The oncogene Musashi1 encodes novel miRNAs in breast cancer.

RNA-binding protein Musashi1 (MSI1) shows an increased expression level in several cancers and has been introduced as a prognostic marker in some malignancies. It is expected that if any miRNA is encoded by this gene, it might have a role in cancer development or could be considered as a prognostic biomarker. Accordingly, in this study, we aimed to find novel miRNA(s) inside the intronic regions of the MSI1 gene. Here, we report two novel miRNAs within intron 4 of MSI1 gene, named MSM2 and MSM3, which were selected among several miRNA precursors predicted by bioinformatic studies. For experimental analysis, corresponding precursor miRNAs were transfected into HEK293T cells and exogenous expression of the mature miRNAs were detected. Two mature miRNAs, MSM3-3p and MSM3-5p were generated by MSM3 precursor and one, MSM2-5p was derived from MSM2. Besides, endogenous expression of MSM2-5p and MSM3-3p was detected in MCF-7 and SH-SY5Y cell lines. Expression of both mature miRNAs was also detected in clinical samples of breast cancer. Additionally, the interaction between the MSM3-3p and 3'UTR region of PDE11A was confirmed by dual luciferase assay. Overall, our data demonstrated that MSI1 gene encodes two novel miRNAs in breast cancer cells.

Resolving the challenge of insoluble production of mature human growth differentiation factor 9 protein (GDF9) in E. coli using bicistronic expression with thioredoxin.

Growth differentiation factor 9 (GDF9) is an oocyte-derived protein with fundamental functions in folliculogenesis. While the crucial contributions of GDF9 in follicular survival have been revealed, crystallographic studies of GDF9 structure have not yet been carried out, essentially due to the insoluble expression of GDF9 in E. coli and lack of appropriate source for structural studies. Therefore, in this study, we investigated the impact of different expression rate of bacterial thioredoxin (TrxA) using bicistronic expression constructs to induce the soluble expression of mature human GDF9 (hGDF9) driven by T7 promoter in E. coli. Our findings revealed that in BL21(DE3), the high rate of TrxA co-expression at 30 °C was sufficiently potent for the soluble expression of hGDF9 and reduction of inclusion body formation by 4 fold. We also successfully confirmed the bioactivity of the purified soluble hGDF9 protein by evaluation of follicle-stimulating hormone receptor gene expression in bovine cumulus cells derived from small follicles. This study is the first to present an effective approach for expression of bioactive form of hGDF9 using TrxA co-expression in E. coli, which may unravel the current issues regarding structural analysis of hGDF9 protein and consequently provide a better insight into hGDF9 functions and interactions.

Melatonin accelerates the developmental competence and telomere elongation in ovine SCNT embryos.

SCNT embryos suffer from poor developmental competence (both in vitro and in vivo) due to various defects such as oxidative stress, incomplete epigenetic reprogramming, and flaws in telomere rejuvenation. It is very promising to ameliorate all these defects in SCNT embryos by supplementing the culture medium with a single compound. It has been demonstrated that melatonin, as a multitasking molecule, can improve the development of SCNT embryos, but its function during ovine SCNT embryos is unclear. We observed that supplementation of embryonic culture medium with 10 nM melatonin for 7 days accelerated the rate of blastocyst formation in ovine SCNT embryos. In addition, the quality of blastocysts increased in the melatonin-treated group compared with the SCNT control groups in terms of ICM, TE, total cell number, and mRNA expression of NANOG. Mechanistic studies in this study revealed that the melatonin-treated group had significantly lower ROS level, apoptotic cell ratio, and mRNA expression of CASPASE-3 and BAX/BCL2 ratio. In addition, melatonin promotes mitochondrial membrane potential and autophagy status (higher number of LC3B dots). Our results indicate that melatonin decreased the global level of 5mC and increased the level of H3K9ac in the treated blastocyst group compared with the blastocysts in the control group. More importantly, we demonstrated for the first time that melatonin treatment promoted telomere elongation in ovine SCNT embryos. This result offers the possibility of better development of ovine SCNT embryos after implantation. We concluded that melatonin can accelerate the reprogramming of telomere length in sheep SCNT embryos, in addition to its various beneficial effects such as increasing antioxidant capacity, reducing DNA damage, and improving the quality of derived blastocysts, all of which led to a higher in vitro development rate.

Site-specific integration as an efficient method for production of recombinant human hyaluronidase PH20 in semi-adherent cells.

PH20 is a hyaluronidase enzyme that can hydrolyze the glycosidic bond in hyaluronic acid as the major proteoglycan found in extracellular matrices. In the present study, we constructed and characterized two donor plasmids, one of them with one and the second with two PH20 expression cassettes. The expression vectors were site specifically integrated into the genome of HEK293T cells using PhiC31 integrase system to develop HEK293T stable cell lines secreting His-tagged recombinant human PH20 (rhPH20) in the culture supernatant. The produced rhPH20 was quantified using ELISA and turbidimetric assay tests, and its catalytic activity was also assessed by treating the mouse cumulus-oocyte complexes. Our results showed that the secreted rhPH20 in the culture supernatant had the specific activity of 16,660 IU/mg and the recombinant enzyme was able to remove the cumulus cells from oocytes. The results also indicated that phiC31 enzyme inserted the PH20-expressing donor vectors into the specific pseudo attP sites including 10q21.2 and 20q11.22 in the genome of the target cells with different copy numbers. Taken together, our findings demonstrate that PhiC31 integrase system is able to be applied as a robust tool for efficient production and secretion of soluble and active rhPH20 by HEK293T cells as a semi-adherent human cell line. KEY POINTS: • Efficient production of human recombinant PH20 in a semi-adherent human cell line • Successful application of PhiC31 integrase system for generation of stable recombinant clones • Use of a human cell line for expression of a recombinant human protein due to complex and efficient post-translational modifications and protein folding.

Smart co-delivery of miR-34a and cytotoxic peptides (LTX-315 and melittin) by chitosan based polyelectrolyte nanocarriers for specific cancer cell death induction.

A novel polyelectrolyte nanocarrier was synthesized via layer-by-layer self-assembly of polycationic and polyanionic chains. The nanocarrier is composed of polyglutamate grafted chitosan core, dextran sulfate as a complexing agent, and polyethyleneimine shell decorated with folic acid. This polyelectrolyte complex has unique physicochemical properties so that the core is considered as an efficient carrier for LTX-315 and melittin peptides, and the shell is suitable for delivery of miR-34a. The spherical nanocarriers with an average size of 123 ± 5 nm and a zeta potential of -36 ± 1 mV demonstrated controlled-release of gene and peptides ensured a synergistic effect in establishing multiple cell death pathways on chemoresistance human breast adenocarcinoma cell line, MDA-MB-231. In vitro cell viability assays also revealed no cytotoxicity for the nanocarriers, and an IC50 of 15 µg/mL and 150 µg/mL for melittin and LTX-315, respectively, after 48 h, whereas co-delivery of melittin with miR-34a increased smart death induction by 54%.

Assessment of MUSASHI 1 and MUSASHI 2 expression in spermatozoa and testicular tissue.

MUSASHI (MSI) family plays the main role in the spermatogenesis process. The purpose of this study was the assessment of sperm MSI1 and MSI2, and sperm functional tests in infertile men (n = 30) with varicocele and fertile men (n = 30). Furthermore, MSI1 and MSI2 proteins were assessed in testicular tissue of azoospermic men (n = 9) as well as epididymal spermatozoa and testis of mice. Expression of MSI1 and MSI2 was assessed at RNA and protein levels in human spermatozoa. Sperm concentration and motility were significantly lower, while abnormal sperm morphology, lipid peroxidation, DNA fragmentation and protamine deficiency were significantly higher in men with varicocele compared to fertile individuals. Any significant difference was not observed in the expression of MSI1 and MSI2 mRNA between the two groups. Unlike MSI1 protein that was not detectable in humans, the relative expression of MSI2 protein was similar in varicocele and fertile individuals. The expression level of both Msi1 and Msi2 proteins was also observable in mouse spermatozoa. No significant relationship was observed between sperm functional parameters with expression of these genes. The data of this study demonstrated that although MSI1 and MSI2 play important roles during spermatogenesis, their relative expression in spermatozoa was not affected by varicocele.

Improving germline transmission efficiency in chimeric chickens using a multi-stage injection approach.

Although different strategies have been developed to generate transgenic poultry, low efficiency of germline transgene transmission has remained a challenge in poultry transgenesis. Herein, we developed an efficient germline transgenesis method using a lentiviral vector system in chickens through multiple injections of transgenes into embryos at different stages of development. The embryo chorioallantoic membrane (CAM) vasculature was successfully used as a novel route of gene transfer into germline tissues. Compared to the other routes of viral vector administration, the embryo's bloodstream at Hamburger-Hamilton (HH) stages 14-15 achieved the highest rate of germline transmission (GT), 7.7%. Single injection of viral vectors into the CAM vasculature resulted in a GT efficiency of 2.7%, which was significantly higher than the 0.4% obtained by injection into embryos at the blastoderm stage. Double injection of viral vectors into the bloodstream at HH stages 14-15 and through CAM was the most efficient method for producing germline chimeras, giving a GT rate of 13.6%. The authors suggest that the new method described in this study could be efficiently used to produce transgenic poultry in virus-mediated gene transfer systems.

10-hydroxy-2-decenoic acid a bio-immunomodulator in tissue engineering; generates tolerogenic dendritic cells by blocking the toll-like receptor4.

Dendritic cells (DCs), in response to the biomaterials, utilize toll-like receptors (TLRs) to become mature or tolerogenic through TLRs-dependent signaling pathways, especially TLR4. Regarding the physicochemical properties of biomaterials, some of such signaling pathways are activated. Unsaturated fatty acids have been explored as an antagonist for TLRs and lead to the tolerogenic phenotype of DCs. Here we showed that, although cultured DCs on both chitosan and Alginate-polyethyleneimine (Alg-PEI) films became fully mature, 10-hydroxy-2-decanoic acid (10-HDA), an unsaturated fatty acid found in royal jelly, led to the tolerogenic immunophenotype of DCs on both films. The cultured cells on the films possessed iDCs-like morphology in the presence of 10-HDA. Moreover, 10-HDA expressed lower levels of CD80, CD83, CD86, and HLA-DR, a higher level of IL-10, and lower level of IL-12 in the cultured DCs on both films. Furthermore, HEK293T cells expressing only TLR4 (HEK-TLR4 cells) were co-cultured with LPS, a specific agonist for TLR4, and 10-HDA. The 10-HDA significantly reduced the expression of tumor necrosis factor-a (TNF-α) in the HEK-TLR4 cells compared to treated only with LPS. These findings indicate that the 10-HDA acts as an antagonist of TLR4; therefore, potentially can be used in autoimmune diseases and preventing the rejection of biomaterials implantation and allograft transplantation.

Increased expression of MUSASHI1 in epithelial breast cancer cells is due to down regulation of miR-125b.

BACKGROUND: Musashi1 (MSI1) is an oncogenic protein with a crucial role in the proliferation and characteristics of the epithelial cells in breast cancer. The change in expression of MSI1 has a role in solid tumor progression. There are different factors that regulate MSI1 expression in various cancer tissues including microRNAs which are considered as one of the most important of these factors. The aim of our study is identification of the molecular cause of maximal expression of MSI1 in epithelial breast cancer cell lines. RESULTS: Among predicted microRNAs, miR-125b, miR-637 and miR-802 were able to significantly reduce the luciferase activity. In addition, the relative expression of these three miRNAs were measured in the cancerous cell lines that results showed a significant reduction in expression of all microRNAs. On the other hand, only the overexpression of miR-125b caused a change in the expression pattern of MSI1 in breast epithelial cancer cell lines. Accordingly, our results demonstrated that the exogenous expression of miR-125b decreased not only the MSI1 protein but also expression of epithelial markers in breast cancer cells. CONCLUSIONS: The results of luciferase reporter assay showed that MSI1 is a direct target for miR-125b in epithelial breast cancer cells. Moreover, higher amount of MSI1 in those cell lines seems due to the reduced amount of miR-125b, which is responsible for epithelial features of those kinds of cancer cells. Therefore, the modulation of miR-125b may be a potential approach to help to combat against epithelial breast tumors.

Do neprilysin inhibitors walk the line? Heart ameliorative but brain threatening!

Sacubitril/valsartan (Entresto™; LCZ696) is the first angiotensin receptor-neprilysin inhibitor (ARNI) drug approved by the US and EU for heart failure (HF) and especially recommended for hypertensive HF (HHF). Sacubitril inhibits the enzyme neprilysin (NEP) which produces both beneficial and adverse effects in the human body. While LCZ696 causes beneficial cardiovascular effects, it may induce memory and cognitive dysfunction, or even exacerbate Alzheimer's disease (AD). This article reviewed data reported by experimental and clinical studies that examined NEP inhibitors and their dementia-related side effects. Based on the literature, LCZ696 increases the risk of memory and cognitive dysfunctions, and clinical trials failed to show compelling evidence for LCZ696 safety for the brain. Together, it was concluded that more experimental and clinical studies with particular focus on LCZ696 side effects on ß-amyloid (Aß) degradation are needed to assess LCZ696 safety for the cognitive function, especially in case of long-term administration.

Intracellular functions of RNA-binding protein, Musashi1, in stem and cancer cells.

RNA-binding protein, musashi1 (MSI1), is a main protein in asymmetric cell division of the sensory organ precursor cells, whereas its expression is reported to be upregulated in cancers. This protein is a critical element in proliferation of stem and cancer stem cells, which acts through Wnt and Notch signaling pathways. Moreover, MSI1 modulates malignancy and chemoresistance of lung cancer cells via activating the Akt signaling. Due to the main role of MSI1 in metastasis and cancer development, MSI1 would be an appropriate candidate for cancer therapy. Downregulation of MSI1 inhibits proliferation of cancer stem cells and reduces the growth of solid tumors in several cancers. On the other hand, MSI1 expression is regulated by microRNAs in such a way that several different tumor suppressor miRNAs negatively regulate oncogenic MSI1 and inhibit migration and tumor metastasis. The aim of this review is summarizing the role of MSI1 in stem cell proliferation and cancer promotion.

Hypoxia-related long noncoding RNAs are associated with varicocele-related male infertility.

One of the main molecular causes that contributes to varicocele-related male infertility is excess production of reactive oxygen species (ROS). It is believed that hypoxia is an important stimulator of ROS in this condition. Recently, the significant roles of long non-coding RNAs (lncRNAs) in hypoxia response have emerged. Despite the investigation of hypoxia, there is scant information about the role of hypoxia-responding lncRNAs in varicocele-related male infertility. In the present study, we deduced eight hypoxia-responding lncRNAs based on high-throughput RNA sequencing data from two Gene Expression Omnibus (GEO) datasets. We used qRT-PCR to assess the expression levels of some of these lncRNAs in 42 ejaculated spermatozoa samples from 25 infertile men with varicocele and 17 fertile men as controls. We identified significant increases in expression levels of hypoxia-related lncRNAs, MIR210HG and MLLT4-AS1 in ejaculated spermatozoa of infertile men with varicocele. These lncRNAs also showed significant positive correlations with ROS levels and meaningful negative correlations with sperm parameters (count and motility). Besides, in silico studies identified several hypoxia response elements (HREs) within selected lncRNAs promoters. Delineation of hypoxia-related lncRNAs in varicocele-related infertility provides a valuable insight into male infertility.

Stabilizing osmolytes' effects on the structure, stability and function of tc-tenecteplase: A one peptide bond digested form of tenecteplase.

Organic osmolytes, as major cellular compounds, cause protein stabilization in the native form. In the present study, the possible chaperone effects of the three naturally occurring osmolytes on the two-chain form of tenecteplase (tc-TNK), a recombinant, genetically engineered mutant tissue plasminogen activator, have been explored by using circular dichroism, steady-state fluorescence, UV-Visible spectroscopy, and in silico experiments. The tc-TNK is derived from the one-chain protein upon disruption of one peptide bond. Thermal denaturation experiments showed a slightly more stabilizing effect of the three co-solvents on the single-chain TNK (sc-TNK) in comparison to that on tc-TNK. Unlike single-chain tenecteplase, the two-chain form undergoes reversible denaturation which is somehow perturbed in some cases as the result of the presence of osmolytes. Very minor changes in the secondary structure and the tertiary structure were observed. The molecular dynamics simulations and comparative structural analysis of catalytic domain of the protein in the single-chain and two-chain forms in pure water, mannitol/water, trehalose/water, and sucrose/water showed that while the stabilizing effect of the three osmolytes on tc-TNK might be induced by preferential accumulation of these molecules around the nonpolar and aromatic residues, that is to say, fewer water-hydrophobic residues' interactions in tc-TNK, sc-TNK is stabilized by preferential exclusion effect.

An efficient method for bacterial production and activity assessment of recombinant human insulin like growth factor 1.

Human insulin like growth factor 1 directs physiological roles in cellular proliferation and differentiation process. The protein is considered as an important therapeutic target with clinical significance. In this study, to avoid production of human insulin like growth factor 1 as inclusion body, the thioredoxin was used as a solubilizing fusion tag. The expression of fusion human insulin like growth factor 1 was carried out in E. coli Rosetta-gami by transformation of pET-32b contained functional elements. The evaluation of different conditions involving protein expression including IPTG concentration, temperature and post induction time showed that 0.1 mM IPTG at 34 °C for 4 h was the optimum condition. The isolated fusion protein was purified using nickel affinity purification and digested by entrokinase to produce mature recombinant protein without any additional tag. The accuracy of produced recombinant protein was confirmed by western blot analysis. Biological activity of produced recombinant human insulin like growth factor 1 was determined by its proliferation effects on MCF-7 cells, expansion of bovine granulosa cells and activation of PI3K/Akt signaling pathway in these cells. The present study provides a simple and efficient method for high-level production of soluble, active recombinant human insulin like growth factor 1 in E. coli.

Fine-tuning the physicochemical properties of peptide-based blood-brain barrier shuttles.

N-methylation is a powerful method to modify the physicochemical properties of peptides. We previously found that a fully N-methylated tetrapeptide, Ac-(N-MePhe)4-CONH2, was more lipophilic than its non-methylated analog Ac-(Phe)4-CONH2. In addition, the former crossed artificial and cell membranes while the latter did not. Here we sought to optimize the physicochemical properties of peptides and address how the number and position of N-methylated amino acids affect these properties. To this end, 15 analogs of Ac-(Phe)4-CONH2 were designed and synthesized in solid-phase. The solubility of the peptides in water and their lipophilicity, as measured by ultra performance liquid chromatography (UPLC) retention times, were determined. To study the permeability of the peptides, the Parallel Artificial Membrane Permeability Assay (PAMPA) was used as an in vitro model of the blood-brain barrier (BBB). Contrary to the parent peptide, the 15 analogs crossed the artificial membrane, thereby showing that N-methylation improved permeability. We also found that N-methylation enhanced lipophilicity but decreased the water solubility of peptides. Our results showed that both the number and position of N-methylated residues are important factors governing the physicochemical properties of peptides. There was no correlation between the number of N-methylated amide bonds and any of the properties measured. However, for the peptides consecutively N-methylated from the N-terminus to the C-terminus (p1, p5, p11, p12 and p16), lipophilicity correlated well with the number of N-methylated amide bonds and the permeability of the peptides. Moreover, the peptides were non-toxic to HEK293T cells, as determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay.

Blastocyst Formation Rate and Transgene Expression are Associated with Gene Insertion into Safe and Non-Safe Harbors in the Cattle Genome.

Integration target site is the most important factor in successful production of transgenic animals. However, stable expression of transgene without disturbing the function of the host genome depends on promoter methylation, transgene copy number and transcriptional activity in integration regions. Recently, new genome-editing tools have made much progress, however little attention has been paid to the identification of genomic safe harbors. The aim of the present study was to evaluate the effect of insertion site, promoter and copy number of transgene on the production of embryos from cattle fibroblast cells following somatic cell nuclear transfer (SCNT). So, three donor vectors were constructed with EGFP gene under control of different promoters. Each vector was integrated into safe and non-safe harbors in the genome using phiC31 integrase. Transgenic clones with a single copy of each vector were isolated. Each clone was analyzed to find site and frequency of integration, expression level and promoter methylation before SCNT, as well as transgene expression level and blastocyst formation rate after SCNT. The data obtained demonstrated that BF5, as a safe harbor, not only showed a stable expression, but also the rate of in vitro-produced embryos from BF5-clones are similar to that of non-transfected cells.

Production of Minicircle DNA Vectors Using Site-Specific Recombinases.

Minicircle DNA vectors are plasmid derivatives free of bacterial elements. These vectors are mostly provided from common plasmids via recombination by site-specific recombinases in E. coli. Absence of bacterial backbone in minicircle vectors results in high-level and persistent expression of transgene in comparison with conventional plasmids and provides promising vehicles for gene therapy and vaccination. Here we describe the production of replicative minicircle DNA vectors using the PBAD/araC system expressing ΦC31 integrase in E. coli.

Rational Development of A Polycistronic Plasmid with A CpG-Free Bacterial Backbone as A Potential Tool for Direct Reprogramming.

OBJECTIVE: Induced pluripotent stem cells are generated from somatic cells by direct reprogramming. These reprogrammed pluripotent cells have different applications in biomedical fields such as regenerative medicine. Although viral vectors are widely used for efficient reprogramming, they have limited applications in the clinic due to the risk for immunogenicity and insertional mutagenesis. Accordingly, we designed and developed a small, non-integrating plasmid named pLENSO/Zeo as a 2A-mediated polycistronic expression vector. MATERIALS AND METHODS: In this experimental study, we developed a single plasmid which includes a single expression cassette containing open reading frames of human LIN28, NANOG, SOX2 and OCT4 along with an EGFP reporter gene. Each reprogramming factor is separated by an intervening sequence that encodes a 2A self-processing peptide. The reprogramming cassette is located downstream of a CMV promoter. The vector is easily propagated in the E. coli GT115 strain through a CpG-depleted vector backbone. We evaluated the stability of the constructed vector bioinformatically, and its ability to stoichiometric expression of the reprogramming factors using quantitative molecular methods analysis after transient transfection into HEK293 cells. RESULTS: In the present study, we developed a nonviral episomal vector named pLENSO/ Zeo. Our results demonstrated the general structural stability of the plasmid DNA. This relatively small vector showed concomitant, high-level expression of the four reprogramming factors with similar titers, which are considered as the critical parameters for efficient and consistent reprogramming. CONCLUSION: According to our experimental results, this stable extrachromosomal plasmid expresses reliable amounts of four reprogramming factors simultaneously. Consequently, these promising results encouraged us to evaluate the capability of pLENSO/Zeo as a simple and feasible tool for generation of induced pluripotent stem cells from primary cells in the future.

Super magnetic nanoparticles NiFe2O4, coated with aluminum-nickel oxide sol-gel lattices to safe, sensitive and selective purification of his-tagged proteins.

Super magnetic nanoparticle NiFe2O4 with high magnetization, physical and chemical stability was introduced as a core particle which exhibits high thermal stability (>97%) during the harsh coating process. Instead of multi-stage process for coating, the magnetic nanoparticles was mineralized via one step coating by a cheap, safe, stable and recyclable alumina sol-gel lattice (from bohemite source) saturated by nickel ions. The TEM, SEM, VSM and XRD imaging and BET analysis confirmed the structural potential of NiFe2O4@NiAl2O4 core-shell magnetic nanoparticles for selective and sensitive purification of His-tagged protein, in one step. The functionality and validity of the nickel magnetic nanoparticles were attested by purification of three different bioactive His-tagged recombinant fusion proteins including hIGF-1, GM-CSF and bFGF. The bonding capacity of the nickel magnetics nanoparticles was studied by Bradford assay and was equal to 250 ± 84 µg Protein/mg MNP base on protein size. Since the metal ion leakage is the most toxicity source for purification by nickel magnetic nanoparticles, therefor the nickel leakage in purified final protein was determined by atomic absorption spectroscopy and biological activity of final purified protein was confirmed in comparison with reference. Also, in vitro cytotoxicity of nickel magnetic nanoparticles and trace metal ions were investigated by MTS assay analysis. The results confirmed that the synthesized nickel magnetic nanoparticles did not show metal ion toxicity and not affected on protein folding.

Construction of a new minicircle DNA carrying an enhanced green florescent protein reporter gene for efficient expression into mammalian cell lines.

The presence of a bacterial backbone in conventional eukaryotic expression plasmids may cause undesirable effects by triggering the immune responses in mammals and repression of episomal transgene expression. To avoid these problems, researchers have proposed the use of minicircle DNAs which are episomal vectors that have lost their bacterial backbone using a site-specific recombinase mediated recombination. In the present study, we have constructed a new minicircle DNA vector that carries an enhanced green florescent protein (EGFP) reporter gene using phage ΦC31 integrase-mediated recombination and homing endonuclease ISceI-mediated purification in E. coli. ΦC31 integrase expression was under the control of the araBAD promoter, whereas ISceI endonuclease was controlled by the tac promoter. This vector was transfected into CHO-K1 cells, which showed transient expression of EGFP up to 14 generations. Similar results were obtained upon transient transfection into HEK cells. In addition, PCR results on genomic DNA, demonstrated the EGFP-minicircle was episomal and did not integrate into the host genome. Our constructed parental plasmid expresses EGFP and could be used for the generation of episomal minicircle DNA with intent to carry out transient transfection of interested DNA fragments into the eukaryotic cells for various purposes.