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.

MicroRNA and Male Infertility: A Potential for Diagnosis.

MicroRNAs (miRNAs) are small non-coding single stranded RNA molecules that are physiologically produced in eukaryotic cells to regulate or mostly down-regulate genes by pairing with their complementary base-sequence in related mRNA molecules in the cytoplasm. It has been reported that other than its function in many physiological cell processes, dysregulation of miRNAs plays a role in the development of many diseases. In this short review, the association between miRNAs and some male reproductive disorders is surveyed. Male factor Infertility is a devastating problem from which a notable percentage of couples suffer. However, the molecular mechanism of many infertility disorders has not been clearly elucidated. Since miRNAs have an important role in numerous biological cell processes and cellular dysfunctions, it is of interest to review the related literature on the role of miRNAs in the male reproductive organs. Aberrant expression of specific miRNAs is associated with certain male reproductive dysfunctions. For this reason, assessment of expression of such miRNAs may serve as a suitable molecular biomarker for diagnosis of those male infertility disorders. The presence of a single nucleotide polymorphism (SNP) at the miRNAs' binding site in its targeted mRNA has been reported to have an association with idiopathic male infertility. Also, a relation with male infertility has been shown with SNP in the genes of the factors necessary for miRNA biogenesis. Therefore, focusing on the role of miRNAs in male reproductive disorders can further elucidate the molecular mechanisms of male infertility and generate the potential for locating efficient biomarkers and therapeutic agents for these disorders.

Poly[N-(2-aminoethyl)ethyleneimine] as a new non-viral gene delivery carrier: the effect of two protonatable nitrogens in the monomer unit on gene delivery efficiency.

PURPOSE: The aim of this study was to investigate the in vitro gene delivery efficiency of poly[N-(2-aminoethyl)ethylene-imine](PAEEI), a polymer with a linear Polyethyleneimine (LPEI) backbone and with aminoethyl side groups that has two protonatable nitrogen atoms per monomer unit instead of one as in LPEI (an established gene delivery polymer). Method. PAEEI (Mn=4.5 kDa, Mw= 10 kDa) was synthesized by ring-opening polymerization of N-(2-(1'-aziridino)ethyl)formamide followed by hydrolysis of the amide groups. The buffering capacity of the resulting polymer was determined by acid-base titration and consequently the percentage of the protonated nitrogen atoms was calculated. Polyplexes were prepared separately in buffers with different ionic strength including Hepes buffered saline (150 mM NaCl) and Hepes buffered glucose (5% glucose) and their zeta-potential, hydrodynamic diameter and colloidal stability were measured. Transfection activity (and toxicity in Hela cells) of the polyplexes were done in HeLa, CHO and HEK293T cells. Cell incubations with polyplexes were done both in the presence and absence (HeLa cells) of serum. Results. PAEEEI showed two times more buffering capacity than LPEI. PAEEI-based Polyplexes had about the same size and zeta-potential as those of LPEI, with a higher colloidal stability in saline buffer in continuous particle size measurement. Their transfection activity was slightly higher than 22-kDa LPEI polyplexes whereas their toxicity profiles were similar in cell lines studied. The PAEEI polyplexes showed gene expression activity both in the presence and absence of serum. Conclusion. Paying attention to the fact that LPEI molecules with smaller sizes than 22 kDa show less transfection efficiency than LPEI 22, the effect of smaller size of PAEEI (10 kDa) on the gene delivery efficiency was compensated by its higher buffering capacity due to carrying more protonatable nitrogen per monomeric unit comparing with LPEI (22 kDa). Having slightly higher transfection efficiency and better colloidal stability than PEI-based systems, PAEEI is an attractive candidate for future in vivo gene delivery studies.

Cloning, Expression, and in vitro Functional Activity Assay of phiC31 Integrase cDNA in Escherichia coli.

OBJECTIVE: The aim of present study was cloning and expression of phiC31 integrase cDNA in a bacterial expression vector. Thus, an intra molecular assay vector was applied to show in vitro activity of recombinant protein. MATERIALS AND METHODS: In this experimental study, phiC31 cDNA was subcloned into a prokaryotic expression vector and transformed into E.coli Bl21 (DE3). Recombinant phiC31 integrase was purified form the bacterial cell lysates and its activity was verified by an in vitro functional assessment. RESULTS: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified phiC31 integrase confirmed the size of protein (70 kDa). Finally, the functionality of purified phiC31 integrase was verified. CONCLUSION: The results of this study indicated that the purified integrase has a great potential application for in vitro site-specific integration.

Constructing a Mouse Oct4 Promoter/EGFP Vector, as a Whole-Cellular Reporter to Monitor the Pluripotent State of Cells.

BACKGROUND: The transcription factor Oct-4, is an important marker of undifferentiating level and a key regulating factor for maintenance of pluripotency in cells. Establishment of an Oct-4 promoter-based reporter system is an appropriate tool for monitoring the differentiation of embryonic stem cells both in vivo and in vitro. METHODS: In the present study, we report construction of a recombinant vector, pDB2 Oct4 promoter/EGFP, in which expression of Enhanced Green Fluorescent Protein (EGFP) was controlled by the mouse Oct-4 promoter. RESULTS: In transfected mouse embryonic stem cells with this vector, EGFP was predicted to be specifically expressed in pluripotency state. After transfection, high-level expression of EGFP under the control of Oct-4 promoter was observed in manipulated embryonic stem cells. CONCLUSION: Thus, our new cellular reporter showed that both the properties of embryonic cells and expression the EGFP could be of great help in studying the differentiating and reprogramming mechanisms of mESCs.

Cloning and expression of a human tissue plasminogen activator variant: K2S in Escherichia coli.

The DNA sequence of Kringle-2 and serine protease domains of the human tissue plasminogen activator (reteplase, K2S) was PCR amplified. This product was then cloned into the expression vector pET15b plasmid. The presence of the insert was confirmed by restriction digestion, PCR and determination of the nucleotide sequence. By using isopropyl beta-D thiogalactopyranoside (IPTG), reteplase was induced in E. coli BL21 cells and analyzed using polyacrylamide gel electrophoresis (PAGE).