High level transient gene expression in human lymphoid cells by SV40 large T antigen boost.

High level transient gene expression in lymphoid cells has always been challenging because of the difficulty to efficiently transfect such cells. This has precluded any attempt to clone cDNA encoding proteins by means of their specific biological function in lymphoid cells. We have developed a very efficient transient eukaryotic expression system analogous to the well-known expression system in COS cells. Firefly luciferase and human CD2 genes were used as reporter genes and cloned into the eukaryotic shuttle vector pCDM8 which contains the strong cytomegalovirus promoter and the SV40 origin of replication for autonomous plasmid replication in permissive host cells that express the large SV40 T Antigen. Co-transfection of the reporter plasmids together with an SV40 T Ag expressing plasmid resulted in the several fold amplification of either the Luc activity or the cell surface expression of the CD2 marker in a transient assay. The level of amplification was dependent on the strength of the promoter used to drive the SV40 T Ag expression and was correlated with the extent of autonomous replication of the reporter plasmid in transfected cells. This highly efficient transient gene expression by SV40 T Ag boost was suitable to several human cell lines, making this system of general interest for expression cloning strategies or other gene transfer application that need high level expression.     

Regulated replication of an episomal simian virus 40 origin plasmid in COS7 cells.

The replication of a simian virus 40 (SV40) origin-containing plasmid, pSLneo, stably transfected COS7 cells has been studied. pSLneo contains the SV40 origin of replication and encodes the positive selectable marker for G418 resistance. In transient replication assays, pSLneo replicates to a high copy number in COS7 cells. Uncontrolled SV40 plasmid replication has been reported to be lethal to such transfected cells. Thus, it was anticipated that extensive plasmid replication would preclude isolation of permanent cell lines containing pSLneo. However, significant number of G418-resistant colonies arose after transfection of COS7 cells with pSLneo. Cell lines established from these drug-resistant colonies contained between 100 and 1,000 extrachromosomal pSLneo copies per cell. Episomal plasmid DNA in pSLneo/COS7 lines was stably maintained after 2 months of continuous culture in selective medium. Bromodeoxyuridine labeling and density shift experiments demonstrated that replication of pSLneo closely paralleled that of cellular DNA. On average, plasmid DNA did not replicate more than once during a single cell generation period. Regulation of pSLneo replication appeared to be negatively controlled by a cis-acting mechanism. Endogenous copies of episomal pSLneo remained at a stable low copy number during the simultaneous, high-level replication of a newly transfected plasmid encoding SV40 large T antigen in the same cells. These results indicate that regulated replication of an SV40 origin plasmid can be acquired in a cell and does not require the presence of additional genetic elements. The molecular mechanism by which cells enforce this regulation on extrachromosomal SV40 plasmids remains to be defined.     

Sequence requirements for plasmid nuclear import

The nuclear envelope is a major barrier for nuclear uptake of plasmids and represents one of the most significant unsolved problems of nonviral gene delivery. We have previously shown that the nuclear entry of plasmid DNA is sequence-specific, requiring a 366-bp fragment containing the SV40 origin of replication and early promoter. In this report, we show that, although fragments throughout this region can support varying degrees of nuclear import, the 72-bp repeats of the SV40 enhancer facilitate maximal transport. The functions of the promoter and the origin of replication are not needed for nuclear localization of plasmid DNA. In contrast to the import activity of the SV40 enhancer, two other strong promoter and enhancer sequences, the human cytomegalovirus (CMV) immediate-early promoter and the Rous sarcoma virus LTR, were unable to direct nuclear localization of plasmids. The inability of the CMV promoter to mediate plasmid nuclear import was confirmed by measurement of the CMV promoter-driven expression of green fluorescent protein (GFP) in microinjected cells. At times before cell division, as few as 3 to 10 copies per cell of cytoplasmically injected plasmids containing the SV40 enhancer gave significant GFP expression, while no expression was obtained with more than 1000 copies per cell of plasmids lacking the SV40 sequence. However, the levels of expression were the same for both plasmids after cell division in cytoplasmically injected cells and at all times in nuclear injected cells. Thus, the inclusion this SV40 sequence in nonviral vectors may greatly increase their ability to be transported into the nucleus, especially in nondividing cells.     

Activation of the SV40 late promoter: direct effects of T antigen in the absence of viral DNA replication

We have examined the activation of the SV40 late promoter by inserting the late promoter and the viral origin of replication into chloramphenicol acetyltransferase (CAT) transient expression vectors. Very little late promoter activity was detected in CV-1 cells, compared with high activity in COS cells, in which replication occurs due to endogenous T antigen. Nonreplicative counterparts of these plasmids, containing a mutated origin of replication, produced significantly more late promoter activity in COS cells than any of the plasmids in CV-1 cells. When plasmids were cotransfected into CV-1 cells with a plasmid that supplies T antigen, the nonreplicative plasmid displayed 30% of the activity of the replicative plasmid. Using mutant T antigens unable to replicate viral DNA, late promoter activation occurred only with mutant T antigens that retain DNA binding activity. These results demonstrate that T antigen can substantially stimulate late promoter activity directly and independent of viral DNA replication.     

Transfection of cDNA with G----T point mutation at the cleavage site of insulin receptors to COS 7 cells

To study whether the G----T point mutation of insulin proreceptors at the cleavage site which changed -Arg-Lys-Arg-Arg- to -Arg-Lys-Arg-Ser- caused unprocessed insulin receptors with decreased insulin binding affinity, we performed transfection of cDNA with the mutation in COS 7 cells and examined the expressed insulin receptors. After site-directed mutagenesis, an expression vector pGEM3SV was used to make a plasmid which contained full-length HIRcDNA behind SV40 early promoter. Transfection of normal HIR cDNA produced normal insulin receptors on the plasma membranes in COS 7 cells. However, transfection of cDNA with the mutation resulted in the presence of 210K proreceptors in the plasma membranes with decreased insulin binding ability (35% of normal). These results suggest that the mutation, not the defect of converting enzyme, was the cause for unprocessed insulin proreceptors in the patients with insulin resistance.     

Co-transfected SV40 origin of replication activates expression from SV40 promoterless constructs.

Co-transfection with expression plasmids is widely used to control DNA uptake efficiency in transient transfection experiments. However, a number of problems have been associated with their use. Here, we describe the activation of expression of constructs not containing the simian virus 40 (SV40) origin of replication (ori) by co-transfection in COS-7 cells with plasmids containing the SV40 ori. This effect has consequences for the use of such plasmids to control transfection efficiency.     

The role of plasmid constructs containing the SV40 DNA nuclear-targeting sequence in cationic lipid-mediated DNA delivery

One of the steps that limit transfection efficiency in non-viral gene delivery is inefficient nuclear import of plasmid DNA, once it has been delivered into the cytoplasm. Recently, via microinjection into the cytoplasm and in situ hybridizations into a few cell types, it was shown that a region of Simian virus 40(SV40), specifically a c. 372-bp fragment of SV40 genomic DNA encompassing the SV40 promoter-enhancer-origin of replication (SV40 DTS), could enable the nuclear import of a plasmid carrying these sequences (Dean D.A. Exp. Cell Res. 230 (1997) 293). In this report, we address the issue of the suitability of the SV40 DTS for cationic lipid-mediated gene delivery, and its capacity to improve the efficiency of the transfection process. For this study, we used transient reporter gene expression assays on various cell types. The gene expression from the plasmid constructs carrying the SV40 DTS varied with cell type and plasmid construct used. Such cell-type and plasmid-construct dependency on gene expression from plasmids containing the SV40 DTS suggests that the gene expression from plasmids is not entirely dependent on its ability to enhance the nuclear import of said plasmids.     

Expression vector system based on the chicken beta-actin promoter directs efficient production of interleukin-5

We examined the promoter activity of the 1.3-kb chicken beta-actin gene sequence located between the 5' flanking region and the proximal region of the second exon. This promoter region showed higher promoter activity than the simian virus 40 (SV40) early promoter or the Rous sarcoma virus (RSV) long terminal repeat (LTR) as assayed by transient lacZ gene expression in mouse L cells. Furthermore, replacement of the 3' splice sequence in this promoter by that derived from the rabbit beta-globin gene resulted in a approximately 2.5-fold enhancement in the synthesis of beta-galactosidase (beta Gal). Introduction of the SV40 origin of DNA replication (ori) into the vector carrying this hybrid promoter, which we designate the AG promoter, markedly enhanced the production of beta Gal in an SV40 T antigen-producing cell, BMT10. We have constructed a useful vector containing the strong AG promoter, several unique restriction sites, a SV40 polyadenylation signal and the SV40 ori for transient expression of cDNA in BMT10 or COS cells. We demonstrate the use of this vector for efficient production of interleukin-5 in BMT10 cells.     

Demethylation using the epigenetic modifier, 5-azacytidine, increases the efficiency of transient transfection of macrophages

This study was aimed at developing a method for high-efficiency transient transfection of macrophages. Seven methods were evaluated for transient transfection of murine macrophage RAW 264.7 cells. The highest transfection efficiency was achieved with DEAE-dextran, although the proportion of cells expressing the reporter gene did not exceed 20%. It was subsequently found that the cytomegalovirus plasmid promoter in these cells becomes methylated. When cells were treated with the methylation inhibitor 5-azacytidine, methylation of the plasmid promoter was abolished and a dose-dependent stimulation of reporter gene expression was observed with expression achieved in more than 80% of cells. Treatment of cells with 5-azacytidine also caused increased efficiency of transfection of macrophages with plasmids driven by RSV, SV40, and EF-1alpha promoters and transient transfection of human HepG2 cells. Inhibition of methylation also increased the amount and activity of sterol 27-hydroxylase (CYP27A1) detected in RAW 264.7 cells transfected with a CYP27A1 expression plasmid. Treatment of cells with 5-azacytidine alone did not affect either cholesterol efflux from nontransfected cells or expression of ABCA1 and CYP27A1. However, transfection with CYP27A1 led to a 2- to 4-fold increase of cholesterol efflux. We conclude that treatment with 5-azacytidine can be used for high-efficiency transient transfection of macrophages.     

cis-active elements from mouse chromosomal DNA suppress simian virus 40 DNA replication.

Simian virus 40 (SV40)-containing DNA was rescued after the fusion of SV40-transformed VLM cells with permissive COS1 monkey cells and cloned, and prototype plasmid clones were characterized. A 2-kilobase mouse DNA fragment fused with the rescued SV40 DNA, and derived from mouse DNA flanking the single insert of SV40 DNA in VLM cells, was sequenced. Insertion of the intact rescued mouse sequence, or two nonoverlapping fragments of it, into wild-type SV40 plasmid DNA suppressed replication of the plasmid in TC7 monkey cells, although the plasmids expressed replication-competent T antigen. Rat cells were transformed with linearized wild-type SV40 plasmid DNA with or without fragments of the mouse DNA in cis. Although all of the rat cell lines expressed approximately equal amounts of T antigen and p53, transformants carrying SV40 DNA linked to either of the same two replication suppressor fragments produced significantly less free SV40 DNA after fusion with permissive cells than those transformed by SV40 DNA without a cellular insert or with a cellular insert lacking suppressor activity. The results suggest that two independent segments of cellular DNA act in cis to suppress SV40 replication in vivo, either as a plasmid or integrated in chromosomal DNA.     

Transient expression of murine interferon-alpha genes in mouse and monkey cells

The coding regions of murine interferon-alpha (IFN-alpha) genes were combined with promoter and 3'-noncoding sequences from other eukaryotic genes. Transient expression of these fusion genes was achieved in monkey COS cells and in a mouse cell line (TOP cells) expressing polyoma virus (Py) large T antigen constitutively. The efficiency of the different expression plasmids was determined by measuring the amount of IFN secreted into the medium. Replacement of the 3'-noncoding region of an IFN-alpha gene by that of the rabbit beta-globin gene resulted in a fourfold higher IFN-alpha production. The SV40 early promoter and the Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR) produced similar amounts of IFN-alpha in COS cells. However, a tandem combination of the SV40 enhancer/early promoter and the mouse metallothionein-I promoter appeared fivefold more active than the SV40 early promoter. In TOP cells the MoMLV LTR was found to be threefold more active than the Py early promoter.     

Increased disulphide dimer formation of latent associated peptide fusions of TGF-β by addition of l-cystine

The development of novel protein therapeutics relies on the ability to express appreciable amounts of correctly folded recombinant proteins. Latent IFN-β is engineered using the latency-associated peptide (LAP) of transforming growth factor β1 (TGF-β1) to maintain IFN-β in a biologically inactive form until such time as it is released at sites of inflammation by matrix metalloproteinase activity (see Adams et al., 2003). CHO cells cultured in suspension were used for expression of latent IFN-β to allow medium scale transient transfection. However, the recombinant protein expressed in this system consisted of a mixture of properly linked disulphide dimers and monomers. The ratio of dimer:monomer produced could be significantly altered towards increased dimer production by the addition of l-cystine to the CHO culture medium. The total yield of latent IFN-β was increased by co-transfection of plasmid coding for the simian virus (SV) 40 large T antigen to the plasmid with the SV40 origin of replication expressing latent IFN-β DNA. These results provide valuable new insights for developing protocols to produce substantial quantities of latent cytokine dimers in CHO cells in suspension.     

Expression cloning of cDNAs that render cancer cells resistant to Pseudomonas and diphtheria toxin and immunotoxins.

BACKGROUND: Several immunotoxins in which antibodies are coupled to plant or bacterial toxins are now in clinical trials for the treatment of cancer. One of these is B3-LysPE38 in which MAb B3 which reacts with many human cancers, is coupled with a genetically modified form of Pseudomonas exotoxin (PE). MATERIALS AND METHODS: To investigate how cells can become resistant to PE-derived immunotoxins, we constructed an immunotoxin-sensitive MCF-7 breast cancer cell line that contains SV40 T antigen and allows episomal replication of SV40 origin containing plasmids. We transfected a pCDM8/HeLa cDNA expression library into these cells, thereby causing over-expression of the plasmid-encoded genes. The transfected cells were treated with immunotoxin to select for resistance-mediating plasmids, which were reisolated from these cells and amplified in Escherichia coli. The resulting plasmid pool was transfected into cells for two further rounds of selection and plasmid reisolation. RESULTS: Several plasmids that caused immunotoxin resistance were enriched by this selection procedure. Four plasmids were stably transfected into MCF-7 cells and found to increase their resistance to PE-derived immunotoxins by 5- to 20-fold. These plasmids also confer resistance to native PE and to diphtheria toxin but not to ricin or cycloheximide. Thus, they appear to specifically interfere with the action of ADP-ribosylating toxins. CONCLUSION: Cancer cells can become resistant to immunotoxins by deregulated expression of normal genes. The clinical significance of this type of resistance will be evaluated in clinical trials.     

c-myc protein can be substituted for SV40 T antigen in SV40 DNA replication.

Replicating activity of SV40 origin-containing plasmid was tested in human cells as well as in monkey CosI cells. All the plasmids possessing SV40 ori sequences could replicate, even in the absence of SV40 T antigen, in human HL-60 and Raji cells which are expressing c-myc gene at high level. The copy numbers of the replicated plasmids in these human cells were 1/100 as high as in monkey CosI cells which express SV40 T antigen constitutively. Exactly the same plasmids as the transfected original ones were recovered from the Hirt supernatant of the transfected HL-60 cells. Furthermore, replication of the SV40 ori-containing plasmids in HL-60 cells was inhibited by anti-c-myc antibody co-transfected into the cells. These results indicate that the c-myc protein can be substituted for SV40 T antigen in SV40 DNA replication.     

Electroporation-mediated transfection of mammalian cells with crude plasmid DNA preparations

We designed a simple and reproducible electroporation-mediated transfection procedure with which to screen mammalian expression vector-constructed cDNA libraries. Using a specific chamber composed of five parallel electrodes, the recipient cells can be electroporated separately with 40 plasmid DNA preparations in a single experiment. Over 300 crude plasmids prepared from E. coli (DH-5) carrying a pcD2neo-vector-derived cDNA library were tested. The efficiency of stable transfection by electroporation with crude plasmid DNA preparations was 10-times higher than with the CsCl-purified plasmid DNA. When the crude plasmids were digested with RNase, the efficiency of stable transfection markedly decreased, indicating that the contaminating bacterial RNA in the crude plasmid preparations has a strong carrier effect during the electroporation. Even when salmon sperm DNA or genomic DNA from the recipient cells was used as the carrier of the purified plasmid, the efficiency was not higher than that using the crude preparations. This procedure is useful not only for screening a number of cDNAs but also for routinely introducing biologically active foreign genes into cultured mammalian cells.     

Expression of rat neurofilament proteins NF-L and NF-M in transfected non-neuronal cells

Two cDNA clones fully encoding the rat neurofilament proteins NF-L and NF-M were subcloned into eukaryotic expression vectors behind the strong constitutive viral promoters from SV40 and Rous sarcoma viruses. Transient transfection of L tk- and Cos cell lines with these expression constructs resulted in cells expressing the neurofilament proteins in an intermediate filament-type pattern. Additionally, a putative juxtanuclear organizing center or region was observed in the transfected cells, most noticeable shortly after the transfection procedure. Stable transfections were performed on mouse L tk- and Swiss 3T6 cells using NF-L and NF-M constructs bearing an SV40 early promoter driven neomycin selectable marker. Although G418-resistant clones were recovered with both the NF-L and the NF-M constructs, only clones expressing immunofluorescently stainable amounts of NF-M were detected and established. Immunoelectron microscopic analysis revealed NF-M and vimentin proteins to be colocalized on the same intermediate filaments.