Transfection into Chick Embryos in Ovo/Others by Electroporation

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Transfection into Chick Embryos in Ovo/Others by Electroporation

APPLICATIONS

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)

apli_cuy03a_e

  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 dye.hn: 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

PUBLICATIONS

  • In_ovo
  • Neural_tube

Loss of neuronal activity facilitates surface accumulation of p75NTR and cell death in avian cochlear nucleus

Sato R, Adachi R, Yokoi N, Tsujimura K, Egawa R, Hara Y, Fukata Y, Fukata M, Ogi T, Sone M, Kuba H.

Neurosci Res. 2025 Jan 21:S0168-0102(25)00014-8.

  • In_ovo
  • Forelimb_bud

Generation of a transgenic chicken line with reporters for limb bud mesenchyme and apical ectodermal ridge cells

Atsuta Y, Chen YC, Hattori Y, Takemoto T, Saito D.

Dev Biol. 2025 Jan 8:520:53-61.

  • In_ovo
  • Endoderm

Wide‐ranging migration and destination of early olfactory placode‐derived neurons in chick embryos

Miyakawa M, Murakmai S, Uchiyama Y.

Anat Rec (Hoboken). 2023 Feb;306(2):298-310.

  • In_ovo
  • Neural_tube

CDK5/p35-Dependent Microtubule Reorganization Contributes to Homeostatic Shortening of the Axon Initial Segment

Jahan I, Adachi R, Egawa R, Nomura H, Kuba H.

J Neurosci. 2023 Jan 18;43(3):359-372.

  • In_ovo
  • Neural_tube

Sustained experimental activation of FGF8/ERK in the developing chicken spinal cord models early events in ERK-mediated tumorigenesis

Wilmerding A, Bouteille L, Caruso N, Bidaut G, Etchevers HC, Graba Y, Delfini MC.

Neoplasia. 2022 Feb;24(2):120-132.

  • In_ovo
  • Anterior_primitive_streak

Mechanical Coupling Coordinates the Co-elongation of Axial and Paraxial Tissues in Avian Embryos

Xiong F, Ma W, Bénazéraf B, Mahadevan L, Pourquié O.

Dev Cell. 2020 Nov 9;55(3):354-366.e5.

  • In_ovo
  • Gonadal_epithelium

Oestrogen in the Chick Embryo Can Induce Chromosomally Male ZZ Left Gonad Epithelial Cells to Form an Ovarian Cortex That Can Support Oogenesis

Silvana Guioli, Debiao Zhao, Sunil Nandi, Michael Clinton, Robin Lovell-Badge

Development. 2020 Feb 25;147(4):dev181693.

  • In_ovo
  • Forelimb

L-type Voltage-Gated Ca 2+ Channel Ca V 1.2 Regulates Chondrogenesis During Limb Development

Yuji Atsuta, Reiko R Tomizawa, Michael Levin, Clifford J Tabin

Proc Natl Acad Sci U S A. 2019 Oct 22;116(43):21592-21601.

  • In_Ovo
  • Midgut

Genetic and Mechanical Regulation of Intestinal Smooth Muscle Development

Tyler R Huycke, Bess M Miller, Hasreet K Gill, Nandan L Nerurkar, David Sprinzak, L Mahadevan, Clifford J Tabin

Cell. 2019 Sep 19;179(1):90-105.e21.

  • In_Ovo
  • Ciliary ganglion(CG)

Analysis of Neuro-Neuronal Synapses Using Embryonic Chick Ciliary Ganglion via Single-Axon Tracing, Electrophysiology, and Optogenetic Techniques

Egawa R, Yawo H.

Curr Protoc Neurosci. 2019 Apr;87(1):e64.

  • In_ovo
  • Brain
  • Eye
  • Ex_Ovo
  • Retina

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

Miruna G. Ghinia Tegla, Diego F. Buenaventura, Diana Y Kim, Cassandra Thakurdin, Kevin C. Gonzalez, Mark M Emerson.

bioRxiv. 2019 February 2, https://doi.org/10.1101/538710

  • In_ovo
  • Ex_ovo
  • Limb

Timed Collinear Activation of Hox Genes during Gastrulation Controls the Avian Forelimb Position

Sahasrabudhe A, Ghate K, Mutalik S, Jacob A, Ghose A

Curr Biol. 2019 Jan 7;29(1):35-50.e4.

  • Ex_ovo
  • Endoderm

Molecular control of macroscopic forces drives formation of the vertebrate hindgut

Nerurkar NL, Lee C, Mahadevan L, Tabin CJ.

Nature. 2019 Jan;565(7740):480-484.

  • Ex_ovo

Body Cavity Development Is Guided by Morphogen Transfer between Germ Layers

Schlueter J, Mikawa T.

Cell Rep. 2018 Aug 7;24(6):1456-1463.

  • In_ovo
  • Spinal_cord

Formin-2 regulates stabilization of filopodial tip adhesions in growth cones and affects neuronal outgrowth and pathfinding in vivo.

Sahasrabudhe A, Ghate K, Mutalik S, Jacob A, Ghose A.

Development. 2016 Feb 1;143(3):449-60.

  • In_ovo
  • Forelimb_bud

Specialized filopodia direct long-range transport of SHH during vertebrate tissue patterning.

Sanders TA, Llagostera E, Barna M.

Nature. 2013 May 30;497(7451):628-32

  • In_ovo
  • Spinal_cord

Midline crossing and Slit responsiveness of commissural axons require USP33

Yuasa-Kawada J, Kinoshita-Kawada M, Wu G, Rao Y, Wu JY.

Nat Neurosci. 2009 Sep;12(9):1087-9.

  • In_ovo
  • Somites

In Ovo Electroporation for Targeting the Somitic Mesoderm

Ohata E, Takahashi Y.

Electroporation and Sonoporation in Developmental Biology, 2009.

  • In_ovo
  • Neural_tube

Netrin-1 is a survival factor during commissural neuron navigation.

Furne C, Rama N, Corset V, Chédotal A, Mehlen P.

Proc Natl Acad Sci U S A . 2008 Sep 23;105(38):14465-70.

  • In_ovo
  • Neural_plate
  • Neural_tube

Method for electroporation for the early chick embryo

Hatakeyama J, Shimamura K.

Dev Growth Differ. 2008 Aug;50(6):449-52.

  • Ex_ovo
  • Neural_plate
  • Neural_tube

Apical Accumulation of Rho in the Neural Plate Is Important for Neural Plate Cell Shape Change and Neural Tube Formation

Kinoshita N, Sasai N, Misaki K, Yonemura S.

Mol Biol Cell. 2008 May;19(5):2289-99.

  • In_ovo

Integration of growth and specification in chick wing digit-patterning

Towers M, Mahood R, Yin Y, Tickle C.

Nature. 2008 Apr 17;452(7189):882-6.

  • In_ovo
  • Otocyst

Bmp4 Is Essential for the Formation of the Vestibular Apparatus that Detects Angular Head Movements

Chang W, Lin Z, Kulessa H, Hebert J, Hogan BL, Wu DK.

PLoS Genet. 2008 Apr 11;4(4):e1000050.

  • In_ovo
  • Neural_tube

Expression dynamics of the LIM-homeobox genes, Lhx1 and Lhx9, in the diencephalon during chick development

Sun X, Saitsu H, Shiota K, Ishibashi M.

Int J Dev Biol. 2008;52(1):33-41.

  • In_ovo
  • Brain

In-vivo gene transfer into newly hatched chick brain by electroporation

Yamaguchi S, Katagiri S, Hirose N, Fujimoto Y, Mori M, Fujii-Taira I, Takano T, Matsushima T, Homma KJ.

Neuroreport. 2007 May 28;18(8):735-9.

  • Ex_ovo
  • Anterior_primitive_streak

Dual mode of paraxial mesoderm formation during chick gastrulation

Iimura T, Yang X, Weijer CJ, Pourquié O.

PNAS, Volume 104, Number 8, Pages 2744-2749, 20 February 2007

  • Ex_ovo

Collinear activation of Hoxb genes during gastrulation is linked to mesoderm cell ingression

Iimura T, Pourquié O.

Nature. 2006 Aug 3;442(7102):568-71.

  • In_ovo
  • Endoderm

FGF8 initiates inner ear induction in chick and mouse

Ladher RK, Wright TJ, Moon AM, Mansour SL, Schoenwolf GC.

Genes Dev. 2005 Mar 1;19(5):603-13.

  • In_ovo
  • Neural_tube

Gain- and loss-of-function in chick embryos by electroporation

Nakamura H, Katahira T, Sato T, Watanabe Y, Funahashi J.

Mech Dev. 2004 Sep;121(9):1137-43.

  • In_ovo
  • Neural_tube
  • Brain

RGM and its receptor neogenin regulate neuronal survival

Matsunaga E, Tauszig-Delamasure S, Monnier PP, Mueller BK, Strittmatter SM, Mehlen P, Chédotal A.

Nat Cell Biol. 2004 Aug;6(8):749-55.

  • Ex_ovo
  • Somites

A primer on using in ovo electroporation to analyze gene function

Krull CE.

Dev Dyn. 2004 Mar;229(3):433-9.

  • In_ovo
  • Primitive_cardiac_tube
  • Ex_ovo
  • Cardiac_explants

Dual roles of Sema6D in cardiac morphogenesis through region-specific association of its receptor, Plexin-A1, with off-track and vascular endothelial growth factor receptor type 2

Toyofuku T, Zhang H, Kumanogoh A, Takegahara N, Suto F, Kamei J, Aoki K, Yabuki M, Hori M, Fujisawa H, Kikutani H.

Genes Dev . 2004 Feb 15;18(4):435-47.

  • In_ovo
  • Neural_fold
  • Neural_tube
  • Ex_ovo
  • Neural_tube_explants

Multiple roles of Sox2, an HMG-box transcription factor in avian neural crest development

Wakamatsu Y, Endo Y, Osumi N, Weston JA.

Dev Dyn. 2004 Jan;229(1):74-86.

  • In_ovo
  • Recardiac_mesoderm

Tbx5 specifies the left/right ventricles and ventricular septum position during cardiogenesis

Takeuchi JK, Ohgi M, Koshiba-Takeuchi K, Shiratori H, Sakaki I, Ogura K, Saijoh Y, Ogura T.

Development. 2003 Dec;130(24):5953-64.

  • In_ovo
  • Neural_tube

Gene silencing in chick embryos with a vector-based small interfering RNA system

Katahira T, Nakamura H.

Dev Growth Differ. 2003 Aug;45(4):361-7.

  • In_ovo
  • Limb

Tbx5 and Tbx4 trigger limb initiation through activation of the Wnt/Fgf signaling cascade

Takeuchi JK, Koshiba-Takeuchi K, Suzuki T, Kamimura M, Ogura K, Ogura T.

Development. 2003 Jun;130(12):2729-39.

  • In_ovo
  • Prospective_esophago_tracheobronchial_region

Tbx4-Fgf10 system controls lung bud formation during chicken embryonic development

Sakiyama J, Yamagishi A, Kuroiwa A.

Development. 2003 Apr;130(7):1225-34.

  • In_ovo
  • Brain

Irx4-mediated regulation of Slit1 expression contributes to the definition of early axonal paths inside the retina

Jin Z, Zhang J, Klar A, Chédotal A, Rao Y, Cepko CL, Bao ZZ.

Development. 2003 Mar;130(6):1037-48.

  • In_ovo
  • Brain

The role of Grg4 in tectal laminar formation

Sugiyama S, Nakamura H.

Development. 2003 Feb;130(3):451-62.

  • Ex_ovo
  • Ectoderm

Bimodal functions of Notch-mediated signaling are involved in neural crest formation during avian ectoderm development

Endo Y, Osumi N, Wakamatsu Y.

Development. 2002 Feb;129(4):863-73.

  • In_ovo
  • Retina

The chicken RaxL gene plays a role in the initiation of photoreceptor differentiation

Chen CM, Cepko CL.

Development. 2002 Dec;129(23):5363-75.

  • In_ovo
  • Brain

Targeting Axons to Specific Fiber Tracts In Vivo by Altering Cadherin Expression

Treubert-Zimmermann U, Heyers D, Redies C.

J Neurosci. 2002 Sep 1;22(17):7617-26.

  • In_ovo
  • Limb

Efficient ectopic gene expression targeting chick mesoderm

Oberg KC, Pira CU, Revelli JP, Ratz B, Aguilar-Cordova E, Eichele G.

Dev Dyn. 2002 Jul;224(3):291-302.

  • Ex_ovo
  • Brain

Early subdivisions in the neural plate define distinct competence for inductive signals

Kobayashi D, Kobayashi M, Matsumoto K, Ogura T, Nakafuku M, Shimamura K.

Development . 2002 Jan;129(1):83-93.

  • In_ovo
  • Neural_tube

Inductive signal and tissue responsiveness defining the tectum and the cerebellum

Sato T, Araki I, Nakamura H.

Development 2001 Jul;128(13):2461-9.

  • In_ovo
  • Neural_tube

Misexpression of genes in brain vesicles by in ovo electroporation

Nakamura H, Watanabe Y, Funahashi J.

Dev Growth Differ . 2000 Jun;42(3):199-201.

  • In_ovo

'Shocking' developments in chick embryology: electroporation and in ovo gene expression

Itasaki N, Bel-Vialar S, Krumlauf R.

Nat Cell Biol. 1999 Dec;1(8):E203-7.

Electroporation

Drug Delivery and Transfection

Electro Cell Fusion

Fluorescent Staining

Single-Cell/Micro-Particle Transfer

Cell Freezing

Mechanical Vibration