建立高效稳定的表达系统对于药物重组蛋白的生产至关重要。申请者前期利用核基质附着区（matrix attachment region, MAR）建立了一种新型的CHO细胞表达系统，但此表达系统需要进一步优化，机制尚不清楚。本项目将进一步优化MAR与载体上其他调控元件（如启动子、内含子、IRES等）的分子组装，筛选高效稳定表达载体；利用CRIPSR/Cas 9及同源重组技术定点整合MAR表达载体，构建MAR介导的定点整合CHO表达系统。同时分析转基因拷贝数、组蛋白的乙酰化、磷酸化状态、MAR招募转录因子及染色质构象变化，以验证我们提出的MAR调控机制的染色质重塑假说：MAR能够招募组蛋白乙酰转移酶HAT及其他重塑蛋白以抑制异染色质传播，进而修饰染色质的状态并促进转基因表达。本项目的实现将建立新型的拥有自主知识产权的高效CHO细胞表达系统，理论上为载体的分子组装及阐明MAR调控机制奠定基础。

It is very important to establish the highly efficient and stable expression system for recombinant pharmaceutical protein production. In our previous studies, we have established a new type of CHO cell expression system using matrix attachment regions(MARs). However, this system needs to be further optimized, and the molecular mechanism is still unclear. In this project,we will optimize the molecular assembly of MAR-containing expression vectors with other regulator elements, such as promoter, intron, IRES etc, to screen the efficient and stable expression vectors in CHO cells.We will target the MAR-containing expression vetors in the specific sites of CHO cells using CRIPSR/Cas9 and homologous recombination technology, to construct a site-specific integration CHO expression system mediated by MARs.Furthermore, we will analyze the transgene copy number, histone acetylation, phosphorylation status, MAR recruiting transcription factors and chromatin conformation change. These findings will verify our proposed hypothesis: MARs can recruit the histone acetyltransferase and other remolding proteins,which inhibit heterochromatin spreading and then modify the chromatin state, resulting in promoting the expression level of transgenes.This project will establish a new type of highly efficient and stable expression system with our independent patents, and provide the basis for the molecular assembly of expression vector and understanding MARs’ regulation mechanism.