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查看更多产品信息 Jump-In™TI™ Platform Kit - FAQs (A10897)
13 个常见问题解答
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。
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.
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.
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.