An OriP/EBNA-1-based baculovirus vector with prolonged and enhanced transgene expression

BACKGROUND: The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been explored as a gene delivery vehicle for a variety of mammalian cell lines. However, the transient expression nature due to its incapability to replicate in mammalian cells and insufficient transduction efficiency limit its application. METHODS: Recombinant baculovirus vectors containing genetic elements from Epstein-Barr virus (EBV), OriP and EBNA-1, which are essential for the episomal maintenance of the EBV genome in latently infected cells, were constructed and tested for their ability to sustain and express transgene (enhanced green fluorescence protein (egfp)) in mammalian cells. RESULTS: The recombinant baculovirus containing OriP and EBNA-1 genes driven by the cytomegalovirus (CMV) promoter was capable of persisting in a significant proportion of infected mammalian cells, HEK293, Vero, Cos-7, and Hone-1, without any selective pressure. In HEK293, the expression of EGFP lasted for 60 days with markedly enhanced expression level. The persistence of baculovirus genome correlated with the expression of EBNA-1. CONCLUSIONS: The improved baculovirus vector could mediate prolonged and enhanced foreign gene expression in some mammalian cells. Furthermore, an adequate level of the EBNA-1 protein was essential for the maintenance of the OriP-containing baculovirus genome. The new vector has potential for use in gene therapy.     

Expression of SEAP (secreted alkaline phosphatase) by baculovirus mediated transduction of HEK 293 cells in a hollow fiber bioreactor system

A BacMam baculovirus was designed in our laboratory to express the reporter protein secreted alkaline phosphatase (SEAP) driven by the immediate early promoter of human cytomegalovirus promoter (CMV). In vitro tests have been carried out using this recombinant baculovirus to study the secreted protein in two cell lines and under various culture conditions. The transductions were carried out on two commonly used mammalian cell lines namely the human embryonic kidney (HEK 293A) and Chinese hamster ovary (CHO-K1). Initial studies clearly demonstrated that the transient expression of SEAP was at least 10-fold higher in the HEK 293 cells than the CHO cells under equivalent experimental conditions. Factorial design experiments were done to study the effect of different parameters such as cell density, MOI, and the histone deacetylase inhibitor, trichostatin A concentration. The multiplicity of infection (MOI) and the cell density were found to have the most impact on the process. The enhancer trichostatin A also showed some positive effect. The production of secreted protein in a batch reactor was studied using the Wave disposable bioreactor system. A semi-continuous perfusion process was developed to extend the period of gene expression in mammalian cells using a hollow fiber bioreactor system (HFBR). The growth of cells and viability in both systems was monitored by offline analyses of metabolites. The expression of recombinant protein could be maintained over an extended period of time up to 30 days in the HFBR.     

肿瘤内皮标志物8同型物Ⅰ基因真核表达载体的构建及其在HEK293F细胞中的表达

目的:构建肿瘤内皮标志物8(TEM8)基因真核表达载体,实现TEM8在HEK293F细胞中的外源表达。方法:用PCR技术扩增TEM8基因,经限制性酶切、连接、转化,插入pcDNA3.1(+)-EGFP真核表达载体,并通过脂质体将TEM8表达质粒转染至HEK293F细胞中,Western印迹检测TEM8的表达。结果:PCR扩增得到TEM8基因,构建了真核表达载体pcDNA3.1(+)-TEM8-EGFP并转染HEK293F细胞,经G418加压筛选及有限稀释法得到生长性状良好、表达效率高的单克隆细胞系TEM8-EGFP/HEK293F;Western印迹证明过表达细胞系TEM8-EGFP/HEK293F显著表达TEM8蛋白。结论:构建了表达TEM8的重组HEK293F工程细胞系TEM8-EGFP/HEK293F,为进一步研究TEM8的生理功能奠定了基础。     

基于逆转录病毒载体的外源基因表达系统的评价和应用

逆转录病毒表达系统是基因治疗研究和RNA干扰技术广泛采用的外源基因表达系统。文中以增强型绿色荧光蛋白 (EGFP) 基因的表达水平和稳定性为指标,比较逆转录病毒表达载体pQCXIN和pcDNA3.1(+) 表达质粒介导的外源基因在HEK293细胞和CHO-K1细胞的表达效率。病毒感染HEK293细胞和CHO-K1细胞的相对荧光强度 (Relative fluorescence intensity,RFI) 均约为对应的质粒转染细胞的2倍。多轮反复感染逆转录病毒表达载体能有效提高HEK293细胞表达EGFP的效率。HEK293细胞经4轮病毒感染后的RFI值较1次病毒感染HEK293细胞的RFI值约提高2倍。此外,逆转录病毒表达载体介导的外源基因表达的稳定性优于质粒转染的外源基因表达。采用携带人重组活性蛋白C (Recombinant human activated protein C,rhAPC) 基因的pQCXIN和HEK293细胞进一步验证了逆转录病毒载体介导的外源基因表达效率,构建了rhAPC表达水平为10~15 mg/(106 cells·d) 的HEK293细胞系。研究结果表明,逆转录病毒表达系统是有应用价值的介导外源基因在哺乳动物细胞高效表达的技术途径。     

Development of a Chinese hamster ovary cell line for recombinant adenovirus-mediated gene expression

Recombinant human adenovirus (rhAd) has been used extensively for functional protein expression in mammalian cells including those of human and nonhuman origin. High-level protein production by rhAd vectors is expected in their permissive host cells, such as the human embryonic kidney 293 (HEK293) cell line. This is attributed primarily to the permissiveness of HEK293 to rhAd infection and their ability to support viral DNA replication by providing the missing El proteins. However, the HEK293 cells tend to suffer from cytopathic effect (CPE) as a result of virus replication. Under these circumstances, the host cell function is compromised and the culture viability will be reduced. Consequently, newly synthesized polypeptides may not be processed properly at posttranslational levels. Therefore, the usefulness of HEK293 cells for the expression of complex targets such as secreted proteins could be limited. In the search for a more robust cell line as a production host for rhAd expression vectors, a series of screening experiments was performed to isolate clones from Chinese hamster ovary-K1 (CHO-K1) cells. First, multiple rounds of infection of CHO-K1 cells were performed utilizing an rhAd expressing GFP. After each cycle of infection, a small population of CHO cells with high GFP levels was enriched by FACS. Second, individual clones more permissive to human adenovirus infection were isolated from the highly enriched subpopulation by serial dilution. A single clone, designated CHO-K1-C5, was found to be particularly permissive to rhAd infection than the parental pool and has served as a production host in the successful expression of several secreted proteins.     

稳定表达hHCN2基因 HEK293细胞系的建立

目的：培育稳定表达hHCN2基因的细胞系,建立一种表达研究心肌离子通道的有效模型。方法：通过脂质体转染的方法,将重组pcDNA3-hHCN2真核表达载体导入人胚肾细胞（HEK293细胞）,以G418压力筛选转染细胞,并对其进行全细胞膜片钳记录。结果：经600μg／ml压力筛选后,获得抗性细胞克隆,并用全细胞膜片钳技术记录到克隆hHCN2通道编码电流。结论：本实验采用脂质体转染法成功地培育出G418抗性HEK293细胞。为进一步研究克隆离子通道结构和功能的关系奠定基础。     

A baculovirus superinfection system: efficient vehicle for gene transfer into Drosophila S2 cells

The baculovirus expression vector system is considered to be a safe, powerful, but cell-lytic heterologous protein expression system in insect cells. We show here that there is a new baculovirus system for efficient gene transfer and expression using the popular and genetically well-understood Drosophila S2 cells. The recombinant baculovirus was constructed to carry an enhanced green fluorescent protein under the control of polyhedrin promoter as a fluorescent selection marker in the Sf21 cell line. Recombinant baculoviruses were then used to transduce S2 cells with target gene expression cassettes containing a Drosophila heat shock protein 70, an actin 5C, or a metallothionein promoter. Nearly 100% of the S2 cells showed evidence of gene expression after infection. The time course for the optimal protein expression peaked at 24 to 36 h postinfection, which is significantly earlier than a polyhedrin-driven protein expression in Sf21 cells. Importantly, S2 cells did not appear to be lysed after infection, and the protein expression levels are comparable to those of proteins under the control of polyhedrin promoter in several lepidopteran cell lines. Most surprisingly, S2 cells permit repetitive infections of multiple baculoviruses over time. These findings clearly suggest that this baculovirus-S2 system may effect the efficient gene transfer and expression system of the well-characterized Drosophila S2 cells.     

Baculovirus vectors for efficient gene delivery and expression in mammalian cells

Baculovirus expression vectors are extensively used for the delivery of foreign genes and expression of recombinant proteins in insect and mammalian cells. Modified baculoviruses containing mammalian promoter elements (BacMam viruses) for an efficient transient and stable transduction of diverse mammalian cells ensure a high level of heterologous protein expression both in vitro and in vivo. Recombinant baculovirus vectors containing mammalian expression cassette with cytomegalovirus promoter, green or red fluorescent protein gene, SV40pA polyadenylation signal, and polylinker MCS were constructed for the delivery of genes encoding hepatitis C virus structural proteins into mammalian cells. In HEK293T and Huh7 cells, formation of glycoprotein complexes and HCV4ike particles was observed. A high efficiency of the baculovirus-medi-ated gene transfer and expression of the virus envelope proteins in mammalian cells was demonstrated using fluorescence, flow cytometry, and immunoblot techniques.     

G418抗性HEK293细胞的培育

目的　培育具有G418抗性的HEK2 93细胞 ,用于建立猪内源性反转录病毒感染人HEK2 93细胞的模型。方法　通过脂质体转染的方法 ,将含有neo基因的质粒pIRESneo导入HEK2 93细胞中 ,利用G418的选择特性 ,对转染细胞进行压力筛选 ,并对其进行了PCR鉴定。结果　经 6 0 0 μg ml的G418压力筛选后 ,获得了抗性细胞克隆。抗性细胞的形态和生长速度与筛选前细胞没有差异 ,特异性核苷酸引物检测抗性细胞基因组DNA ,可以扩增出对应的核苷酸片段。结论　成功地培育了G418抗性HEK2 93细胞 ,为建立猪内源性反转录病毒感染人HEK2 93细胞的模型奠定了基础。     

HAX1和EGFP共表达重组腺病毒载体的构建、鉴定

目的构建和鉴定HAX1和EGFP双基因共表达重组腺病毒载体。方法采用DNA重组技术,将目的基因HAX1克隆至含有报告基因EGFP的穿梭质粒pAdTrack—CMV中,并转化于大肠埃希菌DH5a;筛选出重组质粒pAdTrack—CMV—HAX1,并在BJ5183细菌中与pAdEasy-1质粒进行同源重组,产生重组腺病毒载体;用lipofectamine将其转染HEK293细胞,包装携带全长HAX1的重组复制缺陷型腺病毒pad—HAX1-EGFP,酶切和序列测定鉴定;用制备好的Ad—HAX1-EGFP感染HEK293细胞,流式细胞术检测其感染效率,RT—PCR、Western印迹鉴定外源基因HAX1的表达。BrdU检测感染了Ad—HAX1-EGFP的HEK293细胞增殖情况。结果pAdTrack—CMV—HAX1重组质粒构建成功。pAdTrack—CMV—HAX1质粒与pAdEasy-1质粒同源重组后与预期结果相符。构建好的Ad—HAX1-EGFP能有效感染HEK293细胞;外源基因能在239细胞中有效表达。HAX1高表达的HEK293细胞其增殖率得以提高。结论成功构建了表达HAX1和EGFP共表达的重组腺病毒载体,HAX1能够促进结肠癌细胞HEK293细胞的增殖。     

Expression of double foreign protein types following recombinant baculovirus infection of stably transfected Drosophila S2 cells

We sought to develop a platform for simultaneous, regulatable expression of double foreign protein types in cell culture. Drosophila melanogaster Schneider line 2 (S2) insect cells that stably express human erythropoietin (hEPO) were infected with a recombinant baculovirus containing the green fluorescent protein (GFP) gene. Since baculovirus cannot replicate in nonpermissive S2 cells, baculovirus infection did not affect cell growth or viability. Expression of each foreign protein was under the control of the inducible metallothionein (MT) promoter. Addition of copper sulfate to infected, stably transfected cells resulted in simultaneous expression of both GFP and hEPO. Induced hEPO expression profile and levels were similar in both control and infected cells, indicating that baculovirus infection also did not affect expression of stably introduced foreign gene. GFP protein levels were regulated by the infection dose of recombinant baculovirus, while hEPO expression remained constant. hEPO levels were much higher (30-fold) than GFP, indicating plasmid-based introduced gene copies have higher expression than baculovirus-based introduced genes. These data suggest the baculovirus/stable S2 cell system can be used to produce a major target protein by plasmid-based stable transfection, and assistant proteins by recombinant baculovirus infection. Such a system appears to be very attractive as a multiple protein expression platform for engineering metabolic pathways in cell culture.     

A surface-modified baculovirus vector with improved gene delivery to B-lymphocytic cells

A short peptide motif from gp350/220 of Epstein-Barr virus, EDPGFFNVEI, which was known to bind to CD21, a surface protein on B-lymphocyte, was inserted into the baculovirus surface protein gp64. The recombinant virus carrying the hybrid gp64/gp350 gene, vAc-gp350EGFP, was obtained, and the expression of gp64/gp350 protein was confirmed with immunoblot using anti-gp350 antibody. When compared with a control virus with wild type gp64, vAc-gp350EGFP showed increased transduction efficiency in B cell lines Raji, HR1, B95-8, BJAB, and DG75, regardless of their being EBV-positive or EBV-negative. No such increase was seen in non-B cell lines HEK293 and HeLa. When Raji cells were transduced with increased amount of vAc-gp350EGFP, transduction became saturated when the multiplicity of infection was higher than 20pfu/cell. The transduction of Raji cells by vAc-gp350EGFP was dose-dependently inhibited by pre-treatment of cells with anti-CD21 antibody. These results showed that vAc-gp350EGFP entered B cells by interacting with CD21.     

Modulation of oxidative phosphorylation of human kidney 293 cells by transfection with the internal rotenone-insensitive NADH-quinone oxidoreductase (NDI1) gene of Saccharomyces cerevisiae.

In contrast to the mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I), which consists of at least 43 different subunits, the internal rotenone-insensitive NADH-quinone oxidoreductase (Ndi1) of Saccharomyces cerevisiae is a single polypeptide enzyme. The NDI1 gene was stably transfected into the human embryonal kidney 293 (HEK 293) cells. The transfected NDI1 gene was then transcribed and translated in the HEK 293 cells to produce the functional enzyme. The immunochemical and immunofluorescence analyses indicated that the expressed Ndi1 polypeptide was located to the inner mitochondrial membranes. The expression of Ndi1 did not alter the content of existing complex I in the HEK 293 mitochondria, suggesting that the expressed Ndi1 enzyme does not displace the endogenous complex I. The NADH oxidase activity of the NDI1-transfected HEK 293 cells was not affected by rotenone but was inhibited by flavone. The ADP/O ratios coupled to NADH oxidation were lowered from 2.4 to 1.8 by NDI1-transfection while the ADP/O ratios coupled to succinate oxidation (1.6) were not changed. The NDI1-transfected HEK 293 cells were able to grow in media containing a complex I inhibitor such as rotenone and 1-methyl-4-phenylpyridinium ion. The potential usefulness of incorporating the Ndi1 protein into mitochondria of human cells is discussed.     

Spatiotemporal modeling and analysis of transient gene delivery

A quantitative and mechanistic understanding of intracellular transport processes in eukaryotic cells during transient transfection is an important prerequisite for the systematic and specific optimization of transient gene expression procedures for pharmaceutic and industrial protein production. There is evidence that intracellular transport processes during gene delivery and their regulation may have significant influence on the transfection efficiency. This contribution describes a compartmented, spatiotemporally resolved and stochastic modeling approach that describes intracellular transport processes responsible for gene delivery during transient transfection. It enables a detailed prediction and analysis and identification of potential bottlenecks. This model is currently being adapted to a model cell line, HEK293s. The simulated results are compared with experimental quantitative polymerase chain reaction (qPCR) data and confocal imaging data obtained with transfected and stained HEK293 cells. Global parameter estimation is performed to qPCR data based on two different novel plasmid constructs in order to identify candidates for plasmid-specific transport parameter variations. The influence of the specific property of HEK293 cells to grow in clusters is investigated and the impact of active microtubule transport depending on cell morphology and clustering is examined. A general sensitivity analysis allows for the identification of the sensitive parameters.     

Efficient expression screening of human membrane proteins in transiently transfected Human Embryonic Kidney 293S cells

It is often an immense challenge to overexpress human membrane proteins at levels sufficient for structural studies. The use of Human Embryonic Kidney 293 (HEK 293) cells to express full-length human membrane proteins is becoming increasingly common, since these cells provide a near-native protein folding and lipid environment. Nevertheless, the labor intensiveness and low yields of HEK 293 cells and other mammalian cell expression systems necessitate the screening for suitable expression as early as possible. Here we present our methodology used to generate constructs of human membrane proteins and to rapidly assess their suitability for overexpression using transiently transfected, glycosylation-deficient GnT I-HEK 293 cells (HEK 293S). Constructs, in the presence or absence of a C-terminal enhanced green fluorescence protein (EGFP) molecule, are made in a modular manner, allowing for the rapid generation of several combinations of fusion tags and gene paralogues/orthologues. Solubilization of HEK 293S cells, using a range of detergents, followed by Western blotting is performed to assess relative expression levels and to detect possible degradation products. Fluorescence-detection size exclusion chromatography (FSEC) is employed to assess expression levels and overall homogeneity of the membrane proteins, to rank different constructs for further downstream expression trials. Constructs identified as having high expression are instantly suitable for further downstream large scale transient expression trials and stable cell line generation. The method described is accessible to all laboratory scales and can be completed in approximately 3 weeks.     

High-titer, serum-free production of adeno-associated virus vectors by polyethyleneimine-mediated plasmid transfection in mammalian suspension cells

Adeno-associated virus (AAV)-based vectors belong to the most promising gene transfer vectors in clinical studies. To provide vector for late-stage clinical trials as well as for a potential commercial phase, a scalable, cGMP-compliant process is required. Nearly all vector production protocols currently approved in Phase I clinical trials rely on AAV production in adherent HEK 293 cells in the presence of serum. In this study, we present a helper- and serum-free production method of AAV vectors in suspension-adapted HEK 293 cells. The method is based on plasmid transfection with 25 kDa linear polyethyleneimine. Compared to existing methods, our system is highly scalable as cells grow in suspension, does not require animal-derived products or the use of an exogenous virus (adenovirus or baculovirus) and yields genomic titers equal to those obtained in adherent HEK 293 cells in the presence of serum. Most importantly, work load and cost could be dramatically reduced in comparison to earlier methods, when comparing the production of equivalent volumes of cell culture media. Thus, our protocol should appeal to both basic research laboratories and cGMP manufacturing units.     

Induction of the synthesis of a 70,000 dalton mammalian heat shock protein by the adenovirus E1A gene product

We have attempted to determine whether any cellular genes are activated as a result of the action of the adenoviral El A gene. The proteins synthesized in uninfected HeLa cells have been compared to those produced in early adenovirus infected cells. At least one protein, absent from uninfected HeLa cells, was synthesized in large amounts following adenovirus infection. This 70 kd protein was not synthesized in cells infected with the E1A mutant d1312, even when the multiplicity of infection with the mutant was such that the only viral gene not expressed was the E1A gene. Thus the induction of the 70 kd protein requires the expression of the viral E1A gene. The 70 kd protein was also induced by heat shock in uninfected cells. The same 70 kd protein is synthesized in 293 cells, a line of human embryonic kidney cells transformed by a fragment of adenovirus DNA. These cells constitutively express the E1A and E1 B genes.