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.     

Modulating ectopic gene expression levels by using retroviral vectors equipped with synthetic promoters

The human cytomegalovirus and elongation factor 1?? promoters are constitutive promoters commonly employed by mammalian expression vectors. These promoters generally produce high levels of expression in many types of cells and tissues. To generate a library of synthetic promoters capable of generating a range of low, intermediate, and high expression levels, the TATA and CAAT box elements of these promoters were mutated. Other promoter variants were also generated by random mutagenesis. Evaluation using plasmid vectors integrated at a single site in the genome revealed that these various synthetic promoters were capable of expression levels spanning a 40-fold range. Retroviral vectors were equipped with the synthetic promoters and evaluated for their ability to reproduce the graded expression demonstrated by plasmid integration. A vector with a self-inactivating long terminal repeat could neither reproduce the full range of expression levels nor produce stable expression. Using a second vector design, the different synthetic promoters enabled stable expression over a broad range of expression levels in different cell lines.     

A Modular Lentiviral and Retroviral Construction System to Rapidly Generate Vectors for Gene Expression and Gene Knockdown In Vitro and In Vivo

The ability to express exogenous cDNAs while suppressing endogenous genes via RNAi represents an extremely powerful research tool with the most efficient non-transient approach being accomplished through stable viral vector integration. Unfortunately, since traditional restriction enzyme based methods for constructing such vectors are sequence dependent, their construction is often difficult and not amenable to mass production. Here we describe a non-sequence dependent Gateway recombination cloning system for the rapid production of novel lentiviral (pLEG) and retroviral (pREG) vectors. Using this system to recombine 3 or 4 modular plasmid components it is possible to generate viral vectors expressing cDNAs with or without inhibitory RNAs (shRNAmirs). In addition, we demonstrate a method to rapidly produce and triage novel shRNAmirs for use with this system. Once strong candidate shRNAmirs have been identified they may be linked together in tandem to knockdown expression of multiple targets simultaneously or to improve the knockdown of a single target. Here we demonstrate that these recombinant vectors are able to express cDNA and effectively knockdown protein expression using both cell culture and animal model systems.     

Expression cloning of oncogenes by retroviral transfer of cDNA libraries.

a cDNA library transfer system based on retroviral vectors has been developed for expression cloning in mammalian cells. The use of retroviral vectors results in stable cDNA transfer efficiencies which are at least 100-fold higher than those achieved by transfection and therefore enables the transfer and functional screening of very large libraries. In our initial application of retroviral transfer of cDNA libraries, we have selected for cDNAs which induce oncogenic transformation of NIH 3T3 fibroblasts, as measured by loss of contact inhibition of proliferation. Nineteen different transforming cDNAs were isolated from a total of 300,000 transferred cDNA clones. Three of these cDNAs were derived from known oncogenes (raf-1, lck, and ect2), while nine others were derived from genes which had been cloned previously but were not known to have the ability to transform fibroblasts (beta-catenin, thrombin receptor, phospholipase C-gamma 2 and Spi-2 protease inhibitor genes). The Spi-2 cDNA was expressed in antisense orientation and therefore is likely to act as an inhibitor of an endogenous transformation suppressor. Seven novel cDNAs with transforming activities, including those for three new members of the CDC24 family of guanine nucleotide exchange factors, were also cloned from the retroviral cDNA libraries. Retroviral transfer of libraries should be generally useful for cloning cDNAs which confer selectable phenotypes on many different types of mammalian cells.     

Heterologous protein production using euchromatin-containing expression vectors in mammalian cells

Upon stable cell line generation, chromosomal integration site of the vector DNA has a major impact on transgene expression. Here we apply an active gene environment, rather than specified genetic elements, in expression vectors used for random integration. We generated a set of Bacterial Artificial Chromosome (BAC) vectors with different open chromatin regions, promoters and gene regulatory elements and tested their impact on recombinant protein expression in CHO cells. We identified the Rosa26 BAC as the most efficient vector backbone showing a nine-fold increase in both polyclonal and clonal production of the human IgG-Fc. Clonal protein production was directly proportional to integrated vector copy numbers and remained stable during 10 weeks without selection pressure. Finally, we demonstrated the advantages of BAC-based vectors by producing two additional proteins, HIV-1 glycoprotein CN54gp140 and HIV-1 neutralizing PG9 antibody, in bioreactors and shake flasks reaching a production yield of 1 g/l.     

庚型肝炎病毒全长基因在体外的表达

利用第二军医大学微生物学教研室克隆的庚型肝炎病毒 (HGV)全长基因组 (HGVqz) ,构建由不同启动子调控的HGV表达载体 ,将 2种表达载体及HGV全长基因片段进行体外表达 ,探讨此HGV全长基因克隆的功能。利用脂质体将HGV全长基因cDNA以及表达载体转入Changliver或NIH 3T3细胞进行瞬时表达 ,分别提取转染72h后转染细胞的RNA及蛋白质进行RT PCR及Westernblotting ,以检测HGV基因的表达。RT PCR及Westernblotting结果表明 ,HGV全长基因cDNA以及 2种表达载体均可以在体外培养的细胞内表达 ,其表达产物为HGV前体蛋白 ,分子量约为 310ku。第二军医大学微生物学教研室克隆的HGV全长基因具有正确的开读框架和可表达性 ,能表达HGV前体蛋白 ,但在体外培养细胞内不能完成前体蛋白的剪切     

High levels of protein expression using different mammalian CMV promoters in several cell lines

With the recent completion of the human genome sequencing project, scientists are faced with the daunting challenge of deciphering the function of these newly found genes quickly and efficiently. Equally as important is to produce milligram quantities of the therapeutically relevant gene products as quickly as possible. Mammalian expression systems provide many advantages to aid in this task. Mammalian cell lines have the capacity for proper post-translational modifications including proper protein folding and glycosylation. In response to the needs described above, we investigated the protein expression levels driven by the human CMV in the presence or absence of intron A, the mouse and rat CMV promoters with intron A, and the MPSV promoter in plasmid expression vectors. We evaluated the different promoters using an in-house plasmid vector backbone. The protein expression levels of four genes of interest driven by these promoters were evaluated in HEK293EBNA and CHO-K1 cells. Stable and transient transfected cells were utilized. In general, the full-length human CMV, in the presence of intron A, gave the highest levels of protein expression in transient transfections in both cell lines. However, the MPSV promoter resulted in the highest levels of stable protein expression in CHO-K1 cells. Using the CMV driven constitutive promoters in the presence of intron A, we have been able to generate >10 microg/ml of recombinant protein using transient transfections.     

Short-hairpin RNA-mediated gene expression interference in Trichoplusia ni cells

RNA interference (RNAi) is rapidly becoming a valuable tool in biological studies, as it allows the selective and transient knockdown of protein expression. The short-interfering RNAs (siRNAs) transiently silence gene expression. By contrast, the expressed short-hairpin RNAs induce long-term, stable knockdown of their target gene. Trichoplusia ni (T. ni) cells are widely used for mammalian cell-derived glycoprotein expression using the baculovirus system. However, a suitable shRNA expression system has not been developed yet. We investigated the potency of shRNA-mediated gene expression inhibition using human and Drosophila U6 promoters in T. ni cells. Luciferase, EGFP, and beta-N-acetylglucosaminidase (GlcNAcase) were employed as targets to investigate knockdown of specific genes in T. ni cells. Introduction of the shRNA expression vector under the control of human U6 or Drosophila U6 promoter into T. ni cells exhibited the reduced level of luciferase, EGFP, and beta-N-acetylglucosaminidase compared with that of untransfected cells. The shRNA was expressed and processed to siRNA in our vector-transfected T. ni cells. GlcNAcase mRNA levels were down-regulated in T. ni cells transfected with shRNA vectors-targeted GlcNAcase as compared with the control vector-treated cells. It implied that our shRNA expression vectors using human and Drosophila U6 promoters were applied in T. ni cells for the specific gene knockdown.     

Recent patents on alphavirus protein expression and vector production

Alphaviruses contain a single-strand RNA genome that can be modified to express heterologous genes at high levels. Alphavirus vectors can be packaged within viral particles (VPs) or used as DNA/RNA layered systems. The broad tropism and high expression levels of alphavirus vectors have made them very attractive for applications like recombinant protein expression, vaccination or gene therapy. Expression mediated by alphavirus vectors is generally transient due to induction of apoptosis. However, during the last years several non-cytopathic mutations have been identified within the replicase sequence of different alphaviruses, allowing prolonged protein expression in culture cells. Some of these mutants, which have been patented, have allowed the generation of stable cell lines able to express recombinant proteins for extended periods of time in a constitutive or inducible manner. Production of alphavirus VPs usually requires cotransfection of cells with vector and helper RNAs providing viral structural proteins in trans. During this process full-length wild type (wt) genomes can be generated through recombination between different RNAs. Several new strategies to reduce wt virus generation during packaging, optimize VP production, increase packaging capacity, and provide VPs with specific targeting have been recently patented. Finally, hybrid vectors between alphavirus and other types of viruses have led to a number of patents with applications in vaccination, cancer therapy or retrovirus production.     

Combinatorial gene expression using multiple episomal vectors

Episomal vectors offer a powerful alternative to integrative recombination for transgene expression in mammalian cells. In this study, various combinations of G protein-coupled receptors (GPCRs) and the G protein subunit G(i2)alpha, were stably expressed from separate episomal vectors in 293-EBNA (293E) cells. Each episome did not adversely affect the others, as gauged by episomal copy number, steady-state mRNA levels and the presence of functional receptors and G protein. Cell lines expressing genes from multiple autonomously replicating vectors were stable just two weeks after transfection, and remained stable in continuous culture for at least 5months. Co-expression of supplementary G(i2)alpha with receptor amplifies the magnitude of signal transduction thereby permitting the development of more sensitive high throughput functional assays. Given these results, combinatorial transfection is the strategy of choice for generating stable cell lines expressing multiple genes for the study of signal-transduction pathways or the evaluation of receptor ligands.     

Eukaryotic Expression Vectors That Replicate to Low Copy Number in Bacteria: Transient Expression of the Menkes Protein

A set of low copy number plasmid vectors for mammalian gene expression has been constructed. These vectors are derived from the previously described bacterial low copy number expression vectors, pWSK29 and pWKS30, which are present at six to eight copies per cell. The new plasmids also have the following useful properties: (1) they contain antibiotic resistance markers for the selection of stable mammalian cell lines; (2) they have either constitutive or inducible promoters; (3) a chimeric intron, for enhancing gene expression, is present; (4) they contain unique cloning sites; (5) they have an SV40 polyadenylation signal, and a subset of the vectors have an SV40 origin of replication for episomal replication and transient gene expression. A cDNA encoding the Menkes disease protein was cloned into two of these vectors, and transient expression studies in COS-7 cells showed that both constitutive and inducible expression was possible. This set of expression vectors will provide a useful tool for the manipulation, inEscherichia coli,of mammalian genes or cDNAs that are unstable in the high copy number vectors that are currently available.     

A novel affinity gene fusion system allowing protein A-based recovery of non-immunoglobulin gene products

The capacity to produce large amounts of protein in mammalian cells is important in several contexts, including large-scale generation of biologically useful proteins, gene therapy, and transdominant genetics in cultured cells. For transdominant genetics, retroviral vectors are especially useful for delivery of expression libraries. However, even the potent CMV promoter is often unable to stimulate single-copy production of protein beyond the 1 microM level. We have adapted the HIV2/Tat expression system to retroviral vectors to boost expression above levels attainable with CMV promoters. We show that the system produces protein levels in four cell types tested which exceed levels attained by wild-type CMV or modified CMV promoters. In one cell line, the increase is 10-fold above CMV. Coupled with a stable expressed protein, levels of about 4 microM can be produced from presumptive single-copy retroviral transductants, and 30 microM from multicopy transductants.