pFAR plasmids: New Eukaryotic Expression Vectors for Gene Therapy, devoid of Antibiotic Resistance Markers

Efficient production of eukaryotic expression vectors requires the selection of plasmid-containing bacteria. To avoid the risk of dissemination of antibiotic resistance markers, we developed a new system to produce a family of plasmids Free of Antibiotic Resistance genes, called pFARs. The strategy is based on the suppression of a chromosomal nonsense mutation by a plasmid-borne function. The amber mutation was introduced into the Escherichia coli thyA gene that encodes a thymidylate synthase required for dTMP synthesis, resulting in thymidine auxotrophy. In parallel, a small plasmid vector that carries an amber suppressor t-RNA gene was entirely synthesised. The introduction of pFAR plasmids into an optimised thyA mutant restored normal growth to the auxotrophic strain, and led to an efficient production of monomeric supercoiled plasmids, as required for clinical trials. Luciferase activities measured after intramuscular injection and electrotransfer of LUC-encoding pFAR vector were similar to those obtained with a commercial vector containing the same expression cassette. Interestingly, whereas luciferase activities decreased within three weeks after intradermal electrotransfer of conventional expression vectors, sustained levels were observed with the pFAR derivative. Thus, pFAR plasmids represent a novel family of biosafe eukaryotic expression vectors, suitable for gene therapy

A Very Strong Enhancer Is Located Upstream of an Immediate Early Gene of Human Cytomegalovirus

A strong transcription enhancer was identified in the genomic DNA (235 kb) of human cytomegalovirus (HCMV), a ubiquitous and severe pathogen of the herpesvirus group. Cotransfection of enhancerless SV40 DNA with randomly fragmented HCMV DNA yielded two SV40-HCMV recombinant viruses that had incorporated overlapping segments of HCMV DNA to substitute for the missing SV40 enhancer. Within HCMV, these enhancer sequences are located upstream of the transcription initiation site of the major immediate-early gene, between nucleotides -118 and −524. Deletion studies with the HCMV enhancer, which harbors a variety of repeated sequence motifs, show that different subsets of this enhancer can substitute for the SV40 enhancer. The HCMV enhancer, which seems to have little cell type or species preference, is severalfold more active than the SV40 enhancer. It is the strongest enhancer we have analyzed so far, a property that makes it a useful component of eukaryotic expression vectors

Biosynthesis of peptide precursors and protease inhibitors using new constitutive and inducible eukaryotic expression vectors

AbstractA series of expression vectors has been constructed as based on the pML derivative of pBR322. The eukaryotic transcription units employ various promoters followed by polycloning sites for 3–9 commonly used restriction enzymes and are completed by the SV40 polyadenylation sequence. In 4 of the vectors, designed for co-transfection or transient expression studies, only a single transcription unit containing either a constitutive or an inducible promoter was incorporated. The human ubiquitin (UbC) promoter was used as a strong constitutive promoter, while the mouse metallothionein promoter and the promoter of the long terminal repeats of the mouse mammary tumor virus were used as inducible promoters. Another vector contained an additional transcription unit encoding a eukaryotic selection marker, the neomycin resistance encoding gene. The vectors were used in CHO cells and in neuroendocrine CA77 cells to synthesize peptide precursors, protease inhibitors and a protease. It is shown that these vectors are very efficient for the constitutive and inducible expression of nucleotide sequences in both transient and stable transfections of eukaryotic cells

ORFeome-based arrays in eukaryotic expression vectors - a new approach to screen for the function of viral proteins

Since its first description in 1994 by Yuan Chang and Patrick Moore, Kaposi’s sarcoma-associated herpesvirus (KSHV) or Human Herpesvirus 8 (HHV-8) has emerged as a pathogen of international public health importance. It has been detected in biopsies of all forms of Kaposi’s sarcoma (KS), irrespective of geographic origin, age, or gender of the patient. Moreover, KSHV has been shown to be associated with two other diseases, multicentric Castleman’s disease (MCD) and primary effusion lymphoma (PEL). In comparison to alpha and beta-herpesvirinae, the understanding of KSHV-related pathogenesis has been hampered by inefficient virus replication in vitro, poor cell culture systems and the lack of an animal model. Thus, many basic questions concerning the biology of KSHV infection remain open. For example, the primary target cell of KSHV and the function of more than 50% of the viral proteins are still unknown. Since the investigation of viral gene functions by virus mutants did not prove to be very efficient for KSHV, a system for a genome-wide screening of viral gene functions by cloning the complete KSHV ORFeome (all open reading frames) and by generating KSHV arrays in a variety of different expression vectors was established in this project. Very often viruses regulate cellular signalling pathways, which favour viral infection and replication in the host cells. The SRE is a transcription factor binding site present in promoters of many genes involved in cell growth and transformation. In this study, a genome-wide screen for KSHV genes inducing the SRE element and AP-1 was performed. A strong induction of SRE by the latency-associated nuclear antigen 1 (LANA-1) was observed. LANA-1 is a multifunctional protein which interacts with the p53 and RB tumor suppressor proteins. This study reveals several novel functions of LANA-1. LANA-1 led to an activation of the ERK-1/2 MAP kinase, but also bound to the Mediator, a multi-subunit transcriptional coactivator complex for RNA polymerase II, via the ARC92/ACID1 subunit. Since LANA-1 interacted with SRF, one of the two transcription factors binding to the bipartite SRE element, a model for LANA-1 as an adaptor between specific transcription factors and the basal transcriptional machinery was hypothesized

HIV-1 TAT-mediated protein transduction and subcellular localization using novel expression vectors11The nucleotide sequences of vectors pETAT-1/2/11/12, pNB-3/13, pHis-TAT-GFP, pHis-TAT-m-GFP and pHis-GFP have been deposited in GenBank under accession numbers AF525441–525449.

AbstractSeveral novel prokaryotic and eukaryotic expression vectors were constructed for protein transduction and subcellular localization. These vectors employed an N-terminal stretch of 11 basic amino acid residues (47–57) from the human immunodeficiency virus type 1 (HIV-1) TAT protein transduction domain (PTD) for protein translocation and cellular localization. The vectors also contained a six-histidine (His6) tag at the N- or C-terminus for convenient purification and detection, and a multiple cloning site for easy insertion of foreign genes. Some heterologous genes including HSV-TK, Bcl-rambo, Smac/DIABLO and GFP were fused in-frame to TAT PTD and successfully overexpressed in Escherichia coli. The purified TAT-GFP fusion protein was able to transduce into the mammalian cells and was found to locate mainly in the cytosol when exogenously added to the cell culture medium. However, using a transfection system, mammalian-expressed TAT-GFP predominantly displayed a nuclear localization and nucleolar accumulation in mammalian cell lines. This discrepancy implies that the exact subcellular localization of transduced protein may depend on cell type, the nature of imported proteins and delivery approach. Taken together, our results demonstrate that a TAT PTD length of 11 amino acids was sufficient to confer protein internalization and its subsequent cellular localization. These novel properties allow these vectors to be useful for studying protein transduction and nuclear import

<i>Salmonella</i> and Biotechnology

Salmonella strains have been actively studied as live carriers of heterologous antigens for a possible vaccine application. Especially, Salmonella Typhimurium, a facultative anaerobe, has been adapted as an antitumor agent capable of preferentially proliferating within tumors and inhibiting their growth. To enhance the cancer therapeutic efficacy of S. Typhimurium, combinations of gene-directed protein or microRNA therapies and auxotrophic strains of S. Typhimurium have been generated by genetic engineering. Until now, the idea of using bacteria including Salmonella in the treatments of cancer and other diseases has been considered a novel approach. Here, we describe this application based on Salmonella engineering for treatments of cancer or atopic dermatitis

DNA-dependent conversion of Oct-1 and Oct-2 into transcriptional repressors by Groucho/TLE

POU domain proteins contain a bipartite DNA-binding element that can confer allosteric control of coactivator recruitment. Dimerization of Oct-1 and Oct-2 on palindromic response elements results in the conformational dependent inclusion or exclusion of the transcriptional coactivator OBF-1. In this paper, we demonstrate that Oct-1 and Oct-2 can function as transcriptional repressors by recruiting and physically interacting with members of the Grg/TLE family of corepressors. In accordance with a model of DNA induced cofactor assembly, and analogous to the recruitment of the OBF-1 coactivator, the different Grg/TLE members can discriminate between both Oct-1 and Oct-2, and the monomeric or dimeric nature of the POU/DNA complex

A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei

Tissue-specific patterns of methylated deoxycytidine residues in the mammalian genome are preserved by postreplicative methylation of newly synthesized DNA. DNA methyltransferase (MTase) is here shown to associate with replication foci during S phase but to display a diffuse nucleoplasmic distribution in non-S phase cells. Analysis of DNA MTase-β-galactosidase fusion proteins has shown that association with replication foci is mediated by a novel targeting sequence located near the N-terminus of DNA MTase. This sequence has the properties expected of a targeting sequence in that it is not required for enzymatic activity, prevents proper targeting when deleted, and, when fused to β-galactosidase, causes the fusion protein to associate with replication foci in a cell cycle-dependent manner