Construction and utilization of artificial promoters responsive to ultrasound

人工启动子是一种适合于基因治疗载体，因为它可以很容易地整合一个新的性质。特别地，响应性启动子将可用于基因治疗的各个领域，并且具有此类启动子的载体被认为更安全和更有效。启动子由包括与基本转录因子结合的TATA盒和顺式元件的序列组成，与活性转录因子结合。我们尝试通过构建随机组合顺式元件的DNA片段并将其连接到TATA盒来构建感兴趣的人工启动子。使用在各种组织中有活性的转录因子NF-κ B、NF-γ、AP-1和CBF-A的顺式元件构建的11个人工启动子中最强启动子的活性，与SV 40启动子和CMV启动子的启动子活性相当。此外，11个启动子中对X射线最敏感的启动子通过以下方式得到改善： ...更多信息 通过易错PCR引入随机点突变，导致响应于10戈伊X射线照射的基因表达增加20倍。因此，我们将对X射线有反应的人工启动子导入细胞，并以1 W/cm^2的1 MHz超声波照射细胞，以观察超声波照射是否会引起启动子活化，结果显示基因表达增加了2倍。此外，具有点突变的改进的基因表达对超声辐射的响应显示出6倍的增加。然后将改进的启动子稳定地引入细胞的基因组中以建立细胞系。将这种细胞系皮下移植到小鼠身上。虽然在肿瘤形成后暴露于超声照射，但未观察到基因表达的显著增加。因此，我们获得了2个更敏感的启动子，超声辐射显示超过10倍的基因表达增加了62个人工启动子克隆类似于11个人工启动子构建。其中一个通过引入点突变进行了改进，并显示基因表达增加了20倍以上。我们将研究启动子在体内的激活及其对治疗基因表达控制的影响。少

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