Electroporation in the Rodent Embryonic Brain Using Whole Embryo Culture System

Takako Kikkawa1, Masanori Takahashi2, Noriko Osumi1

1 Department of Developmental Neuroscience, United Center for Advanced Research and Translational Medicine (ART), Tohoku University School of Medicine, Sendai, Miyagi, 2 Division of Biology, Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 3.30
DOI:  10.1002/cpns.21
Online Posting Date:  January, 2017
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Abstract

This unit describes basic methods for mammalian whole embryo culture (WEC) using embryonic day 10.5 mouse embryos, including the preparation of high‐quality immediately centrifuged (IC) rat serum that is commonly used for WEC and is essential for normal growth and development of cultured mouse and rat embryos in vitro. An alternative protocol for different stages of rodent embryos is also introduced. Since embryos for WEC are dissected out of the uterus and manipulated under the microscope, one can overcome many of the difficulties of gene delivery encountered using in utero electroporation. A description for a gene transfer method to label neural stem/progenitor cells of the cortical primordium in a highly region‐specific manner is also included. © 2017 by John Wiley & Sons, Inc.

Keywords: whole embryo culture; electroporation; brain development

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Whole Embryo Culture for Embryonic Day 10.5 Mouse
  • Alternate Protocol 1: Time Course and Proper Oxygen Conditions of WEC for Different Stages of Rodent Embryos
  • Support Protocol 1: Preparation of IC Rat Serum
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Whole Embryo Culture for Embryonic Day 10.5 Mouse

  Materials
  • Immediately centrifuged (IC) rat serum (see protocol 3Support Protocol)
  • D‐(+)‐glucose (e.g., Wako Chemicals, cat. no. 041‐00595)
  • Antibiotic‐Antimycotic, 100× (e.g., Gibco, cat. no. 15240062)
  • Pregnant mouse, E10.5
  • Isoflurane
  • 70% ethanol
  • Tyrode's solution (see recipe)
  • Plasmid DNA (see recipe) or Stealth RNAi (e.g., Thermo Fisher Scientific)
  • PBS (see recipe)
  • 4% (w/v) paraformaldehyde (PFA) in PBS
  • 20% (w/v) sucrose
  • Optimal cutting temperature (OCT) compound
  • TBS (see appendix 2A) containing 3% (w/v) bovine serum albumin (BSA) and 0.1% (v/v) Triton X‐100
  • Appropriate primary antibody for protein of interest
  • Appropriate secondary antibody for primary antibody
  • DAPI
  • Mammalian whole embryo culture (WEC) system (e.g., Ikemoto Scientific Technology, cat. no. 10‐0310; Fig. )
  • 0.22‐µm and 0.45‐µm membrane filter, 33 mm diameter (e.g., EMD Millipore, cat. nos. SLGS033SS and SLHA033SS, respectively)
  • Gas mixture cylinder containing the following mixtures:
    • 60% O 2, 5% CO 2, 35% N 2
    • 95% O 2, 5% O 2
  • Water bottle
  • 37°C incubator
  • Glass culture bottle (e.g., Ikemoto Scientific Technology, cat. no. 010‐032‐05)
  • Silicone plug without a hole (e.g., Ikemoto Scientific Technology, cat no. 010‐032‐08)
  • Silicone plug with a hole (e.g., Ikemoto Scientific Technology, cat. no. 010‐032‐07)
  • Aluminum foil
  • Inhalation anesthetic system for mice
  • Scissors, straight and curved
  • Microscissors, straight and curved
  • Curved forceps
  • Fine forceps
  • Sterile disposable petri dishes (90 mm × 20 mm; e.g., Sansei, cat. no. 6‐8663‐02)
  • Stereomicroscope
  • Glass pipet with rubber bulb, 5 mm i.d.
  • Glass needle, for microinjection (1.0 mm o.d., 0.58 mm i.d., 10 cm length; e.g., Sutter Instruments, cat. no. B100‐58‐100)
  • Micropipet puller
  • Electric microinjector system (e.g., Narishige IM‐300)
  • Electrode chamber (20 mm l × 20 mm w × 8 mm h; e.g., BEX, cat. no LF520P20)
  • Electroporator (e.g., BEX CUY21EDIT)
  • Silicone plate with shallow pit
  • Cryostat
  • Confocal microscope

Alternate Protocol 1: Time Course and Proper Oxygen Conditions of WEC for Different Stages of Rodent Embryos

  Materials
  • Sprague Dawley male rat at 6 to 8 month of age (550 to 650 g)
  • Isoflurane
  • 70% ethanol
  • Inhalation anesthetic system for rodents
  • Large scissors
  • Curved forceps
  • 20‐ml syringe
  • 21‐G, 5/8 in. needle
  • 15‐ml disposable tubes, sterile
  • 10‐ml test tubes, sterile
  • Centrifuge
  • 56°C incubator
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Figures

Videos

Literature Cited

Literature Cited
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