Development of a bicistronic expression system in the branchiopod crustacean Daphnia magna.

The viral 2A peptides have recently been used for bicistronic expression in various organisms. In this system, a single mRNA that codes for two proteins flanking the 2A peptide can be translated simultaneously into each protein by ribosomal skipping at this peptide sequence. Here, we tested the function of the Thosea asigna insect virus 2A (T2A) peptide in the branchiopod crustacean Daphnia magna-an emerging model of evolutionary developmental biology. First, we used transgenic Daphnia that expresses a potential bicistronic RNA containing mCherry and histone H2B- green fluorescent protein (GFP) open reading frames upstream and downstream of the T2A sequence, respectively. Microscopic observation revealed difference of localization of the two proteins in the cell, homogenous distribution of mCherry and nuclear localization of H2B-GFP. Second, we changed localization of mCherry from cytoplasm to plasma membrane by attachment of a consensus myristoylation motif in the bicistronic reporter. RNA that codes for this new bicistronic reporter was injected into eggs. At gastrulation stage, we found spectrally distinct fluorescence with enough intensity and resolution to detect membrane localized mCherry and nuclear GFP. These results indicate that the T2A peptide functions in D. magna and T2A-mediated bicistronic expression would be a promising tool for evo-devo studies of this species.

Cytotoxic effect of co-expression of human hepatitis A virus 3C protease and bifunctional suicide protein FCU1 genes in a bicistronic vector.

Recent reports on various cancer models demonstrate a great potential of cytosine deaminase/5-fluorocytosine suicide system in cancer therapy. However, this approach has limited success and its application to patients has not reached the desirable clinical significance. Accordingly, the improvement of this suicide system is an actively developing trend in gene therapy. The purpose of this study was to explore the cytotoxic effect observed after co-expression of hepatitis A virus 3C protease (3C) and yeast cytosine deaminase/uracil phosphoribosyltransferase fusion protein (FCU1) in a bicistronic vector. A set of mono- and bicistronic plasmid constructs was generated to provide individual or combined expression of 3C and FCU1. The constructs were introduced into HEK293 and HeLa cells, and target protein synthesis as well as the effect of 5-fluorocytosine on cell death and the time course of the cytotoxic effect was studied. The obtained vectors provide for the synthesis of target proteins in human cells. The expression of the genes in a bicistronic construct provide for the cytotoxic effect comparable to that observed after the expression of genes in monocistronic constructs. At the same time, co-expression of FCU1 and 3C recapitulated their cytotoxic effects. The combined effect of the killer and suicide genes was studied for the first time on human cells in vitro. The integration of different gene therapy systems inducing cell death (FCU1 and 3C genes) in a bicistronic construct allowed us to demonstrate that it does not interfere with the cytotoxic effect of each of them. A combination of cytotoxic genes in multicistronic vectors can be used to develop pluripotent gene therapy agents.

Design and evaluation of an Edwardsiella tarda DNA vaccine co-encoding antigenic and adjuvant peptide.

Edwardsiella tarda is associated with edwardsiellosis in cultured fish, resulting in heavy losses in aquaculture. So far, different types of vaccine have been attempted against E. tarda. In this study, an optimized eukaryotic expression plasmid was developed and an optimized DNA vaccine co-encoding antigenic and adjuvant peptide using a bicistronic expression system was designed. As a result, a modified plasmid harbored cytomegalovirus (CMV) promoter attached with R region of long terminal repeat from human T-cell leukemia virus type 1 (CMV/R) and woodchuck hepatitis virus post-transcriptional response element (WPRE) component showed an increased antigenic gene expression compared with unmodified plasmid. Moreover, the designed system based on bicistronic system exhibited a stronger ability to express antigenic gene and the RPS achieved 87.3% compared with plasmid encoding antigentic gene. Finally, immunological analysis showed that the DNA vaccine induced both innate and adaptive immune responses. These results suggest that co-encoding antigenic and adjuvant proteins might be an efficient strategy to develop DNA vaccines in aquaculture in the future.

Alternative splicing for tuneable expression of protein subunits at desired ratios.

The controlled expression of two or more proteins at a defined and stable ratio remains a substantial challenge, particularly in the bi- and multispecific antibody field. Achieving an optimal ratio of protein subunits can facilitate the assembly of multimeric proteins with high efficiency and minimize the production of by-products. In this study, we propose a solution based on alternative splicing, enabling the expression of a tunable and predefined ratio of two distinct polypeptide chains from the same pre-mRNA under the control of a single promoter. The pre-mRNA used in this study contains two open reading frames situated on separate exons. The first exon is flanked by two copies of the chicken troponin intron 4 (cTNT-I4) and is susceptible to excision from the pre-mRNA by means of alternative splicing. This specific design enables the modulation of the splice ratio by adjusting the strength of the splice acceptor. To illustrate this approach, we developed constructs expressing varying ratios of GFP and dsRED and extended their application to multimeric proteins such as monoclonal antibodies, achieving industrially relevant expression levels (>1 g/L) in a 14-day fed-batch process. The stability of the splice ratio was confirmed by droplet digital PCR in a stable pool cultivated over a 28-day period, while product quality was assessed via intact mass analysis, demonstrating absence of product-related impurities resulting from undesired splice events. Furthermore, we showcased the versatility of the construct by expressing two subunits of a bispecific antibody of the BEAT® type, which contains three distinct subunits in total.

Engineered Human Adenoviruses of Species B and C Report Early, Intermediate Early, and Late Viral Gene Expression.

Adenoviruses (AdVs) are being developed for oncolytic or vaccination therapy against existing and emerging conditions. Well-characterized replication-competent human and human primate AdVs expressing multiple payloads are desirable, but their replication in rodent models is limited. To score the timing of adenoviral gene expression in cell cultures, we developed fully replication-competent transcriptional reporter viruses for HAdV-C5, -B3, and -B35. The picornavirus-derived 2A sequence, which induces cotranslational peptide splitting and reinitiation (skipping), was linked to GFP and the fused sequence was inserted C-terminal of the early gene E1A, the intermediate early gene protein IX and the late fiber gene. The 2A peptide induced ribosomal skipping during translation of the messenger RNA (mRNA) and gave rise to GFP from the corresponding viral promoters, as shown by immunoblotting and flow cytometry analyses of human and rodent cells. In human cells, both species B and C AdV exhibited highest reporter expression for fiber, followed by protein IX and lowest for E1A. Inoculation with either HAdV-C5 or -B3/35 viruses encoding protein IX- or fiber-GFP gave rise to higher GFP levels in hamster than mouse cells. Remarkably, despite rather low 2A ribosomal skipping efficiency of ∼50% for E1A-2A-GFP, protein IX-2A-GFP, and fiber-2A-GFP, unprocessed protein IX-2A-GFP and fiber-2A-GFP fusion proteins were efficiently incorporated into HAdV-B3 virions, respectively. These data indicate that the B3 C-termini of protein IX and fiber can be considered for retargeting engineered oncolytic or vaccination vectors, or for antigen display. The variable expression levels of transgenes from different subviral promoters may be used to improve oncolytic AdV vectors expressing therapeutic genes.

Characterization of a bicistronic knock-in reporter mouse model for investigating the role of CABLES2 in vivo.

Two members of the CDK5 and ABL enzyme substrate (CABLES) family, CABLES1 and CABLES2, share a highly homologous C-terminus. They interact and associate with cyclin-dependent kinase 3 (CDK3), CDK5, and c-ABL. CABLES1 mediates tumor suppression, regulates cell proliferation, and prevents protein degradation. Although Cables2 is ubiquitously expressed in adult mouse tissues at RNA level, the role of CABLES2 in vivo remains unknown. Here, we generated bicistronic Cables2 knock-in reporter mice that expressed CABLES2 tagged with 3×FLAG and 2A-mediated fluorescent reporter tdTomato. Cables2-3×FLAG-2A-tdTomato (Cables2Tom) mice confirmed the expression of Cables2 in various mouse tissues. Interestingly, high intensity of tdTomato fluorescence was observed in the brain, testis and ovary, especially in the corpus luteum. Furthermore, immunoprecipitation analysis using the brain and testis in Cables2Tom/Tom revealed interaction of CABLES2 with CDK5. Collectively, our new Cables2 knock-in reporter model will enable the comprehensive analysis of in vivo CABLES2 function.

In Vitro Assay for the Evaluation of Cytotoxic Effects Provided by a Combination of Suicide and Killer Genes in a Bicistronic Vector.

When using bicistronic expression constructs the issue arises concerning proper evaluation of the cytotoxic efficiency of a combination of therapeutic genes. For this purpose, an approach can be applied based on the transient transfection of cultured human cells with a specifically designed set of mono- and bicistronic expression constructs and on the comparison of their cytotoxic effects. Here the application of this approach is described using an example of the evaluation of the combined cytotoxic action of bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion protein (FCU1) and hepatitis A virus 3C protease in a bicistronic plasmid construct.

HrpG and HrpV proteins from the Type III secretion system of Erwinia amylovora form a stable heterodimer.

Bacterial type III secretion systems (T3SSs) are specialized multicomponent nanomachines that mediate the transport of proteins either to extracellular locations or directly into eukaryotic host cell cytoplasm. Erwinia amylovora, the main agent of rosaceous plants fireblight disease, employs an Hrp/Hrc1 T3SS to accomplish its pathogenesis. The regulatory network that controls the activation of this T3SS is largely unknown in E. amylovora. However, in Pseudomonas syringae pathovars, the HrpG/HrpV complex has been shown to directly regulate the activity of transcription factor HrpS and consequently the upregulation of the Hrp/Hrc1 T3SS related genes. In this work, we report the successful recombinant production and purification of a stable E. amylovora HrpG/HrpV complex, using pPROpET, a bicistronic expression vector. Furthermore, we present the first solution structure of this complex based on small-angle X-ray scattering data.

Comparative analysis of internal ribosomal entry sites as molecular tools for bicistronic expression.

Internal ribosomal entry sites (IRESes) are sequences that drive cap-independent translation. They are found in some viral and cellular transcripts and they have been extensively used in both basic and applied research for the translation of two or more polypeptides from a single mRNA molecule in eukaryotic cells. Although the most widely used IRES comes from the encephalomyocarditis virus (EMCV), several other viral and cellular IRES elements have been identified and successfully used, including those of the human VCIP gene and the mouse Gtx gene. In this report we have compared the EMCV IRES with the VCIP and the Gtx IRESes, and we provide evidence that by using the EMCV IRES much higher levels of second cistron expression can be achieved.

Insert sequence length determines transfection efficiency and gene expression levels in bicistronic mammalian expression vectors.

Bicistronic expression vectors have been widely used for co-expression studies since the initial discovery of the internal ribosome entry site (IRES) about 25 years ago. IRES sequences allow the 5' cap-independent initiation of translation of multiple genes on a single messenger RNA strand. Using a commercially available mammalian expression vector containing an IRES sequence with a 3' green fluorescent protein fluorescent marker, we found that sequence length of the gene of interest expressed 5' of the IRES site influences both expression of the 3' fluorescent marker and overall transfection efficiency of the vector construct. Furthermore, we generated a novel construct expressing two distinct fluorescent markers and found that high expression of one gene can lower expression of the other. Observations from this study indicate that caution is warranted in the design of experiments utilizing an IRES system with a short 5' gene of interest sequence (<300 bp), selection of single cells based on the expression profile of the 3' optogenetic fluorescent marker, and assumptions made during data analysis.