Transfection into Chick Embryos in Ovo/Others by Electroporation

Transfection into Chick Embryos in Ovo/Others by Electroporation


Misexpression of the gene of interest by in ovo electroporation

  1. Injection of the plasmid solution colored by Fast Green into the central canal.
  2. A pair of electrodes are put on the vitelline membrane overlying the embryos, and a 25V, 50 ms rectangular pulse was charged 4 times.
  3. We can monitor the transfection efficiency by electroporating GFP expression vector.
  4. Strong GFP fluorescence could be detected even 48 hours after electroporation.
  5. Right side of the embryo is transfected, hence the left side could be served as the control. (Dorsal view)

Mechanisms of brain regionalization and neural circuit formation have been studied by misexpression of transcription factors (En1/2, Pax2/5/6, Otx2, Gbx2), secreted factors (Fgf, Shh, semaphoring), signal transduction molecule (Ras, Sprouty2) and receptors (neuropilin). 


Harukazu Nakamura and Yuji Watanabe, Graduate School of Life Sciences, and Institute of Development, Aging and Cancer, Tohoku University
*Development Growth & Differentiation, Volume 42, Issue 3, Page 199-201, June 2000

Knock-down by transfection of shRNA expression vector by in ovo electroporation

  1. shRNA expression vector is electroporated as shown above.
  2. Select target DNA sequence of 19 to 21 mer. Sense and antisense sequence were linked to a nucleotide spacer as a loop and put into expression vector that is driven by U6 or H1 promoter. Commercially available expression vector that dreives expression by mouse U6 promoter is effective in chick embryos.
  3. After transcription, hairpin is digested to form siRNA, and siRNA forms RISK (RNA-induced silencing complex) to dgrade the target mRNA.

24 hours after electroporation

  1. Transfection is monitored by co-electroporated GFP fluorescence.
  2. Application of siRNA against En2 by electroporation shows degradation of En2 mRNA.
  3. Control side

Harukazu Nakamura and Tatsuya Katahira, Graduate School of Life Sciences, and Institute of Development, Aging and Cancer, Tohoku University
*Mechanisms of Development, Volume 121, Issue 9, Pages 1137-1143, September 2004
*Development Growth & Differentiation, Volume 45, Issue 4, Pages 361-367, August 2003

Electroporation for early chick embryos using New's culture (gastrula)


  1. Schema illustrating the system.
  2. Hamburger and Hamilton (HH) stage 4 chick embryo with DNA solution injected to the prospective neural plate region. DNA solution was colored with a FastGreen Hensen’s node, ps: primitive streak, ao: area opaca, ap: area pellucida
  3. Schema illustrating the location of DNA solution between the vitelline membrane and the ectoderm.

Experimental Procedure

  1. Dissect an embryo using a paper ring (dried filter paper) out of the egg.
  2. Rinse gently to remove excess yolk, and place the embryo onto the platform of the cathode chamber.
  3. Inject DNA solution into the space between the ectoderm and vitelline membrane using a glass pipette (for introduction to the ectoderm derivatives).
  4. Adjust the target site right onto the cathode, and set the anode above it. Set the electrode distance at 5mm.
  5. Deliver electric pulses (10V, pulse on 50ms, pulse of 100ms, 5 pulses) using a CUY21 electroporator.
  6. Place the specimen on an agarose-albumin plate, and incubate at 39C

Transgene expression after the electroporation

GFP expression becomes detectable by HH6 (about 5 hours after electroporation) in the neural plate, which spreads throughout the central nervous system at HH17 (Scale bar: 1mm).

  • GFP gene was introduced to the prospective neural plate at HH4, and the embryo was cultured for about 34 hours (HH17 equivalent).
  • GFP expression was occasionally monitored under a fluorescent dissecting microscope.
  • GFP fluorescence was detectable as early as 3 hours after electroporation at the central nervous system and head ectoderm.
  • More than 80% of cells within the transfected area express GFP.

Gene transfection for tissues other than the CNS

Based on the fate map of the chick epiblast, transgenes can be introduced to the somites (A, E), hematopoietic system (B, F), notochord (C, G), or lateral plate mesoderm (D, H).
E-H are the high power views of A-D, respectively.

Kenji Shimamura, Division of Morphogenesis, Institute of Molecular Embryology and Genetics, Kumamoto University



Drug Delivery and Transfection

Electro Cell Fusion

Fluorescent Staining

Single-Cell/Micro-Particle Transfer

Cell Freezing

Mechanical Vibration