High-titer bicistronic retroviral vectors employing foot-and-mouth disease virus internal ribosome entry site.

Bicistronic retroviral vectors were constructed containing the foot-and-mouth disease virus (FMDV) internal ribosome entry site (IRES) followed by the coding region of beta-galactosidase (beta-gal) or therapeutic genes, with the selectable neomycin phosphotransferase gene under the control of the viral long terminal repeat (LTR) promoter. LNFX, a vector with a multiple cloning site 3' to foot-and-mouth disease virus IRES, was used to construct vectors encoding rat erythropoietin (EP), rat granulocyte colony-stimulating factor (G-CSF), human adenosine deaminase (ADA) and beta-gal. In transduced primary rat vascular smooth muscle cells the cytokines were expressed at high levels, similar to those obtained from vectors employing the viral LTR promoter. LNFZ, a vector encoding beta-gal, had a 10-fold increase in titer over that of LNPoZ, a comparable vector containing the poliovirus (Po) internal ribosome entry site. Primary canine vascular smooth muscle cells infected with LNFZ and LNPoZ expressed similar activities of beta-gal and neomycin phosphotransferase (NPT). Overall, these vectors had titers between 10(6) and 2 x 10(7) c.f.u./ml, indicating that foot-and-mouth disease virus IRES provides high-titer bicistronic vectors with high-level two gene expression.     

Generation of mammalian cells stably expressing multiple genes at predetermined levels

Expression of cloned genes at desired levels in cultured mammalian cells is essential for studying protein function. Controlled levels of expression have been difficult to achieve, especially for cell lines with low transfection efficiency or when expression of multiple genes is required. An internal ribosomal entry site (IRES) has been incorporated into many types of expression vectors to allow simultaneous expression of two genes. However, there has been no systematic quantitative analysis of expression levels in individual cells of genes linked by an IRES, and thus the broad use of these vectors in functional analysis has been limited. We constructed a set of retroviral expression vectors containing an IRES followed by a quantitative selectable marker such as green fluorescent protein (GFP) or truncated cell surface proteins CD2 or CD4. The gene of interest is placed in a multiple cloning site 5' of the IRES sequence under the control of the retroviral long terminal repeat (LTR) promoter. These vectors exploit the approximately 100-fold differences in levels of expression of a retrovirus vector depending on its site of insertion in the host chromosome. We show that the level of expression of the gene downstream of the IRES and the expression level and functional activity of the gene cloned upstream of the IRES are highly correlated in stably infected target cells. This feature makes our vectors extremely useful for the rapid generation of stably transfected cell populations or clonal cell lines expressing specific amounts of a desired protein simply by fluorescent activated cell sorting (FACS) based on the level of expression of the gene downstream of the IRES. We show how these vectors can be used to generate cells expressing high levels of the erythropoietin receptor (EpoR) or a dominant negative Smad3 protein and to generate cells expressing two different cloned proteins, Ski and Smad4. Correlation of a biologic effect with the level of expression of the protein downstream of the IRES provides strong evidence for the function of the protein placed upstream of the IRES.     

High-throughput gateway bicistronic retroviral vectors for stable expression in mammalian cells: exploring the biologic effects of STAT5 overexpression

Stable expression of cloned genes in mammalian cells has been achieved in the past by retroviral transduction using bicistronic retroviral vectors. In these vectors, the use of an Internal Ribosome Entry Site (IRES) allows simultaneous expression of a protein of interest and a fluorescence marker. However, traditional cDNA cloning in these vectors is often difficult. Here we report the construction of a high-throughput retroviral vector using the Invitrogen "Gateway" Cloning system. The Gateway recombination sequences (attR) flanking the ccdB and chloramphenicol resistance genes were incorporated at the 5' of the IRES of pMX-IRES-GFP, -CD2, or -CD4 vectors. Through recombination, these vectors can acquire cDNAs coding for genes of interest, which will result in simultaneous expression of the recombined gene and the marker protein. We constructed Gateway bicistronic vectors coding for the erythropoietin receptor (EpoR) and GFP, CD4, or CD2. Epo-dependent proliferation assays and analysis of Jak2-dependent EpoR cell-surface expression showed that these vectors were able to function indistinguishable from the original pMX-EpoR-IRES-GFP. The expression levels of the genes cloned upstream the IRES were proportional to the levels of expression of GFP, which was cloned downstream of the IRES. We used the same approach and generated Ba/F3 cells that overexpress STAT5a, STAT5b, or a constitutively active form of STAT5. Overexpression of STAT5 lead to a significant effect on the intrinsic adherence to plastic of these cells, but did not change their proliferative responses to cytokines. We discuss possible applications of the new vectors for cell signaling and expression cloning.     

The uptake and expression of the factor VIII and reporter genes by vascular cells.

The conditions and efficacy of transfection of vascular cells in primary culture using DEAE-dextran, calcium phosphate and lipofectin have been investigated using chloramphenicol acetyltransferase and luciferase as reporter genes. Subsequently factor VIII was expressed in endothelial and smooth muscle cells. Both reporter genes could be expressed after transfection of umbilical vein endothelial cells, umbilical artery smooth muscle cells and fibroblasts. The expression of both reporter genes in endothelial and smooth muscle cells was highest using lipofectin. After transfection of smooth muscle cells with both full-length and mutant factor VIII genes, factor VIII activity and antigen were secreted into the culture medium, the secretion remaining stable to serial cell passage. The secretion of factor VIII from transfected smooth muscle cells was confirmed by the immunoprecipitation of [35S]methionine labelled protein. Endothelial cells also were successfully transfected with the mutant factor VIII gene.     

pLR: A lentiviral backbone series to stable transduction of bicistronic genes and exchange of promoters

Gene transfer based on lentiviral vectors allow the integration of exogenous genes into the genome of a target cell, turning these vectors into one of the most used methods for stable transgene expression in mammalian cells, in vitro and in vivo. Currently, there are no lentivectors that allow the cloning of different genes to be regulated by different promoters. Also, there are none that permit the analysis of the expression through an IRES (internal ribosome entry site) - reporter gene system. In this work, we have generated a series of lentivectors containing: (1) a malleable structure to allow the cloning of different target genes in a multicloning site (mcs); (2) unique site to exchange promoters, and (3) IRES followed by one of two reporter genes: eGFP or DsRed. The series of the produced vectors were named pLR (for lentivirus and RSV promoter) and were fairly efficient with a strong fluorescence of the reporter genes in direct transfection and viral transduction experiments. This being said, the pLR series have been found to be powerful biotechnological tools for stable gene transfer and expression.     

Retroviral vectors containing putative internal ribosome entry sites: development of a polycistronic gene transfer system and applications to human gene therapy.

Recombinant retroviral vectors producing multicistronic mRNAs were constructed. Picornavirus putative internal ribosome entry sites (IRES) were used to confer cap-independent translation of an internal cistron. Internal cistrons were engineered by ligation of various lengths of the IRES of encephalomyocarditis (EMC) virus or polio virus to the E. coli chloramphenicol acetyltransferase (CAT) gene. The IRES/CAT fusions were introduced into retroviral vectors 3' to the translation stop codon of the neomycin phosphotransferase (NEO) gene, and the molecular constructs transfected into retroviral vector packaging lines. Retroviral vector producer cells efficiently express the internal CAT gene product only when the full length IRES is used. Both the EMC/CAT and polio/CAT retroviral vectors produced high titer vector supernatant capable of productive transduction of target cells. To test the generality of this gene transfer system, a retroviral vector containing an IRES fusion to the human adenosine deaminase (ADA) gene was constructed. Producer cell supernatant was used to transduce NIH/3T3 cells, and transduced cells were shown to express NEO, and ADA. Novel three-gene-containing retroviral vectors were constructed by introducing the EMC/ADA fusion into either an existing internal-promoter-containing vector, or a polio/CAT bicistronic vector. Producer cell clones of the three-gene vectors synthesize all three gene products, were of high titer, and could productively transduce NIH/3T3 cells. By utilizing cap-independent translation units, IRES vectors can produce polycistronic mRNAs which enhance the ability of retroviral-mediated gene transfer to engineer cells to produce multiple foreign proteins.     

Optimization of in vitro vascular cell transfection with non-viral vectors for in vivo applications

BACKGROUND: Syngeneic vascular cells are interesting tools for indirect gene therapy in the cardiovascular system. This study aims to optimize transfection conditions of primary cultures of vascular smooth muscle cells (VSMCs) using different non-viral vectors and zinc as an adjuvant and to implant these transfected cells in vivo. METHODS: Non-liposomal cationic vectors (FuGene 6), polyethylenimines (ExGen 500), and histidylated polylysine (HPL) were used as non-viral vectors in vitro with secreted alkaline phosphatase (SEAP) as reporter gene. Transfection efficiency was compared in cultured rat, rabbit and human VSMCs and fibroblasts. Zinc chloride (ZnCl2) was added to optimize transfection of rat VSMCs in vitro which were then seeded in vivo. RESULTS: Much higher SEAP levels were obtained in rabbit cells with FuGene 6 (p <0.0001) at day 2 than in equivalent rat and human cells. Rat VSMCs transfected in vitro with FuGene 6 and ExGen 500 expressed higher SEAP levels than with HPL. In rat VSMCs, SEAP secretion was more than doubled by addition of 250 microM ZnCl2 (p <0.0001) for all vectors. Seeding of syngeneic VSMCs transfected under optimized conditions (FuGene 6/pcDNA3-SEAP +250 microM ZnCl2) into healthy Lewis rats using various routes or into post-infarct myocardial scar resulted in a peak of SEAP expression at day 2 and detectable activity in the plasma for at least 8 days. CONCLUSIONS: FuGene 6 is an efficient non-viral transfection reagent for gene transfer in somatic smooth muscle cells in vitro and ZnCl2 enhances its efficiency. This increased expression of the transgene product is maintained after seeding in vivo.     

Construction of a bicistronic vector for the co-expression of two genes in Caenorhabditis elegans using a newly identified IRES

The nematode Caenorhabditis elegans is an important model animal for biological research. Currently, transgenic C. elegans strains are mainly generated by injecting DNA encoding a gene of interest, in combination with a reporter gene, into the gonad. With this approach, the interpretation of negative results, such as the failure to observe reporter expression, is frequently required. Single, selectable vectors are urgently required. Internal ribosome entry site (IRES) elements are known to bind the eukaryotic ribosomal translation initiation complex and independently promote translation initiation. Bioinformatic analysis predicted an IRES motif upstream of the start codon of the C. elegans Hsp-3 gene. While this sequence has a Y-shaped double-hairpin secondary structure characteristic of IRES elements, it was unclear if it could function as an IRES. In the present study, this predicted Hsp-3 IRES was incorporated into a bicistronic vector driven by the myo-3 promoter, which allowed co-expression of RFP and GFP genes in the muscle tissue of C. elegans and thereby demonstrated that this IRES element is functional. This vector provides a novel, powerful tool for C. elegans research.     

Construction of Retroviral Vectors with Improved Safety, Gene Expression, and Versatility

Murine leukemia virus (MLV)-based retroviral vectors are the most frequently used gene delivery vehicles. However, the current vectors are still not fully optimized for gene expression and viral titer, and many genetic and biochemical features of MLV-based vectors are poorly understood. We have previously reported that the retroviral vector MFG, where the gene of interest is expressed as a spliced mRNA, is superior in the level of gene expression with respect to other vectors compared in the study. As one approach to developing improved retroviral vectors, we have systematically performed mutational analysis of the MFG retroviral vector. We demonstrated that the entire gag coding sequence, together with the immediate upstream region, could be deleted without significantly affecting viral packaging or gene expression. To our knowledge, this region is included in all currently available retroviral vectors. In addition, almost the entire U3 region could be replaced with the heterologous human cytomegalovirus immediately-early promoter without deleterious effects. We could also insert internal ribosome entry sites (IRES) and multicloning sites into MFG without adverse effects. Based on these observations, we have constructed a series of new, improved retroviral constructs. These vectors produced viral titers comparable to MFG, expressed high levels of gene expression, and stably transferred genes to the target cells. Our vectors are more convenient to use because of the presence of multicloning sites and IRESs, and they are also more versatile because they can be readily converted to various applications. Our results have general implications regarding the design and development of improved retroviral vectors for gene therapy.     

Picornavirus internal ribosome entry site elements can stimulate translation of upstream genes

Certain viral and cellular mRNAs initiate translation cap-independently at internal ribosome entry site (IRES) elements. Picornavirus IRES elements are widely used in dicistronic or multicistronic vectors in gene therapy, virus replicon systems, and analysis of IRES function. In such vectors, expression of the upstream gene often serves as internal control to standardize the readings of IRES-driven downstream reporter activity. Picornaviral IRES elements translate optimally at up to 120 mM K(+) concentration, whereas genes used as upstream reporters usually have lower salt optima when present in monocistronic mRNAs. However, here we show that such reporter genes are efficiently translated at higher K(+) concentrations when placed upstream of a functional picornavirus IRES. This translation enhancement occurs in cis, is independent of the nature of the first reporter and of second reporter translation, and is conferred by the IRESs of picornaviruses but not of hepatitis C virus. A defective picornavirus IRES with a deletion killing IRES activity but leaving the binding site for initiation factor eIF4G intact retains translation enhancement activity. Translation enhancement on a capped mRNA is disabled by m(7)GDP. In addition, the C-terminal fragment of eIF4G can confer translation enhancement also on uncapped mRNA. We conclude that whenever eIF4F has been captured to a dicistronic mRNA by binding to a picornavirus IRES via its eIF4G moiety, it can be provided in cis to the 5'-end of the RNA and there stimulate translation initiation, either by binding to the cap nucleotide using its eIF4E moiety or by binding to the RNA cap-independently using its eIF4G moiety.     

SM22alpha promoter targets gene expression to vascular smooth muscle cells in vitro and in vivo

BACKGROUND: Gene transfer into vascular smooth muscle cells (vsmcs) holds promise for studying the pathogenesis of arterial disorders. However, a potential limitation of vectors with heterologous promoters is organ toxicity resulting from unrestricted transgene expression. Vascular smooth muscle cell-specific gene expression could increase the safety of vectors for vascular diseases. MATERIALS AND METHODS: To develop vectors that target gene expression to vsmcs, we constructed vectors encoding human placental alkaline phosphatase (hpAP) and chloramphenicol transferase (CAT) driven by a 441-bp region of the murine SM22alpha promoter (AdSM22alpha-hpAP). RESULTS: Transfection of AdSM22alpha-hpAP into vascular and nonvascular cells resulted in the expression of alkaline phosphatase (AP) in primary arterial and venous smcs, but not in primary endothelial cells or National Institutes of Health (NIH) 3T3 cells. Expression of AP was observed on 32.5 +/- 1.4% of primary pig vsmcs-infected AdSM22alpha-hpAP at a multiplicity of infection (MOI) of 500; whereas, infection with AdCMV-hpAP resulted in 100 +/- 0.0% expression at a MOI of 250. In vitro, expression from the heterologous cytomegalovirus (CMV) promoter was approximately 10(3)-fold higher in vsmcs, compared with the SM22alpha promoter. Following introduction of AdSM22alpha-hpAP vectors into balloon-injured pig arteries, AP recombinant protein was detected in neointimal (2.23 +/- 1.14%) and medial (0.56 +/- 0.21%) smcs, but not in endothelial or adventitial cells. In contrast, AdCMV-hpAP vectors led to AP expression in intimal endothelial and smcs cells (39.14 +/- 10.09%) and medial smcs (2.84 +/- 1.05%). AP expression was not observed in endothelial or vsmcs following transfection with the control vector, adenoviral vector lacking E1 (AddeltaE1). CONCLUSIONS: The SM22alpha promoter programs recombinant gene expression exclusively to vascular smcs in vitro and in vivo. Although expression levels are lower than with heterologous promoters, these vectors may provide a safe and effective tool for gene therapy of vascular diseases.     

Transient gene expression mediated by integrase-defective retroviral vectors

Nonintegrating retroviral vectors were produced from a Moloney murine leukemia virus (MoMLV)-based retroviral vector system by introducing a point mutation into the integrase (IN) gene of the packaging plasmid. The efficacy of IN-defective retroviral vectors was measured through the transient expression of ZsGreen or luciferase in human cell lines. The IN-defective retroviral vectors could transduce target cells efficiently, but their gene expression was transient and lower than that seen with the integrating vectors. IN-defective retroviral vector gene expression decreased to background levels in fewer than 10 days. Southern blot analysis of transduced K562 cells confirmed the loss of a detectable vector sequence by 15 days. The residual integration activity of the IN-defective vector was 1000- to 10,000-fold lower than that of the integrating vector. These results demonstrate that the IN-defective retroviral vectors can provide a useful tool for efficient transient gene expression targeting of primary hematopoietic stem cells and lymphoid cells.     

Translational efficiency of BVDV IRES and EMCV IRES for T7 RNA polymerase driven cytoplasmic expression in mammalian cell lines

Mammalian T7 polymerase-based cytoplasmic expression systems are common tool for molecular studies. The majority of these systems include the internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). To carry out a cap-independent translation process, this type of IRES might require the expression of an extensive array of host factors, what is a disadvantage. Other IRESes might be less dependent on the host cell factors, but their biology is characterized to a lesser degree. Here, we compare the translational efficiencies of bovine viral diarrhea virus (BVDV) IRES with that of ECMV. Both IRESes were tested in reporter vectors containing the T7 promoter, an IRES of choice and the coding sequence of the enhanced green fluorescent protein (EGFP). To provide for the expression of T7 RNA polymerase, the corresponding gene was isolated from Escherichia coli and inserted into pCDNA3.1-Hygro(+). After co-transfection of the T7 RNA polymerase encoding vector with either of the two IRES-containing reporter vectors into T7 baby hamster kidney (T7-BHK), human embryonic kidney (HEK) 293T, chinese hamster ovary (CHO) and HeLa cells, the translational efficiency of the reporter construct was studied by fluorescence microscopy and flow cytometry. In T7-BHK, HEK 293T and HeLa cells the translational efficiency of BVDV IRES was two to three times higher than that of EMCV IRES. In CHO cells, BVDV IRES and EMCV IRES were equally efficient. An analysis of the secondary structure of respective mRNAs showed that their ΔG values were–544.00 and–469.40 kcal/mol for EMCV IRES and BVDV IRES harboring molecules, respectively. As EMCV IRES-containing mRNA is more stable, it is evident that other, still unidentified factors should be held responsible for the enhanced translational efficiency of BDVD IRES. Taken together, our results indicate the potential of BVDV IRES as a replacement for EMCV IRES, which is now commonly used for T7 polymerase driven cytoplasmic expression of genes of interest or virus cDNA rescue experiments.