Plasmid Vector System Research Articles

Rational design of unrestricted pRN1 derivatives and their application in the construction of a dual plasmid vector system for Saccharolobus islandicus.

Saccharolobus islandicus REY15A represents one of the very few archaeal models with versatile genetic tools, which include efficient genome editing, gene silencing, and robust protein expression systems. However, plasmid vectors constructed for this crenarchaeon thus far are based solely on the pRN2 cryptic plasmid. Although this plasmid coexists with pRN1 in its original host, early attempts to test pRN1-based vectors consistently failed to yield any stable host-vector system for Sa. islandicus. We hypothesized that this failure could be due to the occurrence of CRISPR immunity against pRN1 in this archaeon. We identified a putative target sequence in orf904 encoding a putative replicase on pRN1 (target N1). Mutated targets (N1a, N1b, and N1c) were then designed and tested for their capability to escape the host CRISPR immunity by using a plasmid interference assay. The results revealed that the original target triggered CRISPR immunity in this archaeon, whereas all three mutated targets did not, indicating that all the designed target mutations evaded host immunity. These mutated targets were then incorporated into orf904 individually, yielding corresponding mutated pRN1 backbones with which shuttle plasmids were constructed (pN1aSD, pN1bSD, and pN1cSD). Sa. islandicus transformation revealed that pN1aSD and pN1bSD were functional shuttle vectors, but pN1cSD lost the capability for replication. These results indicate that the missense mutations in the conserved helicase domain in pN1c inactivated the replicase. We further showed that pRN1-based and pRN2-based vectors were stably maintained in the archaeal cells either alone or in combination, and this yielded a dual plasmid system for genetic study with this important archaeal model.

Review of knockout technology approaches in bacterial drug resistance research.

Gene knockout is a widely used method in biology for investigating gene function. Several technologies are available for gene knockout, including zinc-finger nuclease technology (ZFN), suicide plasmid vector systems, transcription activator-like effector protein nuclease technology (TALEN), Red homologous recombination technology, CRISPR/Cas, and others. Of these, Red homologous recombination technology, CRISPR/Cas9 technology, and suicide plasmid vector systems have been the most extensively used for knocking out bacterial drug resistance genes. These three technologies have been shown to yield significant results in researching bacterial gene functions in numerous studies. This study provides an overview of current gene knockout methods that are effective for genetic drug resistance testing in bacteria. The study aims to serve as a reference for selecting appropriate techniques.

Transgene Expression and Transposition Efficiency of Two-Component Sleeping Beauty Transposon Vector Systems Utilizing Plasmid or mRNA Encoding the Transposase

The use of two-component transposon plasmid vector systems, namely, a transposase construct and a donor vector carrying the gene of interest (GOI) can accelerate the development of recombinant cell lines. However, the undesired stable transfection of the transposase construct and the sustained expression of the enzyme can cause genetic instability due to the re-mobilization of the previously transposed donor vectors. Using a Sleeping Beauty-derived vector system, we established three recombinant cell pools and demonstrate stable integration of the transposase construct and sustained expression of the transposase over a period of 48 days. To provide an alternative approach, transcripts of the transposase gene were generated in vitro and co-transfected with donor vector plasmid at different ratios and mediating high GOI copy number integrations and expression levels. We anticipate that the use of transposase mRNA will foster further improvements in future cell line development processes.

Mapping the Domain Structure and Aggregation Propensity of Proteins Using a Gateway Plasmid Vector System.

Some proteins represent members of conserved families, meaning that their domain structure can be easily predicted by comparison to homologous proteins whose structures have been solved experimentally. Many other proteins, however, do not share significant detectable homology with other proteins, often as results of high amounts of coiled-coil structure and/or intrinsically unstructured regions. These proteins include many whose aggregation is linked to human disease.Here we present a refined and reliable workflow for identifying the domains of such proteins, through cloning of multiple alternative fragments, and testing whether they form soluble, folded structures when expressed as recombinant peptides in E. coli, through the use of size exclusion chromatography. By using Gateway recombination for cloning, these fragments can then be rapidly transferred to alternate vectors for testing in mammalian cells. We then specifically illustrate its use for proteins that form pathological aggregates in disease, mapping not just their basic domain structures but also the specific subdomains responsible for aggregation.

Production of neoagarooligosaccharides by probiotic yeast Saccharomyces cerevisiae var. boulardii engineered as a microbial cell factory

BackgroundSaccharomyces cerevisiae var. boulardii is a representative probiotic yeast that has been widely used in the food and pharmaceutical industries. However, S. boulardii has not been studied as a microbial cell factory for producing useful substances. Agarose, a major component of red macroalgae, can be depolymerized into neoagarooligosaccharides (NAOSs) by an endo-type β-agarase. NAOSs, including neoagarotetraose (NeoDP4), are known to be health-benefiting substances owing to their prebiotic effect. Thus, NAOS production in the gut is required. In this study, the probiotic yeast S. boulardii was engineered to produce NAOSs by expressing an endo-type β-agarase, BpGH16A, derived from a human gut bacterium Bacteroides plebeius.ResultsIn total, four different signal peptides were compared in S. boulardii for protein (BpGH16A) secretion for the first time. The SED1 signal peptide derived from Saccharomyces cerevisiae was selected as optimal for extracellular production of NeoDP4 from agarose. Expression of BpGH16A was performed in two ways using the plasmid vector system and the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system. The production of NeoDP4 by engineered S. boulardii was verified and quantified. NeoDP4 was produced by S. boulardii engineered using the plasmid vector system and CRISPR-Cas9 at 1.86 and 0.80 g/L in a 72-h fermentation, respectively.ConclusionsThis is the first report on NAOS production using the probiotic yeast S. boulardii. Our results suggest that S. boulardii can be considered a microbial cell factory to produce health-beneficial substances in the human gut.Graphical abstract

Design and Evaluation of AgoshRNAs with 3'-Terminal HDV Ribozymes to Enhance the Silencing Activity.

Since the first application of RNA interference (RNAi) in mammalian cells, the expression of short hairpin RNA (shRNA) molecules for targeted gene silencing has become a benchmark technology. Plasmid and viral vector systems can be used to express shRNA precursor transcripts that are processed by the cellular RNAi pathway to trigger sequence-specific gene knockdown. Intensive RNAi investigations documented that only a small percentage of computationally predicted target sequences can be used for efficient gene silencing, in part because not all shRNA designs are active. Many factors influence the shRNA activity and guidelines for optimal shRNA design have been proposed. We recently described an alternatively processed shRNA molecule termed AgoshRNA with a ~18base pairs (bp) stem and a 3-5 nucleotides(nt) loop. This molecule is alternatively processed by the Argonaute (Ago) protein into a single guide RNA strand that efficiently induces the RNAi mechanism. The design rules proposed for regular shRNAs do not apply to AgoshRNA molecules and therefore new rules had to be defined. We optimized the AgoshRNA design and managed to create a set of active AgoshRNAs targeted against the human immunodeficiency virus (HIV). In an attempt to enhance the silencing activity of the AgoshRNA molecules, we included the hepatitis delta virus (HDV) ribozyme at the 3' terminus, which generates a uniform 3' end instead of a 3' U-tail of variable length. We evaluated the impact of this 3'-end modification on AgoshRNA processing and its gene silencing activity and we demonstrate that this novel AgoshRNA-HDV design exhibits enhanced antiviral activity.

Establishment of plasmid vector and allelic exchange mutagenesis systems in a mycobacterial strain that is able to degrade polycyclic aromatic hydrocarbon.

Plasmid vector and allelic exchange mutagenesis systems were established for the genetic analysis of a phenanthrene-degrading mycobacterial strain, Mycobacterium sp. EPa45. Successful application of these systems revealed the necessity of the EPa45 phdI gene for the degradation of 1-hydroxy-2-naphthoate, which has been proposed to be an intermediate product in the degradation pathway of phenanthrene.

Historical Events That Spawned the Field of Plasmid Biology.

This chapter revisits the historical development and outcome of studies focused on the transmissible, extrachromosomal genetic elements called plasmids. Early work on plasmids involved structural and genetic mapping of these molecules, followed by the development of an understanding of how plasmids replicate and segregate during cell division. The intriguing property of plasmid transmission between bacteria and between bacteria and higher cells has received considerable attention. The utilitarian aspects of plasmids are described, including examples of various plasmid vector systems. This chapter also discusses the functional attributes of plasmids needed for their persistence and survival in nature and in man-made environments. The term plasmid biology was first conceived at the Fallen Leaf Lake Conference on Promiscuous Plasmids, 1990, Lake Tahoe, California. The International Society for Plasmid Biology was established in 2004 (www.ISPB.org).

Gateway®-compatible plant transformation vectors.

Studies in functional genomics and crop improvement programs often rely on the introduction and expression of transgenes in plants. There are two essential components required for in planta transgene expression, a plasmid vector on which the transgene sequence is carried and a delivery system capable of transferring the vector to the target cells. Agrobacterium-mediated plant transformation and the binary plasmid vector system is the preferred method of transgene delivery. The cloning technologies used for DNA manipulation underpin many of these studies. Increased demand for efficient high-throughput transformation systems is driving forward improvements in gene cloning techniques. This chapter gives an overview of Gateway(®)-compatible binary vectors for use in Agrobacterium-mediated transformation systems. It describes a fast, efficient, and robust cloning protocol for the production of an over-expression binary vector using Gateway(®) recombinational cloning.

A Mini-intronic Plasmid (MIP): A Novel Robust Transgene Expression Vector In Vivo and In Vitro

The bacterial backbone (BB) sequences contained within a canonical plasmid DNA dampen exogenous transgene expression by tenfold to 1,000-fold over a period of a few weeks following transfection into quiescent tissues such as the liver. Minicircle DNA vectors devoid of bacterial plasmid backbone sequences overcome transgene silencing providing persistent transgene expression. Because, we recently established that the length rather than sequence of the DNA flanking the transgene expression cassette is the major parameter affecting transgene silencing, we developed an alternative plasmid propagation process in which the essential bacterial elements for plasmid replication and selection are placed within an engineered intron contained within the eukaryotic expression cassette. As with the minicircle vector, the mini-intronic plasmid (MIP) vector system overcomes transgene silencing observed with plasmids but in addition provides between 2 and often 10 times or higher levels of transgene expression compared with minicircle vectors containing the same expression cassette in vivo and in vitro. These improved plasmids will benefit all studies involving gene transfer/therapy approaches.

Development of Transgenic Papaya throughAgrobacterium-Mediated Transformation

Transgenic papaya plants were regenerated from hypocotyls and immature zygotic embryo after cocultivation with Agrobacterium tumefaciens LBA-4404 carrying a binary plasmid vector system containing neomycin phosphotransferase (nptII) gene as the selectable marker and β-glucuronidase (GUS) as the reporter gene. The explants were co-cultivated with Agrobacterium tumefaciens on regeneration medium containing 500 mg/L carbenicillin + 200 mg/L cefotaxime for one week. The cocultivated explants were transferred into the final selection medium containing 500 mg/L carbenicillin + 200 mg/L cefotaxime + 50 mg/L kanamycin for callus induction as well as plant regeneration. The callus derived from the hypocotyls of Carica papaya cv. Shahi showed the highest positive GUS activities compared to Carica papaya cv. Ranchi. The transformed callus grew vigorously and formed embryos followed by transgenic plantlets successfully. The result of this study showed that the hypocotyls of C. papaya cv. Shahi and C. papaya cv. Ranchi are better explants for genetic transformation compared to immature embryos. The transformed C. papaya cv. Shahi also showed the maximum number of plant regeneration compared to that of C. papaya cv. Ranchi.

Design and characterization of a plasmid vector system capable of rapid generation of antibodies of multiple isotypes and specificities

We present a detailed method for constructing a vector system composed of plasmids encoding immunoglobulin genes that have been constructed in such a fashion so as to allow for the generation and secretion of antibodies of multiple specificities and isotypes via a rapid and easy cloning-and-ligation scheme. Restriction sites within each plasmid allow for the removal of variable domains, constant domains, leader sequences, or the entire immunoglobulin gene. Degenerate primers are used to clone variable regions from hybridoma cDNAs, allowing for the creation of antibodies with varying binding specificities. Sequence-specific primers are used to clone antibody constant domains, allowing for the creation of antibodies of multiple isotypes from a variety of lineages. A high-efficiency leader sequence has been inserted into the start of each gene to improve secretion. Antibodies constructed through this system are efficiently secreted by transfected cells, and are fully functional in antigen-specific binding assays.

Contribution ofrpoB2RNA Polymerase β Subunit Gene to Rifampin Resistance inNocardiaSpecies

Nocardia species are gram-positive environmental saprophytes, but some cause the infectious disease nocardiosis. The complete genomic sequence of Nocardia farcinica IFM 10152 has been determined, and analyses indicated the presence of two different RNA polymerase beta subunit genes, rpoB and rpoB2, in the genome (J. Ishikawa, A. Yamashita, Y. Mikami, Y. Hoshino, H. Kurita, K. Hotta, T. Shiba, and M. Hattori, Proc. Natl. Acad. Sci. USA 101:14925-14930, 2004). These genes share 88.8% identity at the nucleotide level. Moreover, comparison of their amino acid sequences with those of other bacterial RpoB proteins suggested that the nocardial RpoB protein is likely to be rifampin (RIF) sensitive, whereas RpoB2 protein contains substitutions at the RIF-binding region that are likely to confer RIF resistance. Southern analysis indicated that rpoB duplication is widespread in Nocardia species and is correlated with the RIF-resistant phenotype. The introduction of rpoB2 by using a newly developed Nocardia-Escherichia coli shuttle plasmid vector and transformation system conferred RIF resistance to Nocardia asteroides IFM 0319T, which has neither RIF resistance nor rpoB duplication. Furthermore, unmarked rpoB2 deletion mutants of N. farcinica IFM 10152 showed no significant resistance to RIF. These results indicated the contribution of rpoB2 to RIF resistance in Nocardia species. Since this is the first example of genetic engineering of the Nocardia genome, we believe that this study, as well as our determination of the N. farcinica genome sequence, will be a landmark in Nocardia genetics.

Electrogene transfer of an Epstein–Barr virus‐based plasmid replicon vector containing the diphtheria toxin A gene suppresses mammary carcinoma growth in SCID mice

Experimental mammary cancer therapy in mice was conducted using electrogene transfer of a non-viral EBV-based plasmid vector (reduced size of the oriP gene), containing the DT-A gene. Because the EBV-based plasmid vector exhibits high transfer efficiency and strong persistent transgene expression due to autonomous replication in human cells, it is particularly suitable as a tool for cancer gene therapy. In vitro, 79% of MDA-MB231 human mammary carcinoma cells died as a result of the EBV-based vector containing DT-A (pEB-DTA) by 48 h after transfection. DNA synthesis was also significantly decreased as compared to levels with a control vector. In vivo, mammary tumors induced by inoculation of SCID mice with MDA-MB231 cells were subsequently treated by direct injection of pEB-DTA vector or pEB-GFP vector as a control once a week for 5 weeks. After each injection, the tumors were subjected to in vivo electrogene transfer. Significantly reduced tumor volumes were observed for the pEB-DTA group in experimental week 1 and thereafter throughout the study. At necropsy, strong and extent expression of GFP was still observed in tumors receiving pEB-GFP 6 days after the last electrogene transfer. The ratio of histological necrotic area to viable area was significantly increased in the pEB-DTA-treated tumors, where levels of apoptosis were significantly higher than those observed in the pEB-GFP group. DNA synthesis showed a tendency to decrease in the pEB-DTA group but this was not significant. The incidence and multiplicity of lung metastasis were similar between the groups. There was also no difference in the density of microvessels between groups. We therefore conclude that the EBV-based plasmid vector system combined with in vivo electrogene transfer can result in efficient gene transfection and that the non-viral replicon vector containing DT-A suppresses tumor growth due to apoptotic cell death in this model.

406. Determination of the Most Efficient Vector System for Gene Transduction into Dendritic Cells (DC)

Background: It has been well established that the specialized antigen presenting cells (APC), dendritic cells (DC), play a pivotal role in initiating a primary immune response. Although it has been reported that several vector systems including Adenoviral vector, HVJ liposome, inactivated HVJ vector, and electroporation-based plasmid vector systems, are able to transduce the genes into the DC in mouse and human, this is not the case with rat DC. In addition to this, it appears that the reported studies were not always employing highly purified DC and, to our best knowledge, there has been no direct evidence to support the view that currently used-vector systems are able to effectively transduce the gene into DC. Inasmuch as most, if not all, DC or DC-related gene-transfer studies are performed by employing heterogeneous cell populations, it is important to determine whether and to what extent each vector system is able to transduce the gene into DC.

Further development of the cassette-based pYC plasmid system by incorporation of the dominant hph, nat and AUR1-C gene markers and the lacZ reporter system.

Dominant selection markers encoding hygromycin B phosphotransferase (hph), nourseothricin N-acetyltransferase (nat) and a mutant inositol phosphoceramide synthase (AUR1-C) were all incorporated into the pYC yeast plasmid vector system, thus expanding this system with possible alternatives to the use of G418 resistance. We found the markers to be of use not only in standard laboratory strains of Saccharomyces cerevisiae but also in an industrial strain of S. carlsbergensis (syn. of S. pastorianus) brewing yeast as well as in Saccharomyces kluyveri. As the pYC system contains means of counter-selection for plasmid loss and loop-out of integrated plasmids, it now provides ample opportunities for genetic manipulation of industrial and non-conventional yeasts when the URA3 marker and FOA counter-selection is not an option. Furthermore, the lacZ system for analyzing gene expression was included in the system.

Interplay of SOS induction, recombinant gene expression, and multimerization of plasmid vectors in Escherichia coli.

Using pBR322- and pUC-derived plasmid vectors, a homologous (Escherichia coli native esterase) and three heterologous proteins (human interleukin-2, human interleukin-6, and Zymomonas levansucrase) were synthesized in E. coli IC2015(recA::lacZ) and GY4786 (sfiA::lacZ) strains. Via time-course measurement of beta-galactosidase activity in each recombinant culture, the SOS induction was estimated in detail and the results were systematically compared. In recombinant E. coli, the SOS response did not happen either with the recombinant insert-negative plasmid backbone alone or the expression vectors containing the homologous gene. Irrespective of gene expression level and toxic activity of synthesized foreign proteins, the SOS response was induced only when the heterologous genes were expressed using a particular plasmid vector, indicating strong dependence on the recombinant gene clone and the selection of a plasmid vector system. It is suggested that in recombinant E. coli the SOS response (i.e., activation of recA expression and initial sfiA expression) may be related neither to metabolic burden nor toxic cellular event(s) by synthesized heterologous protein, but may be provoked by foreign gene-specific interaction between a foreign gene and a plasmid vector. Unlike in E. coli XL1-blue(recA(-)) strains used, all expression vectors encoding each of the three heterologous proteins were multimerized in E. coli IC2015 strains in the course of cultivation, whereas the expression vectors containing the homologous gene never formed the plasmid multimers. The extent of multimerization was also dependent on a foreign gene insert in the expression vector. As a dominant effect of the SOS induction, recombinant plasmid vectors used for heterologous protein expression appear to significantly form various multimers in the recA(+) E. coli host.

Delivery of protein antigens and DNA by virulence-attenuated strains of Salmonella typhimurium and Listeria monocytogenes

Two different plasmid-vector systems were developed which allow the efficient production and presentation of protein antigens in antigen-presenting cells (APC) by means of virulence-attenuated bacteria. The first antigen-delivery system is based on the secretion machinery of the Escherichia coli hemolysin (HlyA-type I secretion system), which transports proteins, possessing the specific HlyA secretion signal (HlyA s) at the C-terminus, across both membranes of gram-negative bacteria. This system functions in all gram-negative bacteria that possess the TolC-analogous protein in the outer membrane. This outer membrane protein is necessary for the stable anchoring of the type I secretion apparatus in the cell envelope. Suitable HlyA s-fused antigens are secreted with high efficiency by E. coli and by virulence-attenuated strains of Salmonella, Shigella, Vibrio cholerae and Yersinia enterocolitica. The other vector system expresses the heterologous antigen under the control of an eukaryotic promoter in a similar fashion as in plasmids commonly used for vaccination with naked DNA. This plasmid DNA is introduced into APCs with the help of virulence-attenuated self-destructing Listeria monocytogenes mutants. After synthesis of the heterologous protein, epitopes of the antigen are presented by the APC together with MHC class I molecules. This system functions in macrophages and dendritic cells in vitro and can also be used in a modified form in animal models.

Non-replicating Epstein-Barr Virus-based Plasmids Extend Gene Expression and Can Improve Gene Therapy in Vivo

To date, no gene transfer vector has produced prolonged gene expression following a single intravenous injection and then efficiently re-expressed the delivered gene following repeated systemic injection into immunocompetent hosts. To overcome these limitations, a gene therapy regimen using non-replicating Epstein-Barr virus (EBV)-based expression plasmids was developed. One plasmid contains the FR (EBV family of repeats) sequence and the expressed gene. The other encodes Epstein-Barr nuclear antigen 1 (EBNA-1), but lacks FR. Although unable to replicate in mice, intravenous co-injection of EBV-based plasmids in cationic liposome-DNA complexes (CLDCs) substantially prolonged luciferase gene expression. The use of a two-vector system limited host exposure to the EBNA-1 gene product. Furthermore, this EBV-based vector system could be intravenously re-injected multiple times into immunocompetent mice without loss of transfection efficiency. Use of this vector system significantly improved the therapeutic efficacy of the biologically important human granulocyte colony-stimulating factor gene. Delivery of the human granulocyte colony-stimulating factor gene in EBV-based plasmids increased circulating white blood counts for at least 2 months following a single CLDC-based intravenous co-injection. Conversely, white blood counts were never elevated following injection of CLDCs lacking EBV-derived elements. Thus, this EBV-based plasmid vector system both markedly prolongs gene expression at therapeutic levels and efficiently and repeatedly re-transfects immunocompetent hosts. These properties of EBV-based plasmid vectors appear to be due, at least in part, to the documented abilities of the EBNA-1 protein both to retain FR-containing DNA intracellularly and within the nucleus and to block anti-EBNA-1 cytotoxic T cell responses.

Improved titers for helper virus-free herpes simplex virus type 1 plasmid vectors by optimization of the packaging protocol and addition of noninfectious herpes simplex virus-related particles (previral DNA replication enveloped particles) to the packaging procedure.

A helper virus-free herpes simplex virus type 1 (HSV-1) plasmid vector system has potential for both gene therapy and physiological studies, but relatively low titers have complicated use of this system. In this article, the packaging efficiency was improved by optimizing the packaging protocol and by adding noninfectious HSV-1-related particles, i.e., previral DNA replication enveloped particles (PREPs), during the packaging procedure. PREPs contain many of the tegument proteins that are thought to enhance an HSV-1 infection. Use of both the optimized packaging protocol and the PREPs resulted in an approximately 50-fold increase in the titer, and five different HSV-1 vectors were packaged using this procedure. A purified vector stock (7.8x10(8) infectious vector particles/ml) was microinjected into the striatum, the rats were sacrificed 4 days after gene transfer, and the brains were found to contain an average of approximately 6740 X-Gal-positive striatal cells. This improved packaging procedure may augment use of this vector system.