Toward an objective evaluation of cell transfection performance

In this study, we considered the interplay between the efficiency and cytotoxicity of multicomponent cationic liposome/DNA complexes, in cell lines of different origin, as NIH 3T3, HEK 293T, N18TG2, and SK-N-SH. We show that both efficiency and cytotoxicity vary considerably depending on used transfection agents and cells lines. Such variations are largely overcome when transfection is evaluated by a parameter, R, that combines the percentages of transfected, nonviable, and nonadherent cells. These findings provide a strong validation of R as an unbiased indicator for transfection performance across cell lines and transfection agents. (C) 2010 American Institute of Physics. [doi: 10.1063/1.3499756

From pluripotency to forebrain patterning: an in vitro journey astride embryonic stem cells

Embryonic stem cells (ESCs) have been used extensively as in vitro models of neural development and disease, with special efforts towards their conversion into forebrain progenitors and neurons. The forebrain is the most complex brain region, giving rise to several fundamental structures, such as the cerebral cortex, the hypothalamus, and the retina. Due to the multiplicity of signaling pathways playing different roles at distinct times of embryonic development, the specification and patterning of forebrain has been difficult to study in vivo. Research performed on ESCs in vitro has provided a large body of evidence to complement work in model organisms, but these studies have often been focused more on cell type production than on cell fate regulation. In this review, we systematically reassess the current literature in the field of forebrain development in mouse and human ESCs with a focus on the molecular mechanisms of early cell fate decisions, taking into consideration the specific culture conditions, exogenous and endogenous molecular cues as described in the original studies. The resulting model of early forebrain induction and patterning provides a useful framework for further studies aimed at reconstructing forebrain development in vitro for basic research or therapy

The readthrough variant of acetylcholinesterase remains very minor after heat shock, organophosphate inhibition and stress, in cell culture and in vivo.

Acetylcholinesterase (AChE) exists in various molecular forms, depending on alternative splicing of its transcripts and association with structural proteins. Tetramers of the 'tailed' variant (AChE(T)), which are anchored in the cell membrane of neurons by the PRiMA (Proline Rich Membrane Anchor) protein, constitute the main form of AChE in the mammalian brain. In the mouse brain, stress and anticholinesterase inhibitors have been reported to induce expression of the unspliced 'readthrough' variant (AChE(R)) mRNA which produces a monomeric form. To generalize this observation, we attempted to quantify AChE(R) and AChE(T) after organophosphate intoxication in the mouse brain and compared the observed effects with those of stress induced by swimming or immobilization; we also analyzed the effects of heat shock and AChE inhibition on neuroblastoma cells. Active AChE molecular forms were characterized by sedimentation and non-denaturing electrophoresis, and AChE transcripts were quantified by real-time PCR. We observed a moderate increase of the AChE(R) transcript in some cases, both in the mouse brain and in neuroblastoma cultures, but we did not detect any increase of the corresponding active enzyme

Surface functionalized multicomponent envelope type nanosystems for improved gene delivery

Multicomponent envelope-type lipid nanosystem (MENS) is an emerging nanoparticle technology. These nanoparticles were assembled according to a recently proposed 'programmed packaging concept' in which various devices that control intracellular trafficking are packed into a single multicomponent envelope-type nanoparticle system. The uptake ability and final intracellular fate of MENS formulations was evaluated by means of confocal laser scannig microscopy. Our results revealed that MENS show a high cellular internalization and that are not specifically targeted to metabolic degradation. These studies will contribute to rationally design novel delivery systems with superior transfection efficiency