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查看更多产品信息 Jump-In™ CHO-K1 Kit - FAQs (A14148)
16 个常见问题解答
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。
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。
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 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.
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 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) onto the genome of the platform line. This integration event also positions the EF1alpha promoter upstream of the blasticidin, neomycin, or eosin 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 the 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.
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.
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.
Use 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-In TI 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, 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.