In Utero Cortical Electroporation of Plasmids in the Mouse Embryo

Meyer-Dilhet, Géraldine; Courchet, Julien

doi:10.1016/j.xpro.2020.100027

Cited by 32 publications

(25 citation statements)

References 24 publications

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“…In utero electroporation is used to induce the expression of target genes in neural precursors to control the activities of neurons. This method is less invasive and more convenient than postnatal injections of viral vectors [ 51 , 52 , 53 , 54 , 55 , 56 ]. In most cases, the plasmid vector (to be expressed) is injected into the ventricles of the embryonic brain.…”

Section: Pros and Cons Of Several Methods For Expressing Opsin In Neu...mentioning

confidence: 99%

“…The target gene can be expressed in a specific neuronal population by controlling the embryonic day when the method is applied. The area of expression can also be controlled by placing bipolar electrodes at specific angles and positions [ 53 , 57 , 58 , 59 , 60 ]. Electroporation can be used in various animals, including mice, rats, ferrets, and cats [ 53 , 54 , 57 , 61 , 62 ].…”

Section: Pros and Cons Of Several Methods For Expressing Opsin In Neu...mentioning

confidence: 99%

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In Utero Electroporation for Manipulation of Specific Neuronal Populations

2022

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The complexity of brain functions is supported by the heterogeneity of brain tissue and millisecond-scale information processing. Understanding how complex neural circuits control animal behavior requires the precise manipulation of specific neuronal subtypes at high spatiotemporal resolution. In utero electroporation, when combined with optogenetics, is a powerful method for precisely controlling the activity of specific neurons. Optogenetics allows for the control of cellular membrane potentials through light-sensitive ion channels artificially expressed in the plasma membrane of neurons. Here, we first review the basic mechanisms and characteristics of in utero electroporation. Then, we discuss recent applications of in utero electroporation combined with optogenetics to investigate the functions and characteristics of specific regions, layers, and cell types. These techniques will pave the way for further advances in understanding the complex neuronal and circuit mechanisms that underlie behavioral outputs.

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Section: Pros and Cons Of Several Methods For Expressing Opsin In Neu...mentioning

confidence: 99%

Section: Pros and Cons Of Several Methods For Expressing Opsin In Neu...mentioning

confidence: 99%

In Utero Electroporation for Manipulation of Specific Neuronal Populations

2022

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“…Given the advantages of the piggyBac transposon system, we tested different transposases for gene manipulation in the developing cortex. For plasmid DNA, such as the piggyBac transposon, a favored delivery method is in utero electroporation (IUE), often performed during mid-gestation between E12 and E15 ( Saito, 2006 ; Meyer-Dilhet and Courchet, 2020 ).…”

Section: The Use Of Piggybac Transposon In the Developing Brain In Vivo —A Call For Codon Optimizationmentioning

confidence: 99%

Non-codon Optimized PiggyBac Transposase Induces Developmental Brain Aberrations: A Call for in vivo Analysis

Vierl

Kaur

Götz

2021

Front. Cell Dev. Biol.

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In this perspective article, we briefly review tools for stable gain-of-function expression to explore key fate determinants in embryonic brain development. As the piggyBac transposon system has the highest insert size, a seamless integration of the transposed sequence into the host genome, and can be delivered by transfection avoiding viral vectors causing an immune response, we explored its use in the murine developing forebrain. The original piggyBac transposase PBase or the mouse codon-optimized version mPB and the construct to insert, contained in the piggyBac transposon, were introduced by in utero electroporation at embryonic day 13 into radial glia, the neural stem cells, in the developing dorsal telencephalon, and analyzed 3 or 5 days later. When using PBase, we observed an increase in basal progenitor cells, often accompanied by folding aberrations. These effects were considerably ameliorated when using the piggyBac plasmid together with mPB. While size and strength of the electroporated region was not correlated to the aberrations, integration was essential and the positive correlation to the insert size implicates the frequency of transposition as a possible mechanism. We discuss this in light of the increase in transposing endogenous viral vectors during mammalian phylogeny and their role in neurogenesis and radial glial cells. Most importantly, we aim to alert the users of this system to the phenotypes caused by non-codon optimized PBase application in vivo.

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“…Timed-pregnant NUAK1 F/F females were mated with Nex CRE/+ ; NUAK1 F/F males. At E15.5, we performed in Utero Cortical Electroporation as described in (Meyer-Dilhet and J. Courchet, 2020). A mix containing 1 μg/μl endotoxin-free plasmid DNA (pCAG-mScarlet-i) plus 0.5% Fast Green (Sigma; 1:20 ratio) was injected into one lateral hemisphere.…”

Section: In Utero Cortical Electroporationmentioning

confidence: 99%

The AMPK-related kinase NUAK1 controls cortical axons branching though a local modulation of mitochondrial metabolic functions

Lanfranchi

Meyer-Dilhet

Reis

et al. 2020

Preprint

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The precise regulation of the cellular mechanisms underlying axonal morphogenesis is essential to the formation of functional neuronal networks. We previously identified the autism-candidate kinase NUAK1 as a central regulator of axon branching in mouse cortical neurons through the control of mitochondria trafficking. How does local mitochondrial position or function regulate axon branching during development? Here, we characterized the metabolic regulation in the developing axon and report a marked metabolic decorrelation between axon elongation and collateral branching. We next solved the cascade of event leading to presynaptic clustering and mitochondria recruitment during spontaneous branch formation. Interestingly and contrary to peripheral neurons, mitochondria are recruited after but not prior to branch formation in cortical neurons. Using flux metabolomics and fluorescent biosensors, we observed that NUAK1 deficiency significantly impairs mitochondrial metabolism and axonal ATP concentration. Upregulation of mitochondrial function is sufficient to rescue axonal branching in NUAK1 null neurons in vitro and in vivo. Altogether, our results indicate that NUAK1 exerts a dual function during axon branching through its ability to control mitochondria distribution and activity, and suggest that a mitochondrial-dependent remodeling of local metabolic homeostasis plays a critical role during axon morphogenesis. RESULTS Cortical axon elongation and collateral branching rely on distinct metabolic pathways

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In Utero Cortical Electroporation of Plasmids in the Mouse Embryo

Cited by 32 publications

References 24 publications

In Utero Electroporation for Manipulation of Specific Neuronal Populations

In Utero Electroporation for Manipulation of Specific Neuronal Populations

Non-codon Optimized PiggyBac Transposase Induces Developmental Brain Aberrations: A Call for in vivo Analysis

The AMPK-related kinase NUAK1 controls cortical axons branching though a local modulation of mitochondrial metabolic functions

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