Study On The Chromosome Advanced Structural Mechanisms Of High-expression By RNAa In Recombinant Protein Drug Producing Cells

Objective:CHO cells are currently the most widely used genetic engineering host cells for reorganized protein drug production,with RNA activation(RNAa)technology targeting exogenous gene expression in CHO cells,which can activate reorganised drug high protein expression,thereby improving protein drug productivity.But the mechanisms of activation of RNAa in the expression carrier are still unclear.This study uses different regulatory components to regulate the intervention of RNSa in COH cells and explores the RNA-activation mechanism in the open chromosomal layer and the three-dimensional structure of chrom.Methods:The OCT4 gene promoter containing proximal enhancers and proximal promoters,SOX2 gene promoter containing Cp G island and TATA box,matrix attachment region,MAR),Ubiquitous chromatin opening elements(UCOE)and Small activating RNA(saRNA)combine to construct vectors with different structures,pOCT4-O38,pSOX2-S278,pOCT4-UCOE-O38,pSOX2-UCOE-S278,pOCT4-MAR-O38,pSOX2-MAR-S278 and so on.Tne six vectors were transferred into CHO cells and screened to statically expressed monoclonal cell lines as the study model.Nucleosome deletion assay,Western Blotting(WB),and Quantitative real-time PCR(qRT-PCR)were used to study the relationship between the formation of open chromatin and the amount of expression during the process of gene expression.Appropriate restriction enzymes were selected according to the target gene region,reverse PCR primers were designed,and 4C experiment combined with high-throughput sequencing technology was used to obtain high-resolution interaction fragment data of the whole genome,and the interaction between the target gene region and the whole genome was analyzed and studied.Through further analysis of 4C data combined with the bioinformatics characteristics of promoters,the influence of RNA regulation on gene expression at the three-dimensional structure level of chromatin was discussed.Results:Open chromatin and the three-dimensional structure of chromatin have been found to have a significant impact on the mechanism of RNA activation and high expression in cells that produce recombinant protein drugs.At distinct promoter areas,saRNA can,when working alone,encourage the formation of open chromatin,hence triggering gene expression.RNAa is not significantly impacted by three-dimensional chromatin interaction.However,when the chromatin regulatory elements MAR and UCOE in traditional recombinant expression vectors were utilized to control the action of saRNA on target genes,they were able to not only alter the degree of open chromatin region and nucleosome aggregation caused by RNAa,but also to induce large-scale three-dimensional interaction,thereby lessening the effect of RNA activation.Conclusion:The creation of open chromatin areas at the two-dimensional level of chromatin,rather than RNA’s comparatively minor impact on the three-dimensional space of chromatin,is the primary cause of the high expression of RNA input genes in recombinant protein drug-producing cells.