实验。一般来说，整合酶表达质粒占转染所需DNA量的大部分。
如果您需要稳定的哺乳动物表达效果，并希望快速获得良好表达的克隆时，我们推荐您使用Jump-In Fast系统。您可通过PhiC31 假（pseudo）-att P位点上的一次或多次整合来获得良好表达的细胞克隆。用户有必要使用Southern杂交来确定整合的数目。如果您需要完成同基因的表达操作，则可选择Jump-In TI系统，这一系统可以确保所有的克隆基因都处于同一整合位点和背景水平，而不会出现染色体位置效应。
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。
我们推荐使用One Shot ccdB Survival 2 T1^R 感受态细胞，货号A10460。该菌株能够耐受ccdB基因的毒性效应。
注意： 请勿使用常规的大肠杆菌克隆株 - 包括TOP10或DH5α - 来进行扩增和培养，因为这些菌株均对ccdB的效应很敏感。
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).
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.
We would recommend engineering an expression marker/reporter in the plasmid used to create the platform line, and then screening the platform line for expression of this marker to identify a high-expressing locus. Otherwise, the process can get quite labor-intensive, as multiple lines would have to be screened after retargeting.
The pJTI Phic31 Int vector does not contain an NLS. Adding an NLS could increase the efficiency of site-specific integration at pseudo attP sites, but there are no data to support it. There is one paper describing the use of an NLS on a PhiC31 integrase vector, but the authors didn't measure integration into pseudo attP sites.
The second step in targeted integration is the retargeting event mediated by the R4 integrase, where the genetic elements of interest are site-specifically transferred from the retargeting expression construct (created using the MultiSite Gateway Pro module) into the genome of the platform line. This integration event also positions the EF1alpha promoter upstream of the blasticidin, neomycin, or Zeocin resistance gene (i.e., “promoterless” selection marker), thus allowing the selection of transformants that are successfully “retargeted” using the appropriate selection agent. Although you select from successfully retargeted clones using blasticidin, Geneticin, or Zeocin antibiotic, you may also perform a nested PCR to amplify the region from the EF1alpha promoter to the appropriate resistance gene. You can amplify the hygromycin resistance gene as a positive control. Similar to the platform line creation, you may also perform a Southern blot analysis with a probe designed for your gene of interest.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
A platform cell line is created when the R4 attP retargeting sequences are site-specifically inserted into the mammalian genome via PhiC31 Int-mediated recombination. In addition to the R4 retargeting sequences, this integration event introduces the hygromycin resistance gene under the control of the HSV TK promoter, and the promoterless Bsd, Neo, or Zeo resistance marker, depending on the platform vector used (i.e., pJTI/Bsd, pJTI/Neo, or pJTI/Zeo). Although you select for transformants carrying the R4 retargeting sequences by their resistance to hygromycin, you may perform PCR analysis to check the integrity of the R4 attP retargeting sequences. For this, we recommend amplifying the region from the R4 attP sequence to the appropriate resistance marker (depending on the platform line used) using the genomic DNA from the platform line. A nested PCR is recommended to reduce the high background you may observe with only primary PCR. Alternatively, you may create a labeled DNA probe by PCR amplifying an approximately 1.5 kb region covering the retargeting sequences, and then perform a Southern blot analysis. The Southern blot will also act as an additional check to verify that only a single copy of the retargeting sequence is integrated into the genome.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
The amount of DNA to be used to obtain single copies should be determined by control experiments done in the absence of integrase. The same amount of DNA that yields less than 5 colonies in the absence of integrase should be used in the presence of integrase. Typically, the integrase expression plasmid makes up most of the amount of DNA used for transfection.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
We recommend using the Jump-In Fast system if you need stable mammalian expression and want to quickly generate well-expressing clones. You can have well-expressing clones with one or more integrations at the PhiC31 pseudo-att P sites. A Southern blot is necessary to confirm the number of integrated events. Use the Jump-InTI system if you need isogenic expression, where every cloned gene would be expressed from the same locus in the same background, with no chromosomal position effects.
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.
We recommend using One Shot ccdB Survival 2 T1R Competent Cells, Cat. No. A10460. This strain is resistant to the toxic effects of the ccdB gene. Note: Do not use general E. coli cloning strains, including TOP10 or DH5alpha, for propagation and maintenance, as these strains are sensitive to ccdB effects.
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.
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.
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.