Transfection into Mouse/Rat Embryos In Utero by Electroporation

NEPA21

Tweezer Electrodes

Cables, Foot Switch & Others

Transfection into embryos: brain, spinal cord, inner ear and more.

Transfection into Mouse/Rat Embryos In Utero by Electroporation

APPLICATIONS

Gene transfer into embryonic brains using in utero electroporation technique

1) Materials

    • In Vivo Electroporator: NEPA21/CUY21 SC/CUY21 EDIT (Nepa Gene Co., Ltd.)
    • In Vivo Electrode: CUY650P3/CUY650P5 (Tweezers w/3mm or 5mm diameter platinum disk electrode, Nepa Gene Co., Ltd.)
    • Aspirator tube assembly (Drummond)
    • Optical fiber light (Technolight, Kenko, #KTS-100RSV)
    • Sterile gauze (K-Pine, 7.5cm x 7.5cm)
    • Surgical instruments: Fine forceps x 2, Surgery scissors x 2, Ring forceps, Needle holder, Surgical tape
    • Nylon suture (Nesco, #HT1605NA75)
    • Silk suture (D&G, #112451)
    • Micropipette for DNA injection (make the micropipette by a micropipette puller)

2) In Utero Electroporation Procedure

  1. A 2 cm midline incision is then made in the abdominal wall along the linea alba using a set of forceps and scissors. A piece of sterile gauze with a hole cut in the center is placed over the incision, and one uterine horn is drawn out through the hole in the gauze.
  2. After observing the orientation of the embryos through the wall of the uterine horn, a micropipette is inserted into the lateral ventricle, and 2-5μl of plasmid solution is injected by expiratory pressure using the aspirator tube assembly. When CAG-EGFP is used, a concentration of 1μg/μl is sufficient to visualize the migrating neurons.
  3. After the injection, DNA solution containing 0.01% FastGreen can be seen through the uterine wall (red arrow).
  4. After soaking the uterine horn with PBS, the head of embryo is pinched with a tweezers-type electrode, and electronic pulses are applied with the electroporator.

Electroporation settings for ICR mice

AgeElectrode
(diameter)		VoltagePulse
On		Pulse
Off		Number
of Pulses
E12.53mm33V30msec970ms4
E13.55mm30V50msec950ms4
E14.55mm33V50msec950ms4
E15-5mm35V50msec950ms4

If viability was prioritized over transfection efficiency, the number of pulses can be changed to two.

The actual current is displayed on the screen of the electroporator (NEPA21/CUY21/CUY21E, Nepa Gene). Make sure that the current would become 30-60mA. The current varies according to how the electrode applied or wetness of the uterine horn. Examine the gap between electrodes and the electrode contact areas to fit the current in the appropriate range. If the current is still above the range after the examination, change the voltage setting.

3) GFP expression

in utero electroporation GFP

CAG-EGFP was injected into the both lateral ventricles of E14.5 mouse embryos and electronic pulses (33V, 50msec) were charged four times. 3 days later, the embryos (E17.5) were fixed and the brains were removed and examined under a fluorescence stereomicroscope (Fig. A).
Fluorescence was observed in the lateral region of the hemisphere onto which the anode had been placed and in the medial region of the opposite hemisphere.

Brains were frozen and sliced and the fluorescent image was obtained with a confocal laser microscope (Fig. B). GFP positive cells into which DNA was transferred at the ventricular zone (VZ) migrated to the intermediate zone (IZ) and cortical plate (CP). The arrowheads show the border between VZ and IZ and the border between IZ and CP. Dashed line show the border of tissues.
VZ: Ventricular Zone, IZ: Intermediate Zone, CP: Cortical Plate

 

Hidenori Tabata and Kazunori Nakajima, Department of Anatomy, Keio University School of Medicine

PUBLICATIONS

  • Mouse_In_Utero
  • E14.0
  • E15.5
  • Lateral_ventricle

Assembly of neuron- and radial glial-cell-derived extracellular matrix molecules promotes radial migration of developing cortical neurons

Mubuchi A, Takechi M, Nishio S, Matsuda T, Itoh Y, Sato C, Kitajima K, Kitagawa H, Miyata S.

Elife. 2024 Mar 21:12:RP92342.

  • Mouse_In_Utero
  • E12.5_E13.0
  • E14.0_E15.0
  • E16.0_E17.0
  • Cerebral_cortex
  • Parietal_lobe_Somatosensory_area

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

Hamada N, Nishijo T, Iwamoto I, Shifman S, Nagata KI.

Cells. 2024 Mar 19;13(6):540.

  • Mouse_In_Utero
  • Lateral_ventricle
  • E14.5

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

Felske T, Tocco C, Péron S, Harb K, Alfano C, Galante C, Berninger B, Studer M.

PLoS Biol. 2023 Aug 8;21(8):e3002237.

  • Mouse_In_Utero
  • E14.0
  • Lateral_ventricle

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

Tabata H, Mori D, Matsuki T, Yoshizaki K, Asai M, Nakayama A, Ozaki N, Nagata KI.

Biomolecules. 2023 Apr 27;13(5):763.

  • Mouse_In_Utero
  • E13.5
  • E14.5
  • Cerebral_ventricles

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

Ortiz-Álvarez G, Fortoul A, Srivastava A, Moreau MX, Bouloudi B, Mailhes-Hamon C, Delgehyr N, Faucourt M, Bahin M, Blugeon C, Breau M, Géli V, Causeret F, Meunier A, Spassky N.

Cell Rep. 2022 Dec 13;41(11):111810.

  • Mouse_In_Utero
  • Cerebral_cortex
  • Parietal_lobe_Somatosensory_area
  • E14.0

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

Scala M, Nishikawa M, Ito H, Tabata H, Khan T, Accogli A, Davids L, Ruiz A, Chiurazzi P, Cericola G, Schulte B, Monaghan KG, Begtrup A, Torella A, Pinelli M, Denommé-Pichon AS, Vitobello A, Racine C, Mancardi MM, Kiss C, Guerin A, Wu W, Gabau Vila E, Mak BC, Martinez-Agosto JA, Gorin MB, Duz B, Bayram Y, Carvalho CMB, Vengoechea JE, Chitayat D, Tan TY, Callewaert B, Kruse B, Bird LM, Faivre L, Zollino M, Biskup S; Undiagnosed Diseases Network; Telethon Undiagnosed Diseases Program; Striano P, Nigro V, Severino M, Capra V, Costain G, Nagata KI.

Brain. 2022 Sep 14;145(9):3308-3327.

  • Mouse_In_Utero
  • Cerebral_cortex
  • E14.5

Tbr1 Misexpression Alters Neuronal Development in the Cerebral Cortex

Crespo I, Pignatelli J, Kinare V, Méndez-Gómez HR, Esgleas M, Román MJ, Canals JM, Tole S, Vicario C.

Mol Neurobiola. 2022 Sep;59(9):5750-5765.

  • Mouse_In_Utero

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

Sapir T, Kshirsagar A, Gorelik A, Olender T, Porat Z, Scheffer IE, Goldstein DB, Devinsky O, Reiner O.

Nat Commun. 2022 Jul 21;13(1):4209.

  • Mouse_In_Utero
  • E13.5
  • E14.5
  • Lateral_ventricle

Endosomal trafficking defects alter neural progenitor proliferation and cause microcephaly

Carpentieri JA, Di Cicco A, Lampic M, Andreau D, Del Maestro L, El Marjou F, Coquand L, Bahi-Buisson N, Brault JB, Baffet AD.

Nat Commun. 2022 Jan 10;13(1):16.

  • Mouse_In_Utero
  • E14.0
  • Lateral_ventricle

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

Sapir T, Kshirsagar A, Gorelik A, Olender T, Porat Z, Scheffer IE, Goldstein DB, Devinsky O, Reiner O.

Nat Commun. 2022 Jul 21;13(1):4209.

  • Mouse_In_Utero
  • E14.0
  • Cerebral_cortex
  • Parietal_lobe_Somatosensory_area

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

Nishikawa M, Ito H, Tabata H, Ueda H, Nagata KI.

Cells. 2022 Feb 16;11(4):696.

  • Mouse_In_Utero
  • Lateral_ventricle

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

Bando Y, Wenzel M, Yuste R.

Nat Commun. 2021 Dec 10;12(1):7229

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

Hayano Y, Ishino Y, Hyun JH, Orozco CG, Steinecke A, Potts E, Oisi Y, Thomas CI, Guerrero-Given D, Kim E, Kwon HB, Kamasawa N, Taniguchi H.

Sci Adv. 2021 Jul 14;7(29):eabf1600.

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

Takechi M, Oshima K, Nadano D, Kitagawa H, Matsuda T, Miyata S.

Biochim Biophys Acta Gen Subj. 2020 Oct;1864(10):129679.

  • Mouse_In_Utero
  • E13.5
  • Cerebral_cortex

Huntington's disease alters human neurodevelopment

Barnat M, Capizzi M, Aparicio E, Boluda S, Wennagel D, Kacher R, Kassem R, Lenoir S, Agasse F, Braz BY, Liu JP, Ighil J, Tessier A, Zeitlin SO, Duyckaerts C, Dommergues M, Durr A, Humbert S.

Science. 2020 Aug 14;369(6505):787-793.

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

Ueda M, Matsuki T, Fukada M, Eda S, Toya A, Iio A, Tabata H, Nakayama A.

Mol Brain. 2020 May 24;13(1):80.

  • Mouse_In_Utero
  • E15.5
  • Lateral_ventricle

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

Taiga Kurihara, Emi Kouyama-Suzuki, Michiru Satoga, Xue Li, Moataz Badawi, Thiha, Deeba Noreen Baig, Toru Yanagawa, Takeshi Uemura, Takuma Mori, Katsuhiko Tabuchi

Biochem Biophys Res Commun. 2020 Apr 9;524(3):621-628.

  • Mouse_In_Utero
  • E14.5_E15.5
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In Vivo Imaging of the Coupling Between Neuronal and CREB Activity in the Mouse Brain

Tal Laviv, Benjamin Scholl, Paula Parra-Bueno, Beth Foote, Chuqiu Zhang, Long Yan, Yuki Hayano, Jun Chu, Ryohei Yasuda

Neuron, 105 (5), 799-812.e5 2020 Mar 4

  • Mouse
  • Otocyst

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

Hiroki Takeda, Toru Miwa, Min Young Kim, Byung Yoon Choi, Yorihisa Orita, Ryosei Minoda

Sci Rep, 9 (1), 17979 2019 Nov 29

  • Mouse_In_Utero
  • E15.0
  • Postnatal_electroporation
  • P2
  • Adult_Mouse_electroporation
  • P21

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

Stoufflet J, Chaulet M, Doulazmi M, Fouquet C, Dubacq C, Métin C, Schneider-Maunoury S, Trembleau A, Vincent P, Caillé I.

Sci Adv. 2020 Sep 2;6(36):eaba3992

  • Mouse_In_Utero
  • E14.5
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A Novel LGI1 Missense Mutation Causes Dysfunction in Cortical Neuronal Migration and Seizures

Feng Liu, Chao Du, Xin Tian, Yuanlin Ma, Bei Zhao, Yin Yan, Zijun Lin, Peijia Lin, Ruijiao Zhou, Xuefeng Wang

Brain Res, 1721, 146332 2019 Oct 15

  • Mouse
  • Embryonic_inner_ear
  • Otocyst

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

Toru Miwa, Ryosei Minoda, Yoshihide Ishikawa, Tomohito Kajii, Yorihisa Orita, Takahiro Ohyama

Biol Open, 8 (8) 2019 Aug 20

  • Mouse_In_Utero
  • E14.5
  • Lateral_ventricle

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

Yu-Jie Lai, Bing-Lin Zhu, Fei Sun, Dong Luo, Yuan-Lin Ma, Bio Luo, Jing Tang, Ming-Jian Xiong, Lu Liu, Yan Long, Xiao-Tong Hu, Ling He, Xiao-Juan Deng, John H Zhang, Jian Yang, Zhen Yan, Guo-Jun Chen

Aging Cell, 18 (4), e12961 Aug 2019

  • Mouse_In_Utero
  • E14.5
  • Cerebral_ventricles

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

Steinecke A, Kurabayashi N, Hayano Y, Ishino Y, Taniguchi H.

Cell Rep, 28 (2), 325-331.e4 2019 Jul 9

  • Mouse_In_Utero
  • E14.5
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Both Excitatory and Inhibitory Neurons Transiently Form Clusters at the Outermost Region of the Developing Mammalian Cerebral Neocortex

Shin M, Kitazawa A, Yoshinaga S, Hayashi K, Hirata Y, Dehay C, Kubo KI, Nakajima K.

J Comp Neurol. 2019 Jul 1;527(10):1577-1597.

  • Mouse_In_Utero
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Pathological mTOR Mutations Impact Cortical Development

Tarkowski B, Kuchcinska K, Blazejczyk M, Jaworski J.

Hum Mol Genet. 2019 Jul 1;28(13):2107-2119.

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Proc Natl Acad Sci U S A. 2019 Jun 25;116(26):13097-13106.

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

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FASEB J. 2019 Mar;33(3):3601-3612.

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                                                                                    Tabata H, Nakajima K.

                                                                                    J Neurosci . 2003 Nov 5;23(31):9996-10001.

                                                                                      The in vivo roles of STEF/Tiam1, Rac1 and JNK in cortical neuronal migration

                                                                                      Kawauchi T, Chihama K, Nabeshima Y, Hoshino M.

                                                                                      EMBO J. 2003 Aug 15;22(16):4190-201.

                                                                                        Serine 732 Phosphorylation of FAK by Cdk5 Is Important for Microtubule Organization, Nuclear Movement, and Neuronal Migration

                                                                                        Xie Z, Sanada K, Samuels BA, Shih H, Tsai LH.

                                                                                        Cell. 2003 Aug 22;114(4):469-82.

                                                                                          Mammalian BarH1 Confers Commissural Neuron Identity on Dorsal Cells in the Spinal Cord

                                                                                          Saba R, Nakatsuji N, Saito T.

                                                                                          J Neurosci . 2003 Mar 15;23(6):1987-91.

                                                                                            Emx2 patterns the neocortex by regulating FGF positional signaling

                                                                                            Fukuchi-Shimogori T, Grove EA.

                                                                                            Nat Neurosci. 2003 Aug;6(8):825-31.

                                                                                              Roles of the Basic Helix-Loop-Helix Genes Hes1 and Hes5 in Expansion of Neural Stem Cells of the Developing Brain

                                                                                              Ohtsuka T, Sakamoto M, Guillemot F, Kageyama R.

                                                                                              J Biol Chem . 2001 Aug 10;276(32):30467-74.

                                                                                              • E12.5_E13
                                                                                              • E14_E15
                                                                                              • E16_17

                                                                                              Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex

                                                                                              Tabata H, Nakajima K.

                                                                                              Neuroscience. 2001;103(4):865-72.

                                                                                              Electroporation

                                                                                              Drug Delivery and Transfection

                                                                                              Electro Cell Fusion

                                                                                              Fluorescent Staining

                                                                                              Single-Cell/Micro-Particle Transfer

                                                                                              Cell Freezing

                                                                                              Mechanical Vibration