Rescue of Synthetic Genomic RNA Analogs of Rabies Virus by Plasmid-Encoded Proteins

Proteins eolirely expressed from cDNA wen used to rescue synthetic RNA genome analogs into infectious defective particles or rabies virus (RV). Synthetic negative-stranded RNAs coßtalning 3' · and S'-terminal RV sequences and tnlßscriptional signal sequences wen transcribed (rom plasmids transfeded into cells expressing 1'7 RNA polymerase (rom recombinant vaccinia virus. After simultaneous expression or RV N, P, and L proteiDS (rom plasmids containing a T7 RNA polymerase promoter, tbe synthetic genomes wen encapsidated. replicated, and transcribed by tbe RV polymerase proteiDS. Insertion or the bac1erial chloramphenicol acetyUransferase gene or l3·galactosidase (IacZ) gene between the 3 ' and 5 ' termini containing transcriptional signal sequenees resulted in transcription of mRNAs and expression of ehloramphenlco

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

Construction of three new Gateway® expression plasmids for Trypanosoma cruzi

We present here three expression plasmids for Trypanosoma cruzi adapted to the Gateway® recombination cloning system. Two of these plasmids were designed to express trypanosomal proteins fused to a double tag for tandem affinity purification (TAPtag). The TAPtag and Gateway® cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid. Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined by western blotting and immunofluorescence microscopy. The third Gateway adapted vector assayed was the inducible pTcINDEX. When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX).Fil: Alonso, Victoria Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmaceuticas. Departamento de Microbiología; ArgentinaFil: Ritagliati, Carla. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Cribb, Pamela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmaceuticas. Departamento de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Serra, Esteban Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; Argentin

Ebola virus VP35 induces high-level production of recombinant TPL-2–ABIN-2–NF-κB1 p105 complex in co-transfected HEK-293 cells

Activation of PKR (double-stranded-RNA-dependent protein kinase) by DNA plasmids decreases translation, and limits the amount of recombinant protein produced by transiently transfected HEK (human embryonic kidney)-293 cells. Co-expression with Ebola virus VP35 (virus protein 35), which blocked plasmid activation of PKR, substantially increased production of recombinant TPL-2 (tumour progression locus 2)–ABIN-2 [A20-binding inhibitor of NF-κB (nuclear factor κB) 2]–NF-κB1 p105 complex. VP35 also increased expression of other co-transfected proteins, suggesting that VP35 could be employed generally to boost recombinant protein production by HEK-293 cells

Inference of plasmid copy number mean and noise from single cell gene expression data

Plasmids are extra-chromosomal DNA molecules which code for their own replication. We previously reported a setup using genes coding for fluorescent proteins of two colors that allowed us, using a simple model, to extract the plasmid copy number noise in a monoclonal population of bacteria [J. Wong Ng et al., Phys. Rev. E, 81, 011909 (2010)]. Here we present a detailed calculation relating this noise to the measured levels of fluorescence, taking into account all sources of fluorescence fluctuations: the fluctuation of gene expression as in the simple model, but also the growth and division of bacteria, the non-uniform distribution of their ages, the random partition of proteins at divisions and the replication and partition of plasmids and chromosome. We show how using the chromosome as a reference helps extracting the plasmid copy number noise in a self-consistent manner.Comment: 9 pages, 3 figures, 2 table

Replication of plasmids derived from Shiga toxinconverting bacteriophages in starved Escherichia coli

The pathogenicity of Shiga toxin-producing Escherichia coli (STEC) depends on the expression of stx genes that are located on lambdoid prophages. Effective toxin production occurs only after prophage induction, and one may presume that replication of the phage genome is important for an increase in the dosage of stx genes, positively influencing their expression. We investigated the replication of plasmids derived from Shiga toxin (Stx)-converting bacteriophages in starved E. coli cells, as starvation conditions may be common in the intestine of infected humans. We found that, unlike plasmids derived from bacteriophage lambda, the Shiga toxin phage-derived replicons did not replicate in amino acid-starved relA+ and relA” cells (showing the stringent and relaxed responses to starvation, respectively). The presence of the stable fraction of the replication initiator O protein was detected in all tested replicons. However, while ppGpp, the stringent response effector, inhibited the activities of the l PR promoter and its homologues from Shiga toxin-converting bacteriophages, these promoters, except for lambda PR, were only weakly stimulated by the DksA protein. We suggest that this less efficient (relative to lambda) positive regulation of transcription responsible for transcriptional activation of the origin contributes to the inhibition of DNA replication initiation of Shiga toxin-converting bacteriophages in starved host cells, even in the absence of ppGpp (as in starved relA” hosts). Possible clinical implications of these results are discussed

The Alliance for Cellular Signaling Plasmid Collection: A Flexible Resource for Protein Localization Studies and Signaling Pathway Analysis

Cellular responses to inputs that vary both temporally and spatially are determined by complex relationships between the components of cell signaling networks. Analysis of these relationships requires access to a wide range of experimental reagents and techniques, including the ability to express the protein components of the model cells in a variety of contexts. As part of the Alliance for Cellular Signaling, we developed a robust method for cloning large numbers of signaling ORFs into Gateway® entry vectors, and we created a wide range of compatible expression platforms for proteomics applications. To date, we have generated over 3000 plasmids that are available to the scientific community via the American Type Culture Collection. We have established a website at www.signaling-gateway.org/data/plasmid/ that allows users to browse, search, and blast Alliance for Cellular Signaling plasmids. The collection primarily contains murine signaling ORFs with an emphasis on kinases and G protein signaling genes. Here we describe the cloning, databasing, and application of this proteomics resource for large scale subcellular localization screens in mammalian cell lines

Development of a technology for expression of recombinant human erythropoietin in cultured mammalian cells using alphavirus expression system

Currently there are no industrial eukaryotic expression systems other than transient expression from plasmids or expression from genes integrated into host genome. Both approaches (use of eukaryotic plasmids or chromosomal integration) suffer from poor scalability and often from poor yields. Although, in laboratory settings, effective means for transducing of cultured cells to express foreign proteins and for high-level transient expression were developed based on viral genomes. We thought to develop a scalable and suitable for industrial application technology for the production of recombinant human erythropoietin (EPO) in mammalian cell cultures using an expression vector based on the genome of RNA virus

Non-viral delivery and optimized optogenetic stimulation of retinal ganglion cells led to behavioral restoration of vision

Stimulation of retinal neurons using optogenetics via use of chanelrhodopsin-2 (ChR2) has opened up a new direction for restoration of vision for treatment of retinitis pigmentosa (RP). Here, we report non-viral in-vivo electroporation of degenerated retina of adult RP-mice with ChR2-plasmids and subsequent in-vivo imaging of retina to confirm expression. Further, we demonstrate that in addition to efficient non-viral delivery of ChR2 to a specific retinal layer, threshold level of stimulation light needs to be delivered onto the retina for achieving successful behavioral outcome. Measurement of intensity of light reaching the retina of RP-mouse models along with geometrical optics simulation of light propagation in the eye is reported in order to determine the stimulating source position for optimal light delivery to the retina. The light-guided navigation of mice with ChR2 expressing retinal ganglion cells was found to be significantly improved over a long distance in correlation with stimulation intensity