权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Construction and utilization of artificial promoters responsive to ultrasound

超声响应人工启动子的构建及利用

基本信息

批准号：
18500374
负责人：
OGAWA Ryohei
金额：
$ 2.34万
依托单位：
University of Toyama
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18500374/
关键词：
Promoter cis-element TATA box error prone PCR ultrasound

项目摘要

An artificial promoter is suitable for gene therapy vectors since it could be easily integrated with a novel property. In particular, a responsive promoter would be useful in various field of gene therapy and vectors with such promoters are considered safer and more efficacious. Promoters are composed of a sequence including the TATA box to which basic transcription factors bind and cis-elements to which active transcription factors bind. We have attempted to construct artificial promoters of interest by constructing DNA fragments of randomly combining cis-elements appropriately selected for a purpose and attaching it to the TATA box.Activity of the strongest promoter of 11 artificial promoters that were constructed using cis-elements of NF-kB, NF-Y, AP-1 and CBF-A, transcription factors that are active in various tissues, was almost equivalent to those of SV40 promoters and CMV promoters. In addition, the most responsive promoter to X-rays out of the 11 promoters was improved through … More introduction of random point mutations by error prone PCR to cause 20-fold increase in gene expression in response to 10 Gy X-ray irradiation.It is known that ultrasound irradiation could induce intracellular oxidative stress that would be a similar intracellular environment caused by X-ray irradiation. We thus introduce the artificial promoter responsive to X-ray into cells and irradiate the cells with 1 MHz ultrasound at 1 W/cm^2 to see if ultrasound irradiation might cause promoter activation, showing 2-fold increase in gene expression. In addition, the improved one with point mutations showed 6-fold increase in gene expression in response to the ultrasound irradiation. The improved promoter was then stably introduced into the genome of a cell to establish cell lines. Such a cell line was subcutaneously transplanted on to mice. Although it was exposed to ultrasound irradiation after tumor formation, significant increase in gene expression was not observed. We therefore obtained 2 more sensitive promoters to ultrasound irradiation showing more than 10-fold increase in gene expression out of 62 artificial promoter clones constructed similarly to the 11 artificial promoters. One of the two was improved with point mutation introduction and showed more than 20-fold increase in gene expression. We are going to examine activation of the promoter in vivo and its effect on the expression control of therapeutic genes. Less

人工启动子是一种适合于基因治疗载体的启动子，因为它可以很容易地与一种新的性质相结合。特别是，反应性启动子将在基因治疗的各个领域中发挥作用，并且具有这种启动子的载体被认为更安全，更有效。启动子由一个序列组成，包括与基本转录因子结合的TATA盒和与活性转录因子结合的顺式元件。我们试图通过构建随机组合顺式元件的DNA片段来构建感兴趣的人工启动子，并将其连接到TATA盒上。利用NF-kB、NF-Y、AP-1和CBF-A等多种组织活性转录因子的顺式元件构建的11个人工启动子中，最强启动子活性与SV40启动子和CMV启动子活性基本相当。此外，在11个启动子中，对x射线反应最灵敏的启动子通过易出错PCR引入更多随机点突变，导致基因表达在10 Gy x射线照射下增加20倍。众所周知，超声照射可以诱导细胞内氧化应激，这与x射线照射引起的细胞内环境类似。因此，我们将对x射线有反应的人工启动子引入细胞，并以1mhz超声以1w /cm^2照射细胞，观察超声照射是否会引起启动子激活，结果显示基因表达增加了2倍。此外，点突变的改良品种在超声照射下基因表达量增加了6倍。然后将改进的启动子稳定地引入细胞基因组以建立细胞系。这种细胞系被皮下移植到小鼠身上。虽然在肿瘤形成后进行超声照射，但未观察到基因表达明显增加。因此，我们获得了2个对超声照射更敏感的启动子，在62个与11个人工启动子相似的人工启动子克隆中，基因表达增加了10倍以上。其中一个通过引入点突变得到了改良，基因表达量增加了20倍以上。我们将在体内研究启动子的激活及其对治疗基因表达控制的影响。少