Electroporation | EPFL Graph Search

Electroporation, or electropermeabilization, is a microbiology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing chemicals, drugs, electrode arrays or DNA to be introduced into the cell (also called electrotransfer). In microbiology, the process of electroporation is often used to transform bacteria, yeast, or plant protoplasts by introducing new coding DNA. If bacteria and plasmids are mixed together, the plasmids can be transferred into the bacteria after electroporation, though depending on what is being transferred, cell-penetrating peptides or cell squeeze could also be used. Electroporation works by passing thousands of volts (~8 kV/cm) across suspended cells in an electroporation cuvette. Afterwards, the cells have to be handled carefully until they have had a chance to divide, producing new cells that contain reproduced plasmids. This process is approximately ten times more

Pum2 and TDP-43 refine area-specific cytoarchitecture post-mitotically and modulate translation of Sox5, Bcl11b, and Rorb mRNAs in developing mouse neocortex

Nagammal Neelagandan

In the neocortex, functionally distinct areas process specific types of information. Area identity is established by morphogens and transcriptional master regulators, but downstream mechanisms driving area-specific neuronal specification remain unclear. Here, we reveal a role for RNA-binding proteins in defining area-specific cytoarchitecture. Mice lacking Pum2 or overexpressing human TDP-43 show apparent 'motorization' of layers IV and V of primary somatosensory cortex (S1), characterized by dramatic expansion of cells co-expressing Sox5 and Bcl11b/Ctip2, a hallmark of subcerebral projection neurons, at the expense of cells expressing the layer IV neuronal marker Ror beta. Moreover, retrograde labeling experiments with cholera toxin B in Pum2; Emx1-Cre and TDP43(A315T) mice revealed a corresponding increase in subcerebral connectivity of these neurons in S1. Intriguingly, other key features of somatosensory area identity are largely preserved, suggesting that Pum2 and TDP-43 may function in a downstream program, rather than controlling area identity per se. Transfection of primary neurons and in utero electroporation (IUE) suggest cell-autonomous and post-mitotic modulation of Sox5, Bcl11b/Ctip2, and Ror beta levels. Mechanistically, we find that Pum2 and TDP-43 directly interact with and affect the translation of mRNAs encoding Sox5, Bcl11b/Ctip2, and Ror beta. In contrast, effects on the levels of these mRNAs were not detectable in qRT-PCR or single-molecule fluorescent in situ hybridization assays, and we also did not detect effects on their splicing or polyadenylation patterns. Our results support the notion that post-transcriptional regulatory programs involving translational regulation and mediated by Pum2 and TDP-43 contribute to elaboration of area-specific neuronal identity and connectivity in the neocortex.

eLIFE SCIENCES PUBL LTD2022

Cellular transfection of small interfering ribonucleic acids. Electroporation as an alternative to liposome-based delivery transfection methods

Guillaume Jacot

RNA interference has become an increasingly important tool for all aspects of molecular biology. Nevertheless, there is a technological challenge for performing gene knock-down with chemical transfection agents, as many relevant cell types are refractory to efficient transport of short interfering RNA (siRNA) into cytosol using these classical carrier-based methods. The aim of this project was to evaluate the potential of electroporation as a generic transfection method and to compare it with a standard and well-established chemical reversetransfection using cationic lipids as carriers for short-interfering RNAs. We have used an automated cell electroporation instrument from a swedish company, Cellectricon. The Cellaxess HT instrument is able to perform electroporation in 384 wells plates. We compared cell viability, transfection efficiency and extend of gene silencing caused by electroporation, with reverse-transfection using Lipofectamine RNAiMAX. Gene knock down efficiency has been measured at the protein function level by targeting ubiquitously expressed genes that are essential to cell survival. Cell viability and transfection efficiency are, then, opposed with this strategy. Results clearly showed a net improvement of "difficult-to-transfect" cell transfection but this improvement comes at the price of consuming up to ten fold more siRNAs. For "normal" cell line, lipofection remains the preferred methodology as it is more efficient and less expensive. Furthermore, the Cellaxess HT instrument has some serious drawbacks that pervert easy and reproducible result acquisition.

2010

Anatomical and functional characterization of neocortical circuits involved in transforming whisker sensory processing into goal-directed licking

Georgios Foustoukos

The choice of an action upon perception of an external stimulus, arriving at a sensory organ of an animal, depends on previous experiences and outcomes throughout its life. In the rodent brain, the underlying mechanisms involved in simple sensorimotor transformations, such as the detection of a whisker stimulus through goal-directed licking, still remain largely unknown. In this thesis, using as a model the mouse somatosensory system, I explored the anatomical and functional properties of neuronal circuits at different stages of this cortical processing. To start with, using state-of-the-art viral tracing techniques, I investigated the thalamocortical circuits relaying sensory signals to the primary and secondary whisker somatosensory cortices (wS1, wS2). Challenging the "classical" views, the results indicated two streams of information carrying whisker-selective tactile signals. The principal trigeminal nucleus (Pr5) innervates the ventral posterior medial nucleus of the thalamus (VPM) and finally reaching layer 4 of wS1 while the spinal trigeminal nucleus (Sp5) through the rostral part of the posterior medial (POm) thalamus drives the layer 4 of wS2. Finally, a caudal part of the POm, which does not receive brainstem input, innervates layer 1 and layer 5A. Apart from their anatomical differences, those pathways conveyed distinct whisker sensory signals during goal-directed behaviors. Afterwards, I studied the cortical control of jaw and tongue movements during licking for rewards, using multisensory and multimotor whisker detection tasks. The data revealed a frontal tongue-jaw primary motor area (tjM1) which is necessary and encodes for directional licking, independently of the sensory stimulus type, shedding light on how the neocortex orchestrates the main motor output of the animal. Subsequently, I focused on changes in the L2/3 neuronal networks of wS1 after learning of a whisker stimulus. Using as a benchmark a novel "fast" learning and reward-dependent whisker detection task, I carried out inactivations of wS1 during different stages of learning and chronic two-photon (2P) calcium imaging in the L2/3 of the C2 barrel column. The inactivation results indicated that wS1 is indispensable for the acquisition of the novel stimulus and the execution of the task at expert levels. Moreover, the neural data suggested a learning-induced and "long-lasting" enhancement in the whisker sensory responses even when animals were unmotivated to lick. At a network level, a re-organization of the neuronal circuits was observed at different timescales with some of the alterations accompanying the rapid changes in the animal behavior. Additionally, the changes in the whisker sensory responses of neurons in wS1, after learning, were projection-pathway specific with wS2-projecting neurons showing higher whisker responses than whisker primary motor cortex (wM1)-projecting ones. In the final part, acknowledging the importance of a better characterization of the cortical-cortical communication of wS1, I described recent technical advancements in neuronal reconstructions. In vivo single-cell electroporation combined with 2P tomography and registration to a digital atlas, demonstrated the diversity of the projection targets of neurons in the L2/3 of wS1. Overall, I presented different results which contribute to a pre-existing body of research and help to decipher fundamentals and yet highly complex neural computations of the mammalian brain.