Targeted in vivo genetic manipulation of the mouse or rat brain by in utero electroporation with a triple-electrode probe

Szczurkowska, Joanna; Cwetsch, Andrzej; Maschio, Marco Dal; Ghezzi, Diego; Ratto, Gian Michele; Cancedda, Laura

doi:10.1038/nprot.2016.014

Cited by 78 publications

(86 citation statements)

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“…In utero electroporation was performed as described previously. 22 Briefly, pregnant mice carrying embryonic day (E)14.5 embryos were anesthetized. One microliter of 2.0 mg/mL vectored DNA in water containing Fast Green (Sigma-Aldrich) was injected into the lateral ventricle of embryonic brains.…”

Section: In Utero Electroporation and Quantificationmentioning

confidence: 99%

Pathologic Active mTOR Mutation in Brain Malformation with Intractable Epilepsy Leads to Cell-Autonomous Migration Delay

Hanai

Sukigara

Dai

et al. 2017

The American Journal of Pathology

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The activation of phosphatidylinositol 3-kinase-AKTs-mammalian target of rapamycin cell signaling pathway leads to cell overgrowth and abnormal migration and results in various types of cortical malformations, such as hemimegalencephaly (HME), focal cortical dysplasia, and tuberous sclerosis complex. However, the pathomechanism underlying abnormal cell migration remains unknown. With the use of fetal mouse brain, we performed causative gene analysis of the resected brain tissues from a patient with HME and investigated the pathogenesis. We obtained a novel somatic mutation of the MTOR gene, having approximately 11% and 7% mutation frequency in the resected brain tissues. Moreover, we revealed that the MTOR mutation resulted in hyperphosphorylation of its downstream molecules, S6 and 4E-binding protein 1, and delayed cell migration on the radial glial fiber and did not affect other cells. We suspect cell-autonomous migration arrest on the radial glial foot by the active MTOR mutation and offer potential explanations for why this may lead to cortical malformations such as HME.

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Section: In Utero Electroporation and Quantificationmentioning

confidence: 99%

Pathologic Active mTOR Mutation in Brain Malformation with Intractable Epilepsy Leads to Cell-Autonomous Migration Delay

Hanai

Sukigara

Dai

et al. 2017

The American Journal of Pathology

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“…LSSmClopHensor was transduced by in utero electroporation in CD-1 mice at embryonic day 15.5 to transfect neuronal progenitors of layer 2/3 pyramidal neurons of the visual cortex (30). Before imaging, mice were anesthetized with an i.p.…”

Section: Methodsmentioning

confidence: 99%

“…In utero electroporation (30,31) applied at embryonic day 15.5 led to the transfection of layer 2/3 pyramidal neurons of the visual cortex as shown in a postnatal day (P) 32 mouse ( Fig. 2 A and B).…”

Section: Significancementioning

confidence: 99%

Simultaneous two-photon imaging of intracellular chloride concentration and pH in mouse pyramidal neurons in vivo

Sato

Artoni

Landi

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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132

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Intracellular chloride ([Cl−] i ) and pH (pH i ) are fundamental regulators of neuronal excitability. They exert wide-ranging effects on synaptic signaling and plasticity and on development and disorders of the brain. The ideal technique to elucidate the underlying ionic mechanisms is quantitative and combined two-photon imaging of [Cl − ] i and pH i , but this has never been performed at the cellular level in vivo. Here, by using a genetically encoded fluorescent sensor that includes a spectroscopic reference (an element insensitive to Cl − and pH), we show that ratiometric imaging is strongly affected by the optical properties of the brain. We have designed a method that fully corrects for this source of error. Parallel measurements of [Cl − ] i and pH i at the single-cell level in the mouse cortex showed the in vivo presence of the widely discussed developmental fall in [Cl − ] i and the role of the K-Cl cotransporter KCC2 in this process. Then, we introduce a dynamic twophoton excitation protocol to simultaneously determine the changes of pH i and [Cl − ] i in response to hypercapnia and seizure activity.I ntracellular ion concentrations are controlled by plasmalemmal transporters and channels, which generate and dissipate ionic electrochemical gradients, respectively (1). In recent years, regulation of the intracellular Cl − concentration ([Cl − ] i ) in neurons has attracted lots of attention, because it is the main ion that carries current across GABA A (and also, glycine) receptors. Changes in [Cl − ] i exert an immediate effect on the reversal potential of GABAergic currents (E GABA ) and, thereby, on the properties of GABA A receptor-mediated transmission (2-4). The "ionic plasticity" of GABAergic signaling involves not only the passive flux of Cl − ions through membrane channels but also, a number of ion transporters that regulate [Cl − ] i . Furthermore, this mechanism is under the control of intracellular signaling cascades that regulate the expression patterns as well as functional properties of ion transporters and channels (5, 6). With regard to long-term ionic modulation of GABAergic transmission, a case in point is the decrease in [Cl − ] i that is generally thought to take place during maturation of most central neurons. According to this widely accepted scenario, the Na-K-2Cl cotransporter NKCC1 accumulates Cl − in immature neurons, thereby promoting depolarizing GABA responses (3, 7-9), which is followed by developmental upregulation of the neuron-specific K-Cl cotransporter KCC2 that is required for the generation of classical hyperpolarizing inhibitory postsynaptic potentials (IPSPs) (10).A wealth of electrophysiological evidence dating back to the work in vivo by Eccles and coworkers (11) has provided evidence for active regulation of [Cl − ] i in mammalian central neurons and its crucial effect on the driving force of Cl − in inhibitory synapses (1). However, thus far, there are no direct data on neuronal [Cl − ] i measured in vivo at the single-cell level in the living brain, and for in...

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“…Flexible control of the cell membrane permeability to impermeable molecules by means of electroporation offers a vast array of applications, including, but not limited to, biotechnology, food processing and medicine (Mahnič-Kalamiza, Vorobiev & Miklavčič, 2014; Yarmush et al, 2014; Kotnik et al, 2015; Golberg et al, 2016b). Current state of the art of the biomedical electroporation involves application of various electrode array configurations (Campana et al, 2016b; Ongaro et al, 2016; Szczurkowska et al, 2016) for both invasive (i.e., tissue ablation Reberšek et al, 2014; Golberg et al, 2016a; Klein et al, 2016) and non-invasive (i.e., transdermal) electroporation applications (Blagus et al, 2013; Zorec et al, 2013; Becker et al, 2014; Chen et al, 2015). However, these applications require direct contact between the applicator, electrodes and the biological sample.…”

Section: Introductionmentioning

confidence: 99%

Membrane permeabilization of mammalian cells using bursts of high magnetic field pulses

Novickij

Dermol

Grainys

et al. 2017

PeerJ

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BackgroundCell membrane permeabilization by pulsed electromagnetic fields (PEMF) is a novel contactless method which results in effects similar to conventional electroporation. The non-invasiveness of the methodology, independence from the biological object homogeneity and electrical conductance introduce high flexibility and potential applicability of the PEMF in biomedicine, food processing, and biotechnology. The inferior effectiveness of the PEMF permeabilization compared to standard electroporation and the lack of clear description of the induced transmembrane transport are currently of major concern.MethodsThe PEMF permeabilization experiments have been performed using a 5.5 T, 1.2 J pulse generator with a multilayer inductor as an applicator. We investigated the feasibility to increase membrane permeability of Chinese Hamster Ovary (CHO) cells using short microsecond (15 µs) pulse bursts (100 or 200 pulses) at low frequency (1 Hz) and high dB/dt (>106 T/s). The effectiveness of the treatment was evaluated by fluorescence microscopy and flow cytometry using two different fluorescent dyes: propidium iodide (PI) and YO-PRO®-1 (YP). The results were compared to conventional electroporation (single pulse, 1.2 kV/cm, 100 µs), i.e., positive control.ResultsThe proposed PEMF protocols (both for 100 and 200 pulses) resulted in increased number of permeable cells (70 ± 11% for PI and 67 ± 9% for YP). Both cell permeabilization assays also showed a significant (8 ± 2% for PI and 35 ± 14% for YP) increase in fluorescence intensity indicating membrane permeabilization. The survival was not affected.DiscussionThe obtained results demonstrate the potential of PEMF as a contactless treatment for achieving reversible permeabilization of biological cells. Similar to electroporation, the PEMF permeabilization efficacy is influenced by pulse parameters in a dose-dependent manner.

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Targeted in vivo genetic manipulation of the mouse or rat brain by in utero electroporation with a triple-electrode probe

Cited by 78 publications

References 50 publications

Pathologic Active mTOR Mutation in Brain Malformation with Intractable Epilepsy Leads to Cell-Autonomous Migration Delay

Pathologic Active mTOR Mutation in Brain Malformation with Intractable Epilepsy Leads to Cell-Autonomous Migration Delay

Simultaneous two-photon imaging of intracellular chloride concentration and pH in mouse pyramidal neurons in vivo

Membrane permeabilization of mammalian cells using bursts of high magnetic field pulses

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