Efficiency of the elongation factor-1alpha promoter in mammalian embryonic stem cells using lentiviral gene delivery systems

The establishment of new technology for genetic modification in human embryonic stem (ES) cell lines has raised great hopes for achieving new ground in basic and clinical research. Recently, lentiviral vector technology has been shown to be highly effective and therefore could emerge as a popular tool for human ES cell genetic modification. The objectives of this study were to evaluate the efficiency of promoters in lentiviral gene delivery systems in mammalian ES cells, including mouse, monkey, and human, and to construct efficient and optimized conditions for lentivirus-mediated transfection systems. Mammalian ES cells were transfected with self-inactivating (SIN) human immunodeficiency virus type-1 (HIV-1)-based lentiviral vectors containing the human polypeptide chain elongation factor-1alpha (EF-1alpha) promoter or cytomegalovirus (CMV) promoter and analyzed by fluorescence-activated cell sorting (FACS) analysis for the expression of the enhanced green fluorescent protein (eGFP) reporter gene. The efficiency of the EF-1alpha promoter was higher than that of the CMV promoter in all ES cells tested. The EF-1alpha promoter efficiently drove gene expression (14.74%) compared with CMV promoter (3.69%) in human ES cells. We generated a stable eGFP+ human ES cell line (CHA3-EGFP human ES cells) that continuously expressed high levels of EGFP ( approximately 95%) from the EF-1alpha promoter and was maintained for up to 60 weeks with undifferentiated proliferation. The established CHA3-EGFP human ES cell lines were characterized as being negative for nondifferentiation markers and teratoma formation. These results imply that genetic modification by lentiviral vectors with specific promoters in ES cells constitute a powerful tool for guided differentiation as well as gene therapy.     

Stable expression of hrGFP by mouse embryonic stem cells: promoter activity in the undifferentiated state and during dopaminergic neural differentiation

Three promoters, cellular polypeptide chain elongation factor 1 alpha (EF1), cytomegalovirus (CMV), and Rous sarcoma virus (RSV) were examined for stable transgene expression in mouse embryonic stem (ES) cells and their progeny during dopaminergic neural differentiation. In undifferentiated ES cells the EF1 promoter was highly effective, while CMV had moderate activity. After 3 months in culture, expression of humanized renilla green fluorescent protein (hrGFP) was unchanged for the EF1 promoter and decreased for CMV. At the nestin-positive stage of differentiation, hrGFP and nestin were colocalized in about 20% of cells for EF1, in contrast to 80% of cells for the CMV promoter. In tyrosine hydroxylase (TH)-positive neurons neither the EF1 nor CMV promoter were effective. The RSV promoter was inactive in undifferentiated, nestin-positive, and TH-positive cells. Thus, EF1 and CMV are effective promoters for transgene expression in undifferentiated ES cells and nestin-positive neural precursors.     

HRE/CMV启动子的克隆和缺氧调控活性的测定

目的： 将具有缺氧调控作用的多种缺氧反应元件 (HRE)与CMV启动子组合，以获得缺氧应答启动子。将荧光素酶报告基因置于缺氧应答启动子的下游，通过检测荧光素酶活性的方法，方便地研究缺氧应答启动子缺氧诱导的作用。方法: 合成多种HRE核苷酸序列，并设立HRE突变对照，克隆入pCI-neo中，构建HRE/CMV启动子。扩增HRE/CMV序列，定向亚克隆入pGL3-Basic中，获得HRE/CMV荧光素酶报告载体。瞬时转染HeLa细胞，在0.1% O₂ 环境下培养细胞，并设立正常氧分压环境对照。检测荧光素酶的相对活性。结果: 成功构建了HRE/CMV荧光素酶报告载体，其中mPGK-HRE/CMV载体表现出很好的缺氧诱导作用和高水平的表达。结论: mPGK-HRE/CMV启动子能够有效地进行缺氧诱导和调控工作。     

Transgenes delivered by lentiviral vector are suppressed in human embryonic stem cells in a promoter-dependent manner

Lentiviruses have been increasingly used for genetic modification of human cells including embryonic stem (ES) cells. Using four ubiquitous promoters--cytomegalovirus (CMV), cytomegalovirus immediate-early enhancer/chicken beta-actin hybrid (CAG), phosphoglycerate kinase (PGK), and human elongation factor-1alpha (EF1alpha)--in a lentiviral vector to drive the expression of the enhanced green fluorescent protein (EGFP) gene in human ES cells and mouse ES cells, we determined the extent of EGFP suppression by assessing the percentage of cells that were transduced with the EGFP gene but did not fluoresce green. A much higher level of transgene suppression was observed in human ES cells as compared to mouse ES cells. The suppression was also highly promoter dependent, leading to inactivation of more than 95% of the EGFP genes under the CMV or CAG promoter while only 55% under the PGK promoter. No promoter-dependent suppression was observed in transient transfection of human ES cells. Thus, the common phenomenon of poor transgene expression in human ES cells may be caused mainly by suppression of the transgene right after transduction and integration. Cautions should be taken to choose the optimal promoter when lentiviruses are used for genetic modification of human ES cells.     

Generation of high-level stable transgene expressing human embryonic stem cell lines using Chinese hamster elongation factor-1 alpha promoter system

The utilization of human embryonic stem cells (hESC) in regenerative medicine largely depends on the development of technologies that will allow efficient genetic manipulation of the cells in vitro. Although a few studies have described the transfection of hESC for generation of reporter lines stably expressing specific transgenes driven by different promoters, the optimal choice of promoter system for driving transgene in hESC has yet to be elucidated. We show for the first time that Chinese hamster elongation factor-1 alpha (CHEF1) promoter robustly drove reporter gene expression higher than the human elongation factor 1 alpha (hEF1 alpha), other constitutive Chinese hamster promoters, human cytomegalovirus (CMV) immediate early enhancer/promoter and SV40 promoters in hESC by quantitative analysis. We also successfully generated stably transfected hESC lines using this CHEF1 promoter system and demonstrated that they continued to express enhanced green fluorescent protein (EGFP) during prolonged undifferentiated proliferation, in differentiated embryoid bodies (EBs), and in teratomas without transgene silencing. By immunofluorescence staining and D ow cytometry analysis, the pluripotent markers, OCT-4, SSEA-4, and TRA-1-60, continued to be expressed in undifferentiated CHEF1-EGFP expressing hESC lines. When the stably transfected hESC were directed to differentiate into neural precursors in vitro, high-level EGFP expression was maintained and co-expression of neural markers, Nestin, and beta-tubulin III was observed. The morphology, karyotype, and telomerase activity of CHEF1-EGFP expressing hESC were normal after >50 continuous passages, and the cells retained the ability to differentiate into derivatives of the three germ layers in vitro as confirmed by RT-PCR analysis and immunocytochemical staining and in vivo teratoma formation. Therefore, stable CHEF1-EGFP hESC lines retained the capability for self-renewal and pluripotency. The novel CHEF1 promoter system described here enables high-level transgene expression in the stably transfected hESC. It may have signi, cant implication for uses in bioprocess development and future development of gene-modified hESC in tissue regeneration and transplantation applications.     

小鼠白蛋白基因启动子的克隆及其在不同细胞系中转录活性的检测

目的：对比小鼠白蛋白（mouse albumin promoter, ALB）启动子调控下的增强型绿色荧光蛋白（enhanced green fluorescent protein, EGFP）在不同细胞系中的转录活性。方法：以小鼠全血基因组DNA为模板,聚合酶链反应（polymerase chain reaction, PCR）扩增ALB启动子序列,克隆至pEGFP-1中,构建重组体pALB-EGFP;在Lipofectamine介导下将pALB-EGFP、pEGFP-N1转染人胎肝细胞L02、人宫颈癌细胞HeLa、人结肠癌细胞SW480、人胰腺癌细胞Bxpc-3;荧光显微镜和流式细胞仪对各转染细胞中EGFP的表达进行检测。结果：pALB-EGFP构建成功;L02转染pALB-EGFP 72 h后,ALB启动子可起始EGFP的表达,转录活性为人巨细胞病毒（cytomegalovirus,CMV）启动子的1/4,其它转染细胞的ALB不能起始EGFP的转录;稳定筛选后,ALB的转录活性达到与CMV相当的水平。结论：构建的重组载体在肝脏来源细胞中具有较高的转录活性, 为建立肝脏特异性表达目的基因的转基因小鼠模型奠定了基础。     

Lentiviral Vectors Mediate Long-Term and High Efficiency Transgene Expression in HEK 293T cells

Objectives:Lentiviral vectors have been used successfully to rapidly produce decigram quantities of active recombinant proteins in mammalian cell lines. To optimize the protein production platform, the roles of Ubiquitous Chromatin Opening Element (UCOE), an insulator, and selected promoters were evaluated based on efficiency and stability of foreign gene expression mediated by lentiviral vectors.Methods: Five lentiviral vectors, pFIN-EF1α-GFP-2A-mCherH-WPRE containing EF1α promoter and HS4 insulator, p''HR.cppt.3''1.2kb-UCOE-SFFV-eGFP containing SFFV promoter and UCOE, pTYF-CMV(β-globin intron)-eGFP containing CMV promoter and β-globin intron, pTYF-CMV-eGFP containing CMV promoter, and pTYF-EF1α-eGFP with EF1α promoter were packaged, titered, and then transduced into 293T cells (1000 viral genomes per cell). The transduced cells were passaged once every three days at a ratio of 1:10. Expression level and stability of the foreign gene, green fluorescence protein (GFP), was evaluated using fluorescent microscopy and flow cytometry. Furthermore, we constructed a hepatitis C virus (HCV) E1 recombinant lentiviral vector, pLV-CMV-E1, driven by the CMV promoter. This vector was packaged and transduced into 293T cells, and the recombinant cell lines with stable expression of E1 protein were established by limiting dilution.Results:GFP expression in 293T cells transduced with the five lentiviral vectors peaked between passages 3 and 5 and persisted for more than 5 weeks. The expression was prolonged in the cells transduced with TYF-CMV (β-globin intron)-eGFP or TYF-CMV-eGFP, demonstrating less than a 50% decrease even at 9 weeks post transduction (p>0.05). The TYF-CMV-eGFP-transduced cells began with a higher level of GFP expression than other vectors did. The percentage of GFP positive cells for any of the five lentiviral vectors sustained over time. Moreover, the survival rates of all transfected cells exceeded 80% at both 5 and 9 weeks post transduction. Surprisingly, neither the HS4 insulator nor the UCOE sequence improved the GFP expression level or stability. Clonal cell lines with HCV E1 gene were generated from LV-CMV-E1 vector-infected 293T cells. A representative recombinant cell line maintained stable E1expression for at least 9 weeks without significant difference in morphology compared with untreated 293T cells.Conclusion: The results suggest that all five vectors can stably transduce 293T cells, producing long term transgene expression with different efficiencies. However, neither the insulator nor the UCOE improved the GFP expression. The vectors containing the promoter CMV or CMV (β-globin intron) generated the highest gene expressions, manifesting as more favorable candidates for recombinant protein production in HEK293T cells.     

不同启动子指导的HSV1-sr39TK/GCV系统对小鼠淋巴细胞的敏感性研究

目的观察慢病毒载体中不同启动子指导的突变型单纯疱疹病毒1型胸苷激酶(HSV1-sr39TK)基因在小鼠T淋巴细胞内的表达差异,并进一步比较对丙氧鸟苷(ganciclovir,GCV)的敏感性。方法用亚克隆技术将PCR扩增的HSV1-sr39TK基因分别连接至慢病毒载体不同启动子后,然后在HSV1-sr39TK基因后以内部核糖体进入位点(internal ribosome entry sites,IRES)连接绿色荧光蛋白(green fluorescent pro-tein,GFP)报告基因;采用三质粒包装系统包装病毒,将病毒感染刀豆蛋白A(concanavalin,ConA)激活的小鼠淋巴细胞,分别用流式细胞术、RT-PCR法鉴定HSV1-sr39TK和GFP基因在小鼠淋巴细胞的表达,用Cell Counting Kit-8(CCK-8)法观察感染不同病毒的淋巴细胞对前体药物GCV的敏感性。结果不同启动子指导的HSV1-sr39TK及GFP基因均可在小鼠淋巴细胞表达,巨细胞病毒(cytomegalovirus,CMV)启动子驱动表达能力最强,磷酸甘油激酶(phosphoglycerokinase,PGK)启动子最弱。感染含CMV启动子病毒的淋巴细胞对GCV的IC50值最小。结论在小鼠淋巴细胞内CMV启动子驱动的慢病毒载体HSV1-sr39TK基因表达最强,对前体药物GCV敏感性最高。