首页|SaRNA:克服重组蛋白药物基因工程中转基因沉默的潜在工具

SaRNA:克服重组蛋白药物基因工程中转基因沉默的潜在工具

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为了研究saRNA(small activating RNA)在重组蛋白药物持续表达中克服转基因沉默效应的潜力，利用两个成熟应用的克服转基因沉默效应的染色质调节元件UCOE(universal chromatin opening element)或MAR(matrix attachment region)作为参照系，与反式作用的saRNA共同靶OCT4基因启动子，利用4C(circular chromo-some conformation capture)技术结合基因表达分析方法表征UCOE或MAR与saRNA靶向在三维染色质层面的相互作用，探讨UCOE或MAR在克服转基因沉默效应中的染色质调控机制与saRNA调节启动子染色质的机制的内在关联。结果表明，UCOE或MAR都能与saRNA通过内在关联的机制使共同靶向的OCT4启动子产生独特的染色质相互作用，其中UCOE与saRNA的相互作用使靶向的OCT4启动子上染色质相互作用片段大幅降低，同时OCT4启动子介导的表达效率显著下降;MAR与saRNA的相互作用使靶向的OCT4启动子上染色质相互作用片段大幅增加，同时相应的OCT4启动子介导的表达效率显著增加。UCOE或MAR对saRNA靶向的OCT4启动子的染色质相互作用片段的影响，与体现UCOE或MAR属性的CG或AT含量无关联，显示UCOE或MAR与saRNA的相互作用可能源于染色质调控的内在机制。结论:UCOE或MAR与saRNA共同作用于同一启动子时，存在染色质三维结构方面的相互干涉并影响下游基因表达，提示saRNA靶向表达载体中启动子可能具有克服转基因沉默的相同机制。
SaRNA:A Potential Tool to Overcome Transgene Silencing in Recombi-nant Protein Drug Gene Engineering
Recombinant protein drugs are currently widely used for the treatment of various important diseases,and the expression of expression vectors carrying exogenous genes in host cells is easily affected by transgenic silencing effects.To explore the potential of small activating RNA(saRNA)in overcoming transgene silencing effects within recombinant protein drug sustained expression,two well-established chromatin regulatory elements capable of overcoming transgene silencing effects,universal chromatin opening ele-ment(UCOE),or matrix attachment region(MAR),were used as references.They were co-targeted with saRNA against the OCT4 gene promoter in an antagonistic manner.By employing 4C(circular chromosome conformation capture)technology combined with gene expression analysis methods,the interactions of UCOE or MAR with saRNA at a three-dimensional chromatin level were charac-terized.This study aimed to investigate the intrinsic relationship between the chromatin regulation mechanisms of UCOE or MAR in overcoming transgene silencing effects and saRNA-mediated chromatin modulation of the promoter.The results indicate that both UCOE and MAR can create unique chromatin interactions with the common target OCT4 promoter through inherent associations with saRNA.The interaction between UCOE and saRNA significantly reduces the chromatin interaction segments on the targeted OCT4 promoter,leading to a notable decrease in the efficiency of OCT4 promoter-mediated expression.On the other hand,the interaction between MAR and saRNA substantially increases the chromatin interaction segments on the targeted OCT4 promoter,resulting in a significant enhancement in OCT4 promoter-mediated expression efficiency.The influence of UCOE or MAR on the chromatin interac-tion segments of the OCT4 promoter targeted by saRNA is independent of the CG or AT content that reflects UCOE or MAR attri-butes,demonstrating that their interaction with saRNA may originate from intrinsic chromatin regulatory mechanisms.In conclusion,the association between UCOE or MAR characterized by chromatin interaction segments and the chromatin regulatory mechanisms of saRNA suggests that promoters within saRNA targeting expression vectors might possess similar mechanisms for overcoming transgene silencing.

Recombinant protein drugsChromatin regulatory sequencesSmall activating RNATransgene silencingChromatin interaction segments4C analysis

舒姣、王宵、乔艳雯、杨丽、肖捷、依光研、李继峰、王斌

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大理大学药学院,云南大理 671000

昆明学院农学与生命科学学院,昆明学院云南省教育厅畜禽疫苗及产业关键技术工程中心,云南昆明 650214

重组蛋白药物 染色质调节序列 小激活RNA 转基因沉默 染色质相互作用片段 4C分析

国家自然科学基金项目国家自然科学基金项目云南省地方高校联合基金项目

317602563260147202001BA070001-217

2024

10.19526/j.cnki.1005-8915.20240402

药物生物技术
中国药科大学,中国医药科技出版社,中国药学会

药物生物技术

CSTPCD
影响因子：0.463
ISSN：1005-8915
年,卷(期)：2024.31(4)