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

人工启动子适用于基因治疗载体，因为它可以容易地与新特性整合，特别地，响应启动子将在基因治疗的各个领域中有用，并且具有此类启动子的载体被认为是更安全和更有效的。我们尝试通过构建随机组合出于目的而适当选择的顺式元件并连接的DNA片段，来构建包含基本转录因子结合的TATA盒和活性转录因子结合的顺式元件的序列。使用在各种组织中有活性的转录因子NF-kB、NF-Y、AP-1和CBF-A的顺式元件构建的11个人工启动子中最强的启动子的活性几乎是相当于 SV40 启动子和 CMV 启动子。此外，通过易错 PCR 引入随机点突变，11 个启动子中对 X 射线反应最灵敏的启动子得到了改进，导致 20 倍的突变。响应 10 Gy X 射线照射，基因表达增加。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，因此我们引入了对 X 射线响应的人工启动子。进入细胞并用 1 W/cm^2 的 1 MHz 超声波照射细胞，看看超声波照射是否会导致启动子激活，显示基因表达增加了 2 倍。此外，改善。一种具有点突变的基因在超声波照射下表现出6倍的增加，然后将改进的启动子稳定地引入细胞基因组中以建立这样的细胞系，并将其皮下移植到小鼠体内。在肿瘤形成后暴露于超声辐射下，没有观察到基因表达的显着增加，因此我们获得了 2 个对超声辐射更敏感的启动子，显示 62 个类似构建的人工启动子克隆的基因表达增加了 10 倍以上。 11 个人工启动子中的一个通过点突变引入进行了改进，并显示出基因表达增加了 20 倍以上，我们将检查启动子的体内激活及其对治疗基因表达控制的影响。