Identification of a specific pseudo attP site for phage PhiC31 integrase in bovine genome

PhiC31 integrase system provides a new platform in various felid of research, mainly in gene therapy and creation of transgenic animals. This system enables integration of exogenous DNA into preferred locations in mammalian genomes, which results in robust, long-term expression of the integrated transgene. Identification of a novel pseudo attP site. Genomic DNA was extracted from primary bovine fetal fibroblast cells, which were stably trans-fected with EGFP and phiC31 integrase cDNAs carrying vectors. An inverse PCR was carried out for production of mini-circle DNAs and followed by sequencing. A new specific pseudo attP site termed BF5 was identified in bovine genome. This site is located in an intergenic AT rich region on chromosome 5 with similar features of other mammalian attP pseudo sites. Furthermore, direct sequencing of generated attL site confirmed that site-specific transgene recombination was occurred at this site. This finding confirmed that phiC31 integrase could be feasible for production of transgenic animals for biotechnological applications

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

Cloning, expression, and in vitro functional activity assay of phiC31 integrase cDNA in Escherichia coli

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. 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. 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. 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

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 ystem is an appropriate tool for monitoring the differentiation of embryonic stem cells both in vivo and in vitro. 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. 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. 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

[Creation of tenecteplase-producing CHO cell line using site-specific integrase from the phage phi C31]

The aim of this study was to produce a stable CHO cell line expressing tenecteplase. In the first step, the tenecteplase coding sequence was cloned in a pDB2 vector containing attB recognition sites for the phage phi C31 integrase. Then, using lipofection, the CHO cells were co-transfected with constructed recombinant plasmid encoding tenecteplase and attB recognition sites and the integrase coding sequence containing pCMV-Int plasmid. As the recombinant plasmid contained the neomycin resistance gene [neo], stable cells were then selected using G418 as an antibiotic. Stable transformed cells were assessed using genomic PCR and RT-PCR. Finally, the functionality of tenecteplase was evaluated on the cell culture media. our results indicated that tenecteplase coding sequence was inserted into the CHO cell genome and was successfully expressed. Moreover, tenecteplase activity assessment indicated the presence of our functional tenecteplase in the cell culture medium. Considering the data obtained from this study, phi C31 integrase can be used for the production of a stable cell line and it be used to introduce ectopic genes into mammalian cells