The creation of a novel NF-κB base promoter to enhance transgene expression in tumor cells
此技術可以在Balb/3T3，A549細胞株上挑選到高表現量，或是具腫瘤專一性的啟動子。雖然目前還有很多序列未定出 (長度大於1 kb)，但是可以看出在腫瘤細胞中具高表現量的兩個啟動子其中有一個有兩個NF-κB結合部位。另一個則無。這說明了腫瘤雖然有高活性的NF-κB，但是其它的轉錄因子或許也是很重要的。利用此種策略不但可以了解腫瘤細胞的最佳啟動子組成，也可以觀測出哪些轉錄因子在腫瘤細胞是重要的。藉此可以更深入的了解腫瘤細胞，進而找到治療癌症更好的方法。|
Tumor-specific promoters are useful tools to accomplish specific expression in targeted tumor cells. However, a low level of gene expression is the chief defect of tumor-specific promoters. We have set up a platform to quickly gain high active and specific promoter library. Recently, many researches have discovered that NF-κB is associated with cancer, including metastasis, tumor proliferation and anti-apoptosis. Therefore, we created a novel NF-κB based promoter library by randomly assembling Sp1, CRE, and MEF-2 binding sites. Furthermore, low expressive promoters may interfere with the selection for the promoter with high expression activity. I used the techniques magnetic beads separation combing with the competitor (MBSCS) to solve this problem. In this experiment, promoter library transfected into Balb/3T3 from this modified separation system could decrease the expression of low expressive promoters by 1.59 % overall. Meanwhile, it increased the expression of medium expressive promoters by 7.99% overall. Therefore, the method can reduce the expression ratio of the low expressive promoters in plasmid. Using this technology, high expressive or tumor-specific promoters were selected from A549 and Balb/3T3 cells. Although their sequences are not fully (over 1kb), there were sequenced still observed some results. There are two high and tumor-specific promoters from separation. One has two binding sites; the other has none. Let us which means other transcription factors may play an important role in cancers except for NF-κB. This strategy not only let us understand the context of high efficient promoter in tumor cells, but also let us knows which factors in the tumor cells are important. This may take advantage of it to more thorough understanding about tumor cells, and find a better treatment for cancer.
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