Evidence for expression of promoterless GFP cassette: Is GFP an ideal reporter gene in biotechnology science?

Green fluorescent protein (GFP) has played an important role in biochemistry and cell biology as a reporter gene. It has been used to assess the potency of promoters for recombinant protein production. This investigation reveals evidences suggesting that the gfp GFP gene (EGFP) could be expressed without the promoter. In a study, a pLenti-F/GFP vector was constructed with the purpose to allow GFP expression in transduced cells but not in packaging cells; however, after transfecting the HEK293T cell line, GFP gene was expressed, compared to pLOX/CWgfp-transfected cells showed expression lag, lower levels and reduced percentage of GFP expression in the cells. This unexpected result could be due to auto transduction in packaging cell, possible retrotransposon activity in the cell line, possible contamination of pLenti-F/GFP with the pLOX/CWgfp and possible presence of a promoter within backbone of the vector. All the possibilities were ruled out. To exclude the possibility that a sequence within the region might act as a promoter, the fragment to be transfected was minimized to a region containing "from the start of the GFP gene to 5'LTR R". The GFP gene was again expressed. Therefore, our findings suggest the EGFP does not need promoter for expression. This should appeal to the researchers designing GFP based assays to evaluate the potency of promoters, since possible aberrant expression may have a potential to influence on the results of a planned experiment.

Shining Light on the Sprout of Life: Optogenetics Applications in Stem Cell Research and Therapy.

Optogenetics is the integration of genetics and optics to achieve gain or loss of function of well-defined events in specific cells of living tissue. As a versatile tool, upon light illumination, it allows fast control of precisely defined events in biological systems from single cell to different parts of whole tissue in freely moving animals. Taking advantage of this method, a multitude of studies have been published to understand brain functions and dysfunctions. Although from the beginning, it has been used to target neurons within the neural networks and to understand how specific neurons contribute to brain function, it gradually has been extended to other fields of biology such as stem cell research and therapy. With a combination of optogenetics and stem cells, new opportunities were opened up in stem cell biology and also its integration in new circuit as a cell-based treatment strategy for more common disorders like neurodegenerative and cardiovascular one. Recently, some studies showed that engineered stem cells expressing exogenous light-activated opsins can be used in stem cell biology including tracking the differentiation of stem cells, functional analysis of embryonic stem cell-derived graft, and testing the functional integration of induced pluripotent stem cell-derived neurons. With the advent of non-invasive approach, such as transcranial excitation or inhibition, optogenetics also holds promise for non-invasive control of engineered stem cell.