In Ovo Electroporation

In Ovo (Chic Embryo) Electroporation
The following information indicates the depth of support we can offer our clients for Chick Embryo EP.
We have been making systems and supporting our clients’s In Ovo EP experiments for over 20 years.
The NEPA21 is our latest system and updates and replaces our old CUY21 series systems

In Ovo Electrode Recommendation:
For In Ovo EP (Mid Brain / Hind Brain), we recommendation:
–    CUY613P1  or
–    CUY611

If, however, one wishes to target a specific (smaller) part of Neural Tube, we recommend a combination of:
–          CUY611 series (or CUY613 series) and
–          CUY614T (For your information, the tungsten tip is very sharp)
The correct combination choice is dependent on the target size.
(Indeed, with the CUY650P1 electrode, the transfection site can be even smaller).

With the CUY611 series, a pair of electrodes (held by an electrode holder CUY580) is moved downwards in a long vertical line by a manipulator and placed on the target part of the embryo.

Some clients, however, who have to contend with a large number of samples on a daily basis find it difficult to manipulate a pair of electrodes vertically.  Such clients prefer to move the pair of electrodes horizontally.  The CUY613 series was designed for such clients.

Generally speaking, the CUY611 series is used for gene transfection into the ectoderm or neural tube of the chick embryo and the CUY613 series is used for gene transfection into the mesoderm and endoderm. Both series require the electrode holder CUY580.

The appropriate electrode for one’s application depends on the size of the target where the genes are to be delivered. If the length of the target is 1mm, the CUY613P1 is appropriate.  However, if one would like to deliver the genes to a more specific target area, we recommend the combination of CUY613 and tungsten needle CUY614T.  Please note the Tip for In Ovo Electroporation.  It describes how a neural tube is placed between the electrodes and then the electrodes are pushed slightly against an embryo, so that the neural tube swells up.  As one will find, this will increase the efficiency of gene transfection.

NEPA21/CUY21 – In Ovo Publication List:
NEPA21 and CUY21 Publication List

(with Parameter Settings and Electrode Recommendations)

In terms of resource information:
The following publication / know-how articles are useful:
–    Gain- and loss-of-function in chick embryos by electroporation (Nakamura et al.)
–    Chapter 6: Method of Electroporation for the Early Chick Embryo (Jun Hatakeyama and Kenji Shimamura)
–    Specialized filopodia direct long-range transport of SHH during vertebrate tissue patterning (Timothy A. Sanders, Esther Llagostera & Maria Barna)

In Gain- and loss-of-function in chick embryos by electroporation (Nakamura et al.) the following electrodes are used:
–    CUY580
–    CUY613P1
–    CUY614 & CUY615 (Replaced by the CUY614T)
The EP Conditions detailed in the publication are for our old CUY21 system.
If you purchase the NEPA21 system, we provide equivalent (and better) NEPA21 multi-pulse EP Conditions.

NEPA21 v BTX for Ex-Ovo Chick Endoderm
As client at the Harvard Medical School compares his BTX system and our NEPA21 system for:
–    Ex-Ovo Chick Endoderm
(He subsequently purchased the NEPA21 system)

To explain the difference between the NEPA21 and BTX ECM 830 devices, please note this link:
–    Measurement and Display of Delivered EP Pulse – Comparison of NEPA21 and BTX ECM830

In Ovo EP Targeting the Somitic Mesoderm
–    Please note the article: In Ovo Electroporation for Targeting the Somitic Mesoderm
–    We recommend our CUY610P4-1

This is a useful link to our website:  In Ovo EP Resource link
Please also note the following links to further information on the NEPA21’s In Ovo capacity

Click here for further:
– Detailed APPLICATION INFORMATION
– ELECTRODE INFORMATION
– NEPA21/CUY21 Publications
  (Where a reference is made to the CUY21 systems (EDIT or SC), the NEPA21 replaces them and can replicate their exact same output pulse form – in addition to its own multi-pulse and polarity-exchange capability).

Cultured Embryos
Publication and reference material:
–    Manipulating gene expressions by electroporation in the developing brain of mammalian embryos
–    NEPA21 and CUY21 Publication List and Electrode Recommedations for Rat  Mouse Cultured Embryo
–    NEPA21 and CUY21 Publication List for IN OVO EP  – with Parameter Settings and Electrode Recommendations
–    Electroporation-mediated gene transfer system applied to cultured CNS neurons
–    Chapter 6: Method of Electroporation for the Early Chick Embryo (Jun Hatakeyama and Kenji Shimamura)

In Ovo – Neural Tube and Brain Regions
Publication and reference material:
–    Gain- and loss-of-function in chick embryos by electroporation
–    Misexpression of genes in brain vesicles by in ovo electroporation
–    Applications for Chick Embryo

In Ovo and Ex Vivo and Ex Ovo Chick Retina EP
Publication and reference material:
–    Electroporation for early chick embryos using News culture (gastrula) – Application note 1.pdf
–    Electroporation-mediated gene transfer system applied to cultured CNS neurons – Application Note 2
–    Bimodal functions of Notch-mediated signaling are involved in neural crest formation during avian ectoderm development
–    Irx4-mediated regulation of Slit1 expression contributes to the definition of early axonal paths inside the retina
–    Ex ovo electroporation for gene transfer into older chicken embryos
–    Electroporation-mediated gene transfer system applied to cultured CNS neurons
–    Chick Embryo Retina EP Diagram

New Cultures
Publication and reference material:
–    Electroporation for early chick embryos using News culture (gastrula) – Application note 1
–    Collinear activation of Hoxb genes during gastrulation is linked to mesoderm cell ingression
–    Bimodal functions of Notch-mediated signaling are involved in neural crest formation during avian ectoderm development
–     Apical Accumulation of Rho in the Neural Plate Is Important for Neural Plate Cell Shape Change and Neural Tube Formation
–    Dual mode of paraxial mesoderm formation during chick gastrulation

Limb embryonic ectoderm
Publication and reference material:
Please note the following articles, especially the first named article
–    Efficient Ectopic Gene Expression Targeting Chick Mesoderm
–    FGF8 initiates inner ear induction in chick and mouse
–    PiggyBac-mediated somatic transformation of the two-spotted cricket, Gryllus bimaculatus
–    Tbx5 and Tbx4 trigger limb initiation through activation of the Wnt/Fgf – Limb_bud_2003
NOTE:
In “Tbx5 and Tbx4 trigger limb initiation through activation of the Wnt/Fgfsignaling cascade”, Takeuchi et al, stage 13 is specifically mentioned.

Chick Limb EP:
We usually recommend the following articles and electrodes used in those articles:
–    Congenic method in the chick limb buds by electroporation (Takayuki Suzuki* and Toshihiko Ogura, 2008)
–    Electroporation into the Limb (Beyond Misexpression) (Chapter 9: Takayuki Suzuki and Toshihiko Ogura)
–    CUY612P5-5-3 (anode electrode) and CUY612P6-3 (cathode electrode) are used in the articles
Technical drawing of both: CUY612P5-5-3 (anode electrode) and CUY612P6-3 (cathode electrode)
–    In Ovo chick limb EP – Parameter Sheet with CUY612P5-5-3 and CUY612P6-31

For your further information:
–          Please note this NEPA21 paper (Nature) Specialized filopodia direct long-range transport of SHH during vertebrate tissue patterning
–          It demonstrates In Ovo EP (forelimb)
–          The publication is by one of our clients and he used our electrodes: the CUY611P7-2  (as the negative electrode) and the CUY611P3-1 (as the positive electrode)
–          Technical drawings of both electrodes: CUY611P7-2  and CUY611P3-1

Please also note:
–          a recent presentation from Harvard Medical School
–          The presentation records their success with our NEPA21 device (in comparison with the BTX device)
–          It is Ex Ovo EP.  They had previously used the BTX ECM830 before purchasing the NEPA21

Chick Neural Tube – Ex Ovo RESULTS
–   with CY701P2E and CUY701P2L electrodes

Comparison of the NEPA21 and BTX ECM830
Measurement and Display of Delivered EP Pulse – Comparison of NEPA21 and BTX ECM830
It demonstrates the superiority of the NEPA21 device and the importance of controlling current rather than voltage.

Focal Electroporation on Slice Cultures and In Utero EP
Publication and reference material:
For Slice Culture
–    Negative electrode: CUY700 series or CUY701 series
–    Positive electrode: CUY614 & 615, CUY611P3-1 or CUY195P0.3
–    Protocols:
–    The Caudal Migratory Stream: A Novel Migratory Stream of Interneurons Derived from the Caudal Ganglionic Eminence in the Developing Mouse Forebrain
–    Electroporation-mediated gene transfer system applied to cultured CNS neurons
–    In utero electroporation

For In Utero
Electrodes: CUY650P3 or CUY650P5
Protocols:
–    Efficient in utero gene transfer system to the developing mouse brain using electroporation: visualization of neuronal migration in the developing cortex

NOTE:
The appropriate electrode size option is dependent on the size of your target.
Kindly note that we are also able to custom-manufacture electrodes to your own specifications.

Protocol Recommendation:

Kindly note: Cooperative action between L-Maf and Sox2 on d-crystallin gene expression during chick lens development.

Specifically, the drawings which demonstrate how to target various different parts of the Optic Vesicles by changing the position of an electrode and combination of electrodes, and

–  An electroporation protocol:

Kindly also note the file titled: ‘In Ovo Electroporation Resource’.  This file includes a ‘tip for in ovo electroporation and various publications with relevant protocol information.  The file includes the following five articles and in ovo electroporation tip:

Re Heart EP, please note the following article:

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

In Ovo Electroporation Resource:

Kindly identify the file name of interest and click on the relevant article to download it.