基因修饰动物微卫星不稳定性发生的生物学效应与机制研究

基因修饰动物常出现与修饰基因无关的表型变化，原因不明。大量研究证实肿瘤伴有微卫星不稳定（MSI）发生，且MSI与肿瘤的预后和药物敏感性高度相关。我们前期研究发现在基因修饰动物和化学物致突变动物中均存在MSI，MSI的发生可引起其他相关基因的表达变化。据此提出假说：基因修饰或肿瘤导致MSI发生，MSI作用于其他相关基因，从而引起该动物出现"预料之外"的表型性状或肿瘤特征改变。本研究拟采用前期筛选出的易发MSI的微卫星位点和敲除基因连锁的微卫星位点对基因敲除动物进行MSI检测，探讨MSI发生规律；在此基础上，利用RT-PCR、Western-blot、点突变、诱发肿瘤等技术手段，从体内和细胞水平研究MSI对相关基因表达水平的影响及其甲基化和磷酸化水平的调控，进一步阐明MSI对相关基因功能及肿瘤特征的影响，为基因修饰动物的生物学特性评价提供理论依据，为肿瘤的治疗和预后判断提供新思路。

Genetically modified animals often show phenotypes that are irrelated to the modified genes for unknown reasons. It has been confirmed that there is significant relationship between tumor and microsatellite instability (MSI), of which MSI was closely related to the prognosis of tumor and to the therapy effects of medicines. In our previous study, MSI was found to be existed in genetically modified mice and chemical induced mutant mice. In addition, the changed expression patterns of related genes could be detected when MSI occurred in genetically modified mice. So we hypothesize that: genetic modification or tumor may induce MSI. MSI has additional effects on related genes and induce unexpected phenotypes or tumor features in animals. In present study, in order to investigate the regulatory mechanism of MSI occurrence, we will detect MSI in knockout mice by microsatellite loci, which were prone to be MSI as demonstrated in our previous study. We will detect microsatellite loci that are linked to genes knocked out in those mice as well. Based on this, we will study the influence of MSI on the expression of related genes, and on the methylation and phosphorylation levels of these genes by RT-PCR, Western-blot, point mutation and inducing tumor by chemicals in vivo and in vitro. Furthermore, we will clarify the effect of MSI on the related gene function and characteristics of tumor. The results of the present study will be a theoretical basement of assessing on biological property of genetically modified animals and provide a creative method to treatment and evaluation on prognosis of tumor.