Efficient co-expression of bicistronic proteins in mesenchymal stem cells by development and optimization of a multifunctional plasmid

Abstract

IntroductionLocal synthesis of interferon within B16 tumors mediates anti-tumor effects. Based on reports that stem cells are recruited to tumors, and because systemic administration of interferon causes dose-limiting undesirable side effects, we wanted to improve the anti-tumor effects of interferon while simultaneously minimizing its systemic side effects by employing mesenchymal stem cells (MSCs) as tumor-localized ectopic producers of interferon. Many vectors exist to fulfill this purpose, but their transfection efficiency and resulting expression levels vary considerably.MethodsTo follow both the recruitment to tumors and the synthesis of interferon by MSCs, we designed a bicistronic vector system that permits fluorescent visualization of vector-transfected and interferon-producing MSCs. We used Mu-IFNαA cDNA as the first cistron and the cherry fluorescent protein cDNA as the second cistron, whose translation requires the internal ribosome entry sequence (IRES) from the encephalomyocarditis virus 5' untranslated region. Observing inconsistent expression of these cistrons in various vectors and cell lines, especially compared with a control plasmid pmaxGFP, we optimized the expression of this bicistronic message by mutating pcDNA3 to facilitate exchange of the promoter and polyadenylation segments controlling both the gene of interest and the eukaryotic antibiotic resistance gene as well as the eukaryotic antibiotic resistance gene itself, and effectively compare the effects of these exchanges, creating plasmid pc3.5.ResultsMurine MSCs stably and ectopically expressing Mu-IFNαA inhibited the establishment of tumors in homogeneic C57/BL6 mice. Mu-IFNαA expressed from the bicistronic message is fully biologically active, but is expressed at only two-thirds of the level observed from a monocistronic message. Cap-dependent translation is threefold more efficient than IRES-driven translation in 293T, B16, and MSC cell lines. Both efficient expression and good transfection efficiency require strong expression of the gene of interest and a chimeric intron. High doses of Mu-IFNαA within tumors inhibited tumor establishment but may not inhibit tumor growth.ConclusionsOur modified vector and its derived plasmids will find use in stem cell therapeutics, gene expression, mRNA regulation, and transcription regulation. Local release of Mu-IFNαA within tumors may differently affect tumor establishment and tumor growth.

Improving the spectral analysis of fluorescence resonance energy transfer in live cells: Application to interferon receptors and Janus kinases

“…Polyethyleneimine (PEI), in the linear 25 KDa form was purchased from Fluka as a powder. Solutions of PEI were prepared by dissolution in water, for 24 h, titration with hydrochloric acid until turbidity has disappeared for 1 h, followed by neutralization with NaOH and filtration; aliquots were stored at –70 °C until needed [11]. Diethyldioctadecylammonium bromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE) were purchased from Sigma–Aldrich as powders, dissolved in absolute ethanol, and stored at –20 °C until used.…”

Ligand-independent interaction of the type I interferon receptor complex is necessary to observe its biological activity

“…Transfections using DDAB:DOPE liposomes and polyethyleneimine (PEI) were performed as described elsewhere [11;19]. …”

Analytical use of multi-protein fluorescence resonance energy transfer to demonstrate membrane-facilitated interactions within cytokine receptor complexes

“…Polyethyleneimine (PEI) as the 25 kDa linear polymer, was purchased from Fluka as a powder. Solutions of PEI were prepared as described previously [32].…”

“…All ligations were performed with T4 DNA Ligase. PCR using either genomic DNA or a human embryonic cDNA library (Strategene, Inc.) as a template source was performed as described elsewhere [6,32]. Plasmids are purified using either a variant of the alkaline lysis method employing 7.5 M ammonium acetate instead of 5 M potassium acetate, or the MaxiPrep plasmid isolation kit (Qiagen, Inc., Valencia, CA).…”