构建诱导型永生化基因整合载体与功能验证  

Construction and function validation of inducible immortalized gene integration vectors

作　　者：岳伟 杨玥 钱宝华 李彦欣[2] 顾海慧 YUE Wei;YANG Yue;QIAN Baohua;LI Yanxin;GU Haihui(Department of Transfusion Medicine,The First Affiliated Hospital of Naval Medical University,Shanghai 200433,China;Pediatric Translational Medicine Institute,Shanghai Children′s Medical Center,School of Medicine,Shanghai Jiao Tong University,National Health Commission Key Laboratory of Pediatric Hematology&Oncology,Shanghai 200127,China)

机构地区：[1]中国人民解放军海军军医大学第一附属医院输血科,上海200433 [2]上海交通大学医学院附属上海儿童医学中心转化所,国家卫健委儿童血液肿瘤重点实验室,上海200000

出　　处：《中国输血杂志》2024年第12期1341-1349,共9页Chinese Journal of Blood Transfusion

基　　金：国家自然科学基金面上项目(81970165,32271007)。

摘　　要：目的构建诱导型永生化基因载体,并将其转染入原代细胞,从而建立条件永生化细胞系,实现原代细胞在体外的持续培养和增殖。方法本研究应用基因同源重组技术,将永生化基因人端粒酶逆转录酶(hTERT)、猴病毒40大T抗原(SV40LT)、急性髓系白血病融合基因NUP98-KDM5A(N/K)和CBFA2T3-GLIS2(C/G),以及原癌基因KRAS的编码序列(CDS)定向插入到四环素(Tet)诱导型真核表达的慢病毒载体pLV2-TRE3GS-EGFP-MCS-3×FLAG-hPGK-Tet-On-SV40-Neo和转座子系统PB-TRE3G-3×FLAG-T2A-Puro-SV40-PA中。通过慢病毒包装及生产、细胞转染、基因mRNA表达检测、Western blotting蛋白表达检测、绿色荧光蛋白观测、细胞增殖检测等实验方法,分别在293T细胞和小鼠胚胎成纤维细胞(MEF)中验证载体的转染效率和Tet诱导表达功能。结果本研究成功构建Tet诱导型慢病毒载体pLV2-Tet-SV40LT、pLV2-Tet-N/K、pLV2-Tet-C/G以及转座子载体PB-Tet-hTERT、PB-Tet-SV40LT、PB-Tet-N/K、PB-Tet-C/G和PB-Tet-KRAS。通过在293T细胞中的转染验证所有目标基因表达均显著上调。在MEF细胞进一步验证了SV40LT和N/K基因具有较强的永生化功能,并通过调节Tet诱导剂的添加控制了细胞的增殖水平。最终,成功构建条件永生化的pLV2-SV40LT-MEF和pLV2-N/K-MEF细胞系。结论Tet诱导型永生化基因整合载体的构建为制备条件永生化原代细胞系和实现体外大量扩增血细胞(如红细胞和血小板)提供了重要的技术基础。Objective To construct inducible immortalization gene vectors for transfection into primary cells,enabling the establishment of a conditionally immortalized cell line that support their sustained cultivation and proliferation in vitro.Methods Using gene homologous recombination technology,the coding sequences(CDS)of immortalization genes-inclu-ding human telomerase reverse transcriptase(hTERT),simian virus 40 large T antigen(SV40LT),acute myeloid leukemia fusion genes NUP98-KDM5A(N/K)and CBFA2T3-GLIS2(C/G),as well as the proto-oncogene KRAS were precisely in-serted into the tetracycline(Tet)-inducible eukaryotic expression lentiviral vector pLV2-TRE3GS-EGFP-MCS-3×FLAG-hPGK-Tet-On-SV40-Neo and the transposon PB-TRE3G-3×FLAG-T2A-Puro-SV40-PA.Lentiviral packaging,cell transfec-tion,mRNA expression analysis,Western blotting for protein detection,green fluorescent protein(GFP)visualization,and cell proliferation assays were conducted to evaluate transfection efficiency and assess the regulatory effects of Tet on gene ex-pression in 293T and MEF cells.Results The Tet-inducible lentiviral vectors pLV2-Tet-SV40LT,pLV2-Tet-N/K,and pLV2-Tet-C/G,along with the transposon vectors PB-Tet-hTERT,PB-Tet-SV40LT,PB-Tet-N/K,PB-Tet-C/G,and PB-Tet-KRAS,were successfully constructed.In 293T cells,the expression levels of all target genes were upregulated after transfection.In MEF cells,the immortalizing functions of SV40LT and N/K were validated.By modulating Tet addition,cell proliferation levels were effectively regulated,leading to the successful establishment of conditionally immortalized pLV2-SV40LT-MEF and pLV2-N/K-MEF cell lines.Conclusion The construction of Tet-inducible immortalizing gene vec-tors provides a technical foundation for establishing conditionally immortalized primary cell lines,thereby facilitating re-search on the large-scale in vitro production and expansion of blood cells,such as erythrocytes and platelets.

关 键 词：永生化 四环素调控系统 慢病毒 转座子 

分 类 号：R331.14[医药卫生—人体生理学] R446.113[医药卫生—基础医学] Q344.1[生物学—遗传学]