A mouse hybrid cell line that supports gene expression from a variety of promoters in amplifiable vectors

Summary A hybrid cell line was constructed by fusion of mouse L-cells with an NIH3T3 cell line derivative containing a hybrid gene consisting of the mouse immunoglobulin kappa (IgK) variable gene promoter linked to theEscherichia coli gpt gene. Such hybrids grew to a much higher density compared to either of the parental cell lines. The utility of this cell line as a host to express foreign genes was tested by the expression of TGF-β cDNA using the cytomegalovirus promoter. The vector also contained the human dihydrofolate reductase (DHFR) gene driven by SV40 early promoter, to allow for the amplification of the transfected gene. Initial transformants, selected at 100 nM methotrexate (MTX), were subsequently selected for resistance to a higher concentration of MTX (2 μM). Such clones expressed an increased level of TGF-β when compared to the initial transformants. Both the initial transformants and the clones with the amplified DHFR gene produced TGF-β in an acid-activatable precursor form. This mouse hybrid host cell line also allowed the expression of foreign genes cloned in an eukaryotic expression vector with the mouse IgK variable region promoter and human growth hormone as the reporter gene, whereas such vectors did not function in CHO cells. The mouse hybrid cell line was also found to be capable of being used with a broad range of promoters.     

High-level expression of secreted glycoproteins in transformed lepidopteran insect cells using a novel expression vector

An expression cassette for continuous high-level expression of secreted glycoproteins by transformed lepidopteran insect cells has been developed as an alternative to baculovirus and mammalian cell expression systems. The expression cassette utilizes the promoter of the silkmoth cytoplasmic actin gene to drive expression from foreign gene sequences, and also contains the ie-1 transactivator gene and the HR3 enhancer region of BmNPV to stimulate gene expression. Using an antibiotic-resistance selection scheme, we have cloned a Bm5 (silkmoth) cell line overexpressing the secreted glycoprotein juvenile hormone esterase (JHE-KK) at levels of 190 mg/L in batch suspension cultures. A baculovirus (AcNPV) expressing the same gene under the control of the p10 promoter of AcNPV produced only 4 mg/L active JHE in static cultures of infected Sf21 cells. A cloned Bm5 cell line overexpressing a soluble isoform of the alpha-subunit of the granulocyte-macrophage colony stimulating factor receptor (solGMRalpha) was also generated and produced five times more solGMRalpha in static cultures than a cloned BHK cell line obtained by transformation with a recombinant expression cassette utilizing the human cytomegalovirus (CMV) enhancer-promoter system. Finally, we show that recombinant protein expression levels in transformed Bm5 cells remain high in serum-free media, that expression is stable even in the absence of antibiotic selection, and that lepidopteran cells other than Bm5 may be used equally efficiently with this new expression cassette for producing recombinant proteins.     

Characterization of promoter function and cell-type-specific expression from viral vectors in the nervous system

Viral vectors have become important tools to effectively transfer genes into terminally differentiated cells, including neurons. However, the rational for selection of the promoter for use in viral vectors remains poorly understood. Comparison of promoters has been complicated by the use of different viral backgrounds, transgenes, and target tissues. Adenoviral vectors were constructed in the same vector background to directly compare three viral promoters, the human cytomegalovirus (CMV) immediate-early promoter, the Rous sarcoma virus (RSV) long terminal repeat, and the adenoviral E1A promoter, driving expression of the Escherichia coli lacZ gene or the gene for the enhanced green fluorescent protein. The temporal patterns, levels of expression, and cytotoxicity from the vectors were analyzed. In sensory neuronal cultures, the CMV promoter produced the highest levels of expression, the RSV promoter produced lower levels, and the E1A promoter produced limited expression. There was no evidence of cytotoxicity produced by the viral vectors. In vivo analyses following stereotaxic injection of the vector into the rat hippocampus demonstrated differences in the cell-type-specific expression from the CMV promoter versus the RSV promoter. In acutely prepared hippocampal brain slices, marked differences in the cell type specificity of expression from the promoters were confirmed. The CMV promoter produced expression in hilar regions and pyramidal neurons, with minimal expression in the dentate gyrus. The RSV promoter produced expression in dentate gyrus neurons. These results demonstrate that the selection of the promoter is critical for the success of the viral vector to express a transgene in specific cell types.     

Expression of green fluorescent protein in oligodendrocytes in a time- and level-controllable fashion with a tetracycline-regulated system

Developments in transgenic technology have greatly enhanced our ability to understand the functions of various genes in animal models and relevant human diseases. The tetracycline (tet)-regulated transactivation system for inducing gene expression allowed us to control the expression of exogenous genes in a temporal and quantitative way. The ability to manipulate a cell-specific promoter enabled us to express one particular protein in a single type of cell. The combination of a tetracycline system and a tissue-specific promoter has led us to the development of an innovative gene expression system, which is able to express genes in a cell type-specific and time- and level-controllable fashion. An oligodendrocyte-specific myelin basic protein (MBP) gene promoter controls the reversed tet-inducible transactivator. The green fluorescent protein (GFP) gene was placed under the control of the human cytomegalovirus (CMV) basic promoter in tandem with seven tet-responsive elements (TRE), binding sites for the activated transactivator. Upon the addition of doxycycline (DOX, a tetracycline derivative), tet transactivators became activated and bound to one or more TRE, leading to the activation of the CMV promoter and the expression of GFP in oligodendrocytes. We have successfully expressed GFP and luciferase at high levels in oligodendrocytes in a time- and dose-dependent fashion. In the absence of DOX, there was almost no GFP expression in oligodendroglial cultures. Graded levels of GFP expression were observed after induction with DOX (0.5 to 12.5 microg/ml). Our data indicate that this inducible gene expression system is useful for the study of gene function in vivo and for the development of transgenic animal models relevant to human diseases such as multiple sclerosis.     

Differential expression of mammalian or viral promoter-driven gene in adherent versus suspension cells

Although expression vectors using viral and mammalian promoters constitutively express genes of interest in adherent cells, few studies have examined whether the function of these vectors in suspended cells, such as in over-agar or soft agar assay (an in vitro cell transformation assay), is as robust as when they are in adherent cells. The selection of appropriate expression vector to optimally express genes in suspended cells would be useful in determining whether these genes play a critical role in maintaining colony formation or cell transformation. To compare promoter-driven expression vector function in adherent versus suspension cells, we performed transient transfection assays using viral (simian virus 40 [SV40] and cytomegalovirus [CMV]) and mammalian (beta-actin) promoters fused to luciferase or beta-galactosidase reporter gene. Over-agar assay was used to suspend cells on top of agar, which allowed cell retrieval and analysis. We found that beta-actin and SV40 promoters exhibited suppressed gene expression of 70 and 56%, respectively, in cells suspended on agar compared with those attached on plates. The suppressed response by the exogenous beta-actin promoter in suspension was consistent with the response of the endogenous beta-actin promoter activity because the steady-state level of beta-actin messenger ribonucleic acid in suspended cells was significantly reduced by 50% relative to that expressed in attached cells. In contrast to SV40 promoter, CMV promoter activity was not decreased in cells suspended in over-agar when compared with adherent cells. These studies show that regardless of mammalian or viral vectors, one cannot assume that all expression vectors behave similarly in both suspension and adherent state.     

Dramatic Increase in Expression of a Transgene by Insertion of Promoters Downstream of the Cargo Gene

For expression of genes in mammalian cells, various vectors have been developed using promoters including CMV, EF-1α, and CAG promoters and have been widely used. However, such expression vectors sometimes fail to attain sufficient expression levels depending on the nature of cargo genes and/or on host cell types. In the present study, we aimed to develop a potent promoter system that enables high expression levels of cargo genes ubiquitously in many different cell types. We found that insertion of an additional promoter downstream of a cargo gene greatly enhanced the expression levels. Among the constructs we tested, C-TSC cassette (C: CMV-RU5′ located upstream; TSC: another promoter unit composed of triple tandem promoters, hTERT, SV40, and CMV, located downstream of the cDNA plus a polyadenylation signal) had the most potent capability, showing far higher efficiency than that of potent conventional vector systems. The results indicate that the new expression system is useful for production of recombinant proteins in mammalian cells and for application as a gene therapeutic measure.     

构建基于杆状病毒的BV-T7杂合表达体系及利用其在哺乳动物和禽类细胞中表达eGFP基因

【目的】研究重组杆状病毒BV-T7杂合表达体系能否有效转导禽类细胞并在禽类细胞中表达外源基因(eGFP),从而构建能在禽类细胞中高效稳定表达外源基因的重组杆状病毒表达系统。【方法】本研究利用Bac-to-Bac杆状病毒表达系统,结合T7表达系统,通过对eGFP表达水平的调控来把握噬菌体T7 RNA聚合酶(T7 RNAP)的功能。利用两支重组杆状病毒,pFastBac-CMV-T7 RNAP重组杆状病毒为哺乳动物细胞启动子CMV调控的噬菌体T7 RNA聚合酶的cDNA;pFB-T7pro-IRES-GFP-T7ter重组杆状病毒为T7启动子控制的eGFP报告基因。将两支重组杆状病毒共同侵染哺乳动物OL(oligodendrocyte)细胞、鸡胚成纤维细胞和鸡胚骨骼肌细胞。【结果】两支重组杆状病毒利用T7启动子和T7 RNAP,在OL细胞、鸡胚成纤维细胞和鸡胚骨骼肌细胞中成功表达eGFP报告基因,而且未引起细胞病变,但在鸡胚原代细胞中eGFP的表达相对弱于在OL细胞中的表达。在OL细胞中重组杆状病毒对细胞的转导效率为59.5%,在鸡胚成纤维细胞和鸡胚骨骼肌细胞中转导效率分别为23.2%和33.1%。【结论】本研究构建的基于杆状病毒、T7RNA聚合酶、T7启动子(BV-T7)杂合表达体系能够在哺乳类细胞及禽类细胞中表达T7 RNAP,并利用T7RNAP继续高效而稳定地表达外源基因。这为难于体外操作的RNA病毒提供了有效的研究方法,并对新型基因工程疫苗的研制提供了一个高效而稳定的表达载体系统。     

Comparison of CMV, RSV, SV40 viral and Vlambda1 cellular promoters in B and T lymphoid and non-lymphoid cell lines.

Determining the activity of viral and cellular regulatory elements in B or T lymphoid cell lines would facilitate appropriate utilization of the regulatory sequences for gene transfer- and expression-dependent applications. We have compared the activity of the CMV, RSV and SV40 viral promoter/enhancers as well as the Vlambda1 cellular promoter, in three B cell lines (REH, SMS-SB, C3P), three T cell lines (CEM, Jurkat, ST-F10), and two non-lymphoid cell lines (K-562, HeLa) using the luciferase reporter gene. In B cell lines, the activity of the CMV promoter/enhancer construct was the highest ranging from 10- to 113-fold greater than that of SV40. In contrast, in T cell lines the RSV promoter/enhancer activity was 11-65-fold higher than that of SV40. The Vlambda1 promoter activity was close to that of SV40 promoter/enhancer in most of the cell lines tested. We conclude that CMV and RSV promoter/enhancers contain stronger regulatory elements than do the SV40 and Vlambda1 for expression of genes in lymphoid cell lines.     

Baculovirus-mediated expression of bacterial genes in dipteran and mammalian cells.

A recombinant baculovirus containing the Escherichia coli chloramphenicol acetyltransferase (CAT) gene under the control of the Rous sarcoma virus long terminal repeat promoter and the E. coli beta-galactosidase gene under the control of the very late baculoviral polyhedrin promoter was used to determine if Autographa californica nuclear polyhedrosis virus, a baculovirus of Lepidoptera, can enter and express viral DNA in dipteran (Drosophila sp.) and mammalian (Mus sp.) cells that are considered refractory to baculovirus replication. Following infection, CAT gene expression was observed in both dipteran and mammalian cells, but expression in the mammalian cell line was less than 0.05% of that observed in either dipteran or lepidopteran cells. Although the level of CAT gene expression was similar in permissive lepidopteran and nonpermissive dipteran cells, expression of beta-galactosidase activity from the late polyhedrin promoter in dipteran or mammalian cells was less than 0.3% of the levels observed in lepidopteran cells. These results indicate that foreign gene expression in nonpermissive cells is promoter dependent and that late viral gene expression is restricted in these cells. The Rous sarcoma virus long terminal repeat allows substantial CAT gene expression in both a D. melanogaster cell line and Aedes aegypti midgut cells. Baculovirus DNA undergoes a limited number of replications in Drosophila cells. The results are relevant to baculovirus host range, the safety of baculoviruses as pesticides, and the development of baculovirus pesticides with expanded host ranges.     

Insulin is secreted upon glucose stimulation by both gastrointestinal enteroendocrine K-cells and L-cells engineered with the preproinsulin gene

Transgenic mice carrying the human insulin gene driven by the K-cell glucose-dependent insulinotropic peptide (GIP) promoter secrete insulin and display normal glucose tolerance tests after their pancreatic p-cells have been destroyed. Establishing the existence of other types of cells that can process and secrete transgenic insulin would help the development of new gene therapy strategies to treat patients with diabetes mellitus. It is noted that in addition to GIP secreting K-cells, the glucagon-like peptide 1 (GLP-1) generating L-cells share/ many similarities to pancreatic p-cells, including the peptidases required for proinsulin processing, hormone storage and a glucose-stimulated hormone secretion mechanism. In the present study, we demonstrate that not only K-cells, but also L-cells engineered with the human preproinsulin gene are able to synthesize, store and, upon glucose stimulation, release mature insulin. When the mouse enteroendocrine STC-1 cell line was transfected with the human preproinsulin gene, driven either by the K-cell specific GIP promoter or by the constitutive cytomegalovirus (CMV) promoter, human insulin co-localizes in vesicles that contain GIP (GIP or CMV promoter) or GLP-1 (CMV promoter). Exposure to glucose of engineered STC-1 cells led to a marked insulin secretion, which was 7-fold greater when the insulin gene was driven by the CMV promoter (expressed both in K-cells and L-cells) than when it was driven by the GIP promoter (expressed only in K-cells). Thus, besides pancreatic p-cells, both gastrointestinal enteroendocrine K-cells and L-cells can be selected as the target cell in a gene therapy strategy to treat patients with type 1 diabetes mellitus.     

Improved efficient gene transfer into insect and mammalian cells by baculovirus vectors

The fragments of genomics DNA of the nuclear polyhedrosis virus (NPV) containing genes of late viral proteins p10, p35, p39, were cloned, the promoter regions of this genes were used to design baculovirus transfer vectors. A double-promoter and triple-promoter baculovirus transfer vectors were obtained. Recombinant baculovirus vectors containing mammalian expression cassette with cytomegalovirus (CMV) promoter, the gene for green or red fluorescent protein, SV40pA and polylinker MCS were constructed for the delivery of foreign genes into mammalian cells.     

The efficiency of different IRESs (Internal Ribosomes Entry Site) in monocistronic mRNAS

The IRES from poliovirus and from encephalomyocarditis virus (EMCV) added between the cap and the AUG initiator codon were strong inhibitors of chloramphenicol acetyltransferase gene expression in three different cell types. The poliovirus IRES also inhibited bGH (bovine growth hormone) cDNA expression in the HC11 mammary cell line when added between the rabbit whey acidic gene promoter and the cDNA whereas the HTLV-1 IRES showed a stimulatory effect in the same situation. RNA stem loops were added before HTLV-1 (SUR) and the BiP (Immunoglobulin heavy-chain Binding Protein) IRESs followed by the firefly luciferase gene under the control of Rous sarcoma virus (RSV) promoter. The RNA loops abolished the expression of the reporter gene almost completely. These data suggest that the different IRESs may favour or inhibit translation of monocistronic mRNA.