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Direct and Efficient Transfection of Mouse Neural Stem Cells And © 2017. Published by The Company of Biologists Ltd | Development (2017) 144, 3968-3977 doi:10.1242/dev.151381 STEM CELLS AND REGENERATION TECHNIQUES AND RESOURCES ARTICLE Direct and efficient transfection of mouse neural stem cells and mature neurons by in vivo mRNA electroporation Stéphane Bugeon1, Antoine de Chevigny1, Camille Boutin1, Nathalie Coré1, Stefan Wild2, Andreas Bosio2, Harold Cremer1,*,‡ and Christophe Beclin1,* ABSTRACT (miRNAs)isnowadaysabroadlyusedapproachinstudiesofthe In vivo brain electroporation of DNA expression vectors is a widely developing and postnatal mouse brain (De Vry et al., 2010). However, used method for lineage and gene function studies in the developing DNA electroporation is of limited efficiency and allows generally only and postnatal brain. However, transfection efficiency of DNA is limited the targeting of embryonic (Saito and Nakatsuji, 2001) and early and adult brain tissue is refractory to electroporation. Here, we postnatal neural stem cell compartments (Boutin et al., 2008, 2010). present a systematic study of mRNA as a vector for acute genetic Postmitotic neurons or adult neural stem cells in the subventricular manipulation in the developing and adult brain. We demonstrate that compartment are highly refractory to DNA electroporation (Barnabé- mRNA electroporation is far more efficient than DNA electroporation, Heider et al., 2008; De la Rossa and Jabaudon, 2014), even though and leads to faster and more homogeneous protein expression some invasive techniques were developed to locally electroporate in vivo. Importantly, mRNA electroporation allows the manipulation of adult mature neurons (Molotkov et al., 2010; Ohmura et al., 2015; ̀ neural stem cells and postmitotic neurons in the adult brain using Pages et al., 2015; Porrero et al., 2016). Ciona Xenopus minimally invasive procedures. Finally, we show that this approach In specific experimental models, such as or Laevis can be efficiently used for functional studies, as exemplified by oocytes, mRNA injection was extensively and efficiently transient overexpression of the neurogenic factor Myt1l and by stably used for ectopic gene expression or knockdown (Bertrand et al., inactivating Dicer nuclease in vivo in adult born olfactory bulb 2003; Mimoto and Christian, 2011). Recently, mRNA transfection interneurons and in fully integrated cortical projection neurons. of combination of different transcription factors has been used in vitro in human cells to reprogram somatic cells into induced KEY WORDS: Adult neurogenesis, Subventricular zone, Olfactory pluripotent stem cells (Luni et al., 2016) and to drive the neuronal bulb, Cortex, Dicer, microRNA pathway differentiation of progenitor cells into specific neuronal cell types (Faedo et al., 2016). Based on these findings, we investigated the INTRODUCTION potential of in vitro-generated mRNAs for functional studies in the Manipulation of gene expression in the brain of living animals mouse brain. We show that mRNA electroporation is considerably represents a key approach in neurobiological research today. Over more efficient than DNA for targeting the postnatal neural stem cell the past decades, mouse genetic approaches have been developed pool and leads to more homogeneous expression levels. mRNA- and widely used to study biological processes either via ectopic/ based protein expression is stable over days. Importantly, mRNA overexpression or gene knockout in transgenic mice. However, the electroporation allows efficient targeting of neural stem cells in the generation of genetically modified mice is a long-term project that adult ventricular/subventricular zone (VZ-SVZ) and even fully requires considerable resources and infrastructure. Moreover, mature neurons integrated in the cortex and other brain structures. overexpression or inactivation of a given gene in a specific tissue/ We demonstrate the usefulness of mRNA electroporation for cell type or at a defined moment implicates complex strategies functional analyses in the adult brain. First, we reveal that involving the concomitant presence of several transgenes in a given transient expression of the neuronal gene Myt1l can drive the animal, rendering this approach expensive and time-consuming. transformation of neural stem cells into proliferative progenitors. Several methods were developed to express transgenes in specific Second, we show that stable recombination of the Dicer locus cell populations of living animals, including neurons. Viral vectors through CRE-mRNA electroporation in adult neural stem cells and have been widely used for in vivo gene function studies or even layer 5 mature cortical neurons interferes with dendritic spine therapeutic approaches (Kotterman and Schaffer, 2014; Papale morphology and density, providing new in vivo evidence for the et al., 2009). Although these tools allow the relatively rapid involvement of the microRNA pathway in synapse regulation. manipulation of defined brain structures, safety issues and immune reactions in the host tissue represent considerable limitations for RESULTS their use (Lowenstein et al., 2007; Sack and Herzog, 2009). Increased efficiency of postnatal electroporation using Electroporation of plasmid DNA expression constructs to induce mRNAs overexpression or knockdown of protein-coding genes or microRNAs Postnatal in vivo brain electroporation allows the transfection of DNA constructs into the stem cell compartment surrounding the walls of 1Aix-Marseille University, CNRS, IBDM UMR 7288, Marseille 13009, France. the lateral ventricles (LV). Position of the electrodes around the head 2Miltenyi Biotec, Bergisch-Gladbach 51429, Germany. of the animal, and therefore the orientation of the electric field, allows *These authors contributed equally to this work specific targeting of the lateral or dorsal aspects of the ventricular wall ‡Author for correspondence ([email protected]) (Boutin et al., 2008; de Chevigny et al., 2012; Fernández et al., 2011) H.C., 0000-0002-8673-5176 (Fig. 1A). Electroporation of the lateral wall of the LV with an expression plasmid coding enhanced green fluorescent protein Received 1 March 2017; Accepted 21 September 2017 (EGFP) (pCAGGS-EGFP, 2 µl of a 5 µg/µl solution) produced at DEVELOPMENT 3968 STEM CELLS AND REGENERATION Development (2017) 144, 3968-3977 doi:10.1242/dev.151381 Fig. 1. EGFP mRNA electroporation efficiently labels the postnatal stem cell compartment. (A,B) Coronal sections of P1 pups electroporated with either a pCAGGS-EGFP plasmid or EGFP mRNAs, and sacrificed at 1 dpe. Dorsal and lateral walls of the lateral ventricle (LV) can be efficiently targeted via mRNA electroporation. Scale bars: 200 µm. LV, lateral ventricle; sp, septum; st, striatum; cc, corpus callosum. (C,D) Higher magnifications showing highly efficient transfection for mRNA electroporation. Dotted lines represent the border between VZ-SVZ and the LV. Scale bars: 30 µm. (E,F) Ventricular lateral wall wholemounts were prepared at 1 dpe after pCAGGS-EGFP or EGFP mRNA electroporation. Scale bars: 400 µm. (G) Higher magnifications of lateral wall wholemounts allowed precise quantification of the transfection efficiency for both conditions. Scale bar: 10 µm. In E-G, ZO1 labels tight junctions and FOP labels basal bodies. (H) Percentage of EGFP+ cells among all the cells lining the lateral wall of the ventricle targeted by pCAGGS-EGFP versus EGFP mRNAs. n=3 animals, three areas/animal, horizontal bars indicate mean; P<0.0001, Wilcoxon rank sum test for means. (I) Color-coded EGFP intensity levels of electroporated cells lining the LV. Scale bar: 30 µm. (J) Quantification of mean cytoplasmic EGFP intensity. Individual points represent cells. Horizontal bars indicate mean, error bars indicate s.d. pCAGGS-EGFP, 0.12±0.01; EGFP mRNA, 0.23±0.004 a.u., n=337 and 616 cells, respectively, from three different animals; ***P<0.0001, Wilcoxon rank sum test for means; ***P<0.0001, Levene’s test for homogeneity of variance. (K) P1 pups were electroporated with either a pCAGGS-EGFP plasmid or EGFP mRNAs, and sacrificed 2 h or 6 h after electroporation. EGFP fluorescence is detectable as early as 2 hpe using mRNA electroporation. Scale bar: 100 µm. (L) P1 CD1 mice were electroporated with EGFP mRNA and SVZ tissues were microdissected at 2 hpe, 1 dpe, 2 dpe and 7 dpe. RNA was then extracted and qRT-PCR carried out. We found very high EGFP mRNA expression at 2 hpe, which dramatically decreased at 1, 2 and 7 dpe: 2 hpe, 83.2±8.4; 1 dpe, 0.1±0.01; 2 dpe, 0.7±0.07; 7 dpe, 1±0.12; n=3 animals. Data are mean±s.e.m. (M) P1 pups were electroporated with EGFP mRNA and fluorescence was then compared at 2 and 7 dpe. EGFP fluorescence is still detectable at 7 dpe. Scale bar: 50 µm. 1 day post-electroporation (dpe) a typical sparse salt-and-pepper labeling of the ventricular walls at 1 dpe (Fig. 1D) compared with labeling with clearly distinguishable individual cells that showed DNA-based transfection. radial glia morphology (Fig. 1A,C). DNA electroporation of the To quantitatively evaluate the targeting efficiency of neural stem dorsal wall led generally to higher EGFP+ cell densities. In both, cells by DNA versus mRNA electroporation, we analyzed lateral and dorsal compartments, EGFP intensity was highly variable ventricular Lateral Wall Whole Mount (LWWM) (Mirzadeh et al., between individual cells (Fig. 1C). 2008) preparations at 1 dpe. Low-magnification views of the lateral We followed the same experimental protocol using in vitro- walls after DNA electroporation showed scattered
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