iPS cells were originally generated using monocistronic retroviral vectors carrying the Yamanaka factors ‘OSKM’. The development of a polycistronic viral vector with OSKM linked by 2A peptides has simplified reprogramming procedure and reduced the risk of multiple proviral integrations and insertional mutagenesis. In this study, we demonstrated the production of the polycistronic lentiviral vector encoding OSKM in a single cassette without a reporter gene or drug-based selection system. Syncytia formations were clearly seen following the co-transfection of a lentiviral plasmid construct with the structural and packaging plasmids. The virion was collected at 48 hours post-transfection. Afterwards, the viral titers were measured by the expression of Sox2 protein from transduced HT1080 cells. Subsequently, Oct4 expression was successfully detected in mouse fibroblasts in the range of 5, 10 and 20 MOIs with expression of 90.7%, 97.5% and 98%, respectively. The results obtained from this study could be used as a model for the production of OSKM lentiviral vector for newcomers to cellular reprogramming research.
The use of in vitromodel for screening pharmacological compounds or natural products has gained global interest. The choice of cells to be manipulated plays a vital role in coming up with the best-suitedmodel for specific diseases, including neurodegenerativediseases (ND). A good in vitro ND model should provide appropriate morphological and molecular features that mimic ND conditions where it can be used to screen potential properties of natural products in addition to unravelling the molecular mechanisms of ND. In this mini review, we intend to demonstrate two prospective stem cell lines as the potential cell source for in vitroND model and compare them to the commonly used cells. The common source of cells that have been usedas the in vitroND models is discussedbefore going into details talking about the two prospective stem cell lines.
Activin proteins are members of the transforming growth factor-β family. Activin A is involved in several biological responses including wound repair, cell death, proliferation and differentiation of many cell types. Biologically active activins consist of homodimers or heterodimers of two beta (β) subunits that are linked together by a single covalent disulphide bond. The subunits in humans are βA, βB, βC and βE. As an example, a combination of two βA subunits will produce a unit of activin A. These proteins are found in most cells of body such as macrophage and activated circulating monocytes. Their role in inflammation can be categorised into two types, either pro- or anti-inflammatory agents, depending on the cell type and phase. Activin signals are kept in balance by antagonist follistatin (Fst), which is a glycoprotein expressed in tissues and encoded by the follistatin gene in humans.
Introduction: MiR-3099 was reported to play a role in neuronal cell differentiation/function in the brain during late embryonic and early neonatal development. To further explore its potential regulatory effects on embryonic brain development, this study aims to construct and validate an expression vector of miR-3099 for future gain-of-function and loss-of-function studies. Methods: pCAG-eGFP vector was modified to include IRES2 and miR-3099 with 150bp upstream and downstream genomic sequences. The newly constructed vector, pCAG-miR-3099-IRES2-eGFP, consists of CAG promoter. The in vitro expression level of miR-3099 was measured using stem-loop RT-qPCR after it was transfected into 293FT cell. Later, the vector was electroporated into the embryonic brain at E15.5. Three days later, the E18.5 embryonic brain was harvested and cryopreserved. Immunohistochemistry was performed by using antibody against eGFP to validate the in utero expression of the transgene in the neocortex of the brain. Results: Our finding showed that, the expression level of miR-3099 was significantly upregulated (p