In Utero（子宮内）胎児へのエレクトロポレーションによる遺伝子導入

NEPA21

Sピンセット電極

ｹｰﾌﾞﾙ･ﾌｯﾄｽｲｯﾁ

アプリケーション
文献

アプリケーション

エレクトロポレーション法によるマウス子宮内胎仔脳への遺伝子導入

１）準備する機械・器具

２）In Uteroエレクトロポレーション

ICRマウスを用いる場合の電気設定条件

妊娠日数	電極径	電圧	パルスオン	パルスオフ	回数
12.5日	3 mm	33 V	30 msec	970 msec	4
13.5日	5 mm	30 V	50 msec	950 msec	4
14.5日	5 mm	33 V	50 msec	950 msec	4
15日～	5 mm	35 V	50 msec	950 msec	4

導入効率よりダメージを少なくする方を優先する場合は、回数を2回に設定。導入効率よりダメージを少なくする方を優先する場合は、回数を2回に設定。

この時、実測の電流値は30mA～60mAとなる（実測値は、電気パルスの後、機械のディスプレーに表示される）。ただし、電極の当て方や子宮の濡れ具合によってもこの値は容易に変動する。まずは、上記の条件で、電流値がこの範囲に入るように電極間の距離や、陰極の子宮に接する面積を検討する。調整しきれない場合には、電圧の設定を変える。

３）GFP発現ベクターを導入した例

胎生14.5日目のICRマウスにおいて、両半球の側脳室にCAG-EGFPを注入し、33V、50msecの電気パルスを4回与えた。 3日後（胎生17.5日目）に胎仔をかん流固定して脳を摘出し、蛍光実体顕微鏡で観察した (図A) 。これらの脳において、一方の大脳半球では内側が、もう一方の半球では外側が遺伝子導入され、GFPによる蛍光をはっきりと認めることができた。
また、これらの脳から凍結切片をつくり、GFPの蛍光を共焦点レーザー顕微鏡で観察した（図B）。脳室帯で遺伝子導入されたGFP陽性細胞が脳の表層側へと移動し、中間帯や皮質板にまで進入してきていることが観察された。矢頭は脳室帯と中間帯、もしくは中間帯と皮質帯との境界を示す。破線は組織の境目を示す。 VZ：脳室帯, IZ：中間帯, CP：皮質板

慶応義塾大学医学部解剖学教室田畑秀典先生・仲嶋一範先生提供
※羊土社「必ず上手くいく遺伝子導入と発現解析プロトコール」転載

In Utero（子宮内）胎児へのエレクトロポレーションによる遺伝子導入

アプリケーション

エレクトロポレーション法によるマウス子宮内胎仔脳への遺伝子導入

１）準備する機械・器具

２）In Uteroエレクトロポレーション

３）GFP発現ベクターを導入した例

文献

Analyses of Conditional Knockout Mice for Pogz, a Gene Responsible for Neurodevelopmental Disorders in Excitatory and Inhibitory Neurons in the Brain

Lmo4 synergizes with Fezf2 to promote direct in vivo reprogramming of upper layer cortical neurons and cortical glia towards deep-layer neuron identities

Histological Analysis of a Mouse Model of the 22q11.2 Microdeletion Syndrome

p53/p21 pathway activation contributes to the ependymal fate decision downstream of GemC1

Variant-specific changes in RAC3 function disrupt corticogenesis in neurodevelopmental phenotypes

Tbr1 Misexpression Alters Neuronal Development in the Cerebral Cortex

Endosomal trafficking defects alter neural progenitor proliferation and cause microcephaly

Heterogeneous nuclear ribonucleoprotein U (HNRNPU) safeguards the developing mouse cortex

Impaired Function of PLEKHG2, a Rho-Guanine Nucleotide-Exchange Factor, Disrupts Corticogenesis in Neurodevelopmental Phenotypes

Simultaneous two-photon imaging of action potentials and subthreshold inputs in vivo

IgSF11 homophilic adhesion proteins promote layer-specific synaptic assembly of the cortical interneuron subtype

A pericellular hyaluronan matrix is required for the morphological maturation of cortical neurons

Huntington's disease alters human neurodevelopment

Knockdown of Son, a mouse homologue of the ZTTK syndrome gene, causes neuronal migration defects and dendritic spine abnormalities

In utero gene transfer system for embryos before neural tube closure reveals a role for Hmga2 in the onset of neurogenesis

DNA Repair Protein RAD51 Enhances the CRISPR/Cas9-mediated Knock-In Efficiency in Brain Neurons

In Vivo Imaging of the Coupling Between Neuronal and CREB Activity in the Mouse Brain

Prenatal Electroporation-Mediated Gene Transfer Restores Slc26a4 Knock-Out Mouse Hearing and Vestibular Function

Primary cilium-dependent cAMP/PKA signaling at the centrosome regulates neuronal migration

A Novel LGI1 Missense Mutation Causes Dysfunction in Cortical Neuronal Migration and Seizures

Role of Dach1 Revealed Using a Novel Inner Ear-Specific Dach1-knockdown Mouse Model

Estrogen Receptor α Promotes Cav1.2 Ubiquitination and Degradation in Neuronal Cells and in APP/PS1 Mice

In Vivo Single-Cell Genotyping of Mouse Cortical Neurons Transfected With CRISPR/Cas9

Both Excitatory and Inhibitory Neurons Transiently Form Clusters at the Outermost Region of the Developing Mammalian Cerebral Neocortex

Pathological mTOR Mutations Impact Cortical Development

SponGee: A Genetic Tool for Subcellular and Cell-Specific cGMP Manipulation

Persistence of Learning-Induced Synapses Depends on Neurotrophic Priming of Glucocorticoid Receptors

Role of Per3, a Circadian Clock Gene, in Embryonic Development of Mouse Cerebral Cortex

MicroRNA-129-5p Is Regulated by Choline Availability and Controls EGF Receptor Synthesis and Neurogenesis in the Cerebral Cortex

Intersectional Monosynaptic Tracing for Dissecting Subtype-Specific Organization of GABAergic Interneuron Inputs

Single cell profiling of CRISPR/Cas9-induced OTX2 deficient retinas reveals fate switch from restricted progenitors

An Optogenetic Approach to Studies of the Mechanisms of Heterosynaptic Plasticity in Neocortical Neurons

Drebrin-like (Dbnl) Controls Neuronal Migration via Regulating N-Cadherin Expression in the Developing Cerebral Cortex

Comparative Evaluation of Genetically Encoded Voltage Indicators

Niche-derived laminin-511 Promotes Midbrain Dopaminergic Neuron Survival and Differentiation Through YAP

PLGF, a placental marker of fetal brain defects after in utero alcohol exposure.

Developmental activities of the complement pathway in migrating neurons.

Huntingtin-Mediated Multipolar-Bipolar Transition of Newborn Cortical Neurons Is Critical for Their Postnatal Neuronal Morphology

In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration.

Early postnatal GABAA receptor modulation reverses deficits in neuronal maturation in a conditional neurodevelopmental mouse model of DISC1.

Developing a De Novo Targeted Knock-In Method Based on in Utero Electroporation Into the Mammalian Brain

Targeted DNA demethylation in vivo using dCas9-peptide repeat and scFv-TET1 catalytic domain fusions.

High-Throughput, High-Resolution Mapping of Protein Localization in Mammalian Brain by In Vivo Genome Editing.

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

Early postnatal GABAA receptor modulation reverses deficits in neuronal maturation in a conditional neurodevelopmental mouse model of DISC1.

CRISPR/Cas9-mediated Gene Knock-Down in Post-Mitotic Neurons

Radmis, a Novel Mitotic Spindle Protein that Functions in Cell Division of Neural Progenitors.

Screening for candidate genes involved in the production of mouse subventricular zone proliferative cells and an estimation of their changes in evolutionary pressure during primate evolution.

Development of cerebellar neurons and glias revealed by in utero electroporation: Golgi-like labeling of cerebellar neurons and glias.

Shootin1 Acts in Concert with KIF20B to Promote Polarization of Migrating Neurons.

Selective and regulated gene expression in murine Purkinje cells by in utero electroporation.

Persistent cortical plasticity by upregulation of chondroitin 6-sulfation.

Imaging brain electric signals with genetically targeted voltage-sensitive fluorescent proteins

Protein phosphatase 2A facilitates axonogenesis by dephosphorylating CRMP2.

Knockdown of DISC1 by in utero gene transfer disturbs postnatal dopaminergic maturation in the frontal cortex and leads to adult behavioral deficits.

Neurotrophin-3 is involved in the formation of apical dendritic bundles in cortical layer 2 of the rat.

Involvement of the cytoplasmic C-terminal domain of connexin43 in neuronal migration.

Electroporation-mediated gene transfer to the developing mouse inner ear.

Labeling embryonic mouse central nervous system cells by in utero electroporation.

Sirt1 contributes critically to the redox-dependent fate of neural progenitors

RECK modulates Notch signaling during cortical neurogenesis by regulating ADAM10 activity

Direct evidence that ventral forebrain cells migrate to the cortex and contribute to the generation of cortical myelinating oligodendrocytes.

Radial Migration of Superficial Layer Cortical Neurons Controlled by Novel Ig Cell Adhesion Molecule MDGA1

In vivo electroporation in the embryonic mouse central nervous system

G Protein betagamma Subunits and AGS3 Control Spindle Orientation and Asymmetric Cell Fate of Cerebral Cortical Progenitors

Birth-date-dependent segregation of the mouse cerebral cortical neurons in reaggregation cultures

Commissural neuron identity is specified by a homeodomain protein, Mbh1, that is directly downstream of Math1

Neuronal generation, migration, and differentiation in the mouse hippocampal primoridium as revealed by enhanced green fluorescent protein gene transfer by means of in utero electroporation

Hes genes regulate size, shape and histogenesis of the nervous system by control of the timing of neural stem cell differentiation

A NUDEL-dependent mechanism of neurofilament assembly regulates the integrity of CNS neurons

Generation of Reelin-Positive Marginal Zone Cells from the Caudomedial Wall of Telencephalic Vesicles

Layering defect in p35 deficiency is linked to improper neuronal-glial interaction in radial migration

Multipolar Migration: The Third Mode of Radial Neuronal Migration in the Developing Cerebral Cortex