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Featured Device

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Product Code: OCH01

Desc:Cell Holder
Applic:Electroporation and Sonoporation


Featured Electrode



Product Code: CUY480G3

Application:
Electrodes for Electro Cell Fusion
Description:
Oval Gold Electrode - 3mm gap, Volume 3.0ml

Information
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Mouse/Rat – Muscle

Muscle In Vivo
For targeting Mouse and Rat Muscle  we recommend three methods:

METHOD 1
This is  our preferred method as it is minimally invasive and only requires inserting the needle electrodes (CUY560-3-0.3 or CUY560-5-0.5) in the muscle above the skin.  (The CUY560-3-0.3 electrode is for smaller size targets).

Since no surgery is involved, the researcher can easily perform electroporation and do so consecutively in a short period of time.  In our opinion, this is the best method.  However, as the volume of muscle affects the resistance value, and thus, actual delivered current value, the researcher needs to practice picking up the same volume of muscle by his/her fingers every time the experiment is performed.

This method uses a DNA injection syringe (as one side of the electrode) together with a needle-type electrode (CUY560-3-0.3 or CUY560-5-0.5) and a C118 Cable:

–    Application Note (explaining the method)
–    Electro-transfer of Plasmid Vector DNA into Muscle  (Chapter22, Satsuki Miyazaki and Jun-ichi Miyazaki)
–    Photo of new muscle results
–    ‘EP with rats limb – photo’
–    Illustrated Applications
 …..When the webpage opens, please Click on the “Muscle” Link
–    C118 Technical Drawing
–    Photo of C118 connection to C115CB Cable

METHOD 2
The second option uses a tweezers-type electrode on the outside of the target’s shaved leg.  In this method, more time and care is required as the target’s skin is cut open and then the muscle is picked up with a tweezer-type electrode.  We recommend either of our CUY650P3 or CUY650P5 electrodes.  This is the method used by Dr. Takeshita in her paper (Toll-Like Receptor Adaptor Molecules Enhance DNA-Raised Adaptive Immune Responses against Influenza and Tumors through Activation of Innate Immunity, Journal of Virology, Volume 80, Number 13, Pages 6218-6224, July 2006) .  She cuts and opens the leg, injects DNA buffer into the exposed muscle, grasps the injected muscle with a tweezers-type electrode (CUY650P5) and then applies square pulses.  If one observes the protocol; the impedance is from 218 ohms to 298 ohms.  When the impedance value is in this range, the current value will be stable and this will lead to the high reproducibility of the experiments.  The impedance is adjusted by changing the position of electrodes slightly before electroporation.

–    a photo showing a shaved and cut leg together with the CUY650P5 tweezers-type electrode.

For your information:
We are aware that some researchers follow a similar methodology but do not cut the skin.
Unfortunately, we do not have comparison data for this variation of the methodology.
Please note the link to the article titled: Simultaneous gene transfer of bone morphogenetic protein (BMP)-2 and BMP-7 by in vivo electroporation induces rapid bone formation and BMP-4 expression, Kawai, BMC Musculoskeletal Disorders, May 2006.
In this article electroporation was conducted without cutting the skin.

As a variation on method 2 one first cuts open the skin of the limb but then a pair of needle electrodes CUY560-5/-10 is injected directly into the muscle. If the one has no previous experience of electroporation with limb, we recommend this method.  Kindly note:‘EP with rats limb – photo’for photos of the relevant methodology/protocol.

 

METHOD 3
This is a method that not too many of our clients use as the CUY692-21 electrode configuration is expensive.
However, it did demonstrate better results than the CUY560-3-0.3 configuration of Method 1.
Note the link to a Photo Result comparing the CUY692-21 v CUY560-3-0.3.

………–    CUY692-21-1 Technical Drawing
There is a further electrode configuration option: The CUY692-21.
The difference between CUY692-21 and CUY692-21-1 is the length of the needles.
For Skin, CUY692-21-1 is recommended.
For Muscle, CUY692-21 is suitable but depending on the target site, the needles could be too long.
However, the length of the needles can be tailored to the client’s specification.
With this method, TE can be improved with the use of hyaluronidase.

In Vivo Muscle Results
Regarding Intra-dermal delivery, please note:
Electroporation-mediated intradermal delivery of DNA vaccines in non human primates

Toll-Like Receptor Adaptor Molecules Enhance DNA-Raised Adaptive Immune Responses against Influenza and Tumors through Activation of Innate Immunity, Journal of Virology, Volume 80, Number 13, Pages 6218-6224, July 2006
Protocol Recommendations available in the following Rat & Mouse Muscle EP Resource
This link includes the following articles:
–  ‘Elevated gastrin secretion by in vivo gene electroporation in skeletal muscle’
–  ‘Anti-monocyte chemoattractant protein-1 gene therapy attenuates pulmonary hypertension in rats’
–  ‘Gene Therapy for Central Diabetes Insipidus: Effective Antidiuresis by Muscle-Targeted Gene Transfer’
–  ‘Protection Against Autoimmune Myocarditis by Gene Transfer of Interleukin-10 by Electroporation’
–  ‘Skeletal muscle targeting in vivo electroporation-mediated HGF gene therapy of bleomycin-induced pulmonary fibrosis in mice’
–  ‘Gene Transfer into muscle by Electroporation in vivo’
–  ‘Toll-Like Receptor Adaptor Molecules Enhance DNA-Raised Adaptive Immune Responses against Influenza and Tumors through Activation of Innate Immunity’
Kindly also note the following EP Protocol for Gene Delivery into Mouse Leg at Yokohama City University (Toll-Like Receptor Adaptor Molecules Enhance DNA-Raised Adaptive Immune Responses against Influenza and Tumors through Activation of Innate Immunity’):

 Target  Impedance  (Ω)    Voltage (V)  Pulse length   (msec)  Pulse interval  (msec)  No. of pulses  Measured Current (A)
 Right leg  240  30  50  100  3  0.18
 3*  0.21
 Left leg  222  30  50  100  3  0.18
 3*  0.2
 Right leg  218  30  50  100  3 0.19
 3*  0.21
 Left leg  277  30  50  100  3 0.15
3* 0.16
Right leg 294 30 50 100 3 0.14
3* 0.17
Left leg 277 30 50 100 3 0.12
3* 0.12
Right leg 264 30 50 100 3 0.15
3* 0.17
Left leg 298 30 50 100 3 0.12
3* 0.13
* Reverse Polarity

 

=====================================

Some further In Vivo electrode recommendations and resource information for In Utero EP with the CUY21 and NEPA21 systems.

Re: Muscles In Vivo:

For targeting mice and rats:

We recommend CUY560-3-0.3 and CUY560-5-0.5. I have attached the protocol.  (For your information, the CUY560-3-0.3 is for smaller size targets).

We also recommend getting CUY650P3 or CUY650P5.

 

Muscle:

–          Electro-transfer of Plasmid Vector DNA into Muscle  (Chapter22, Satsuki Miyazaki and Jun-ichi Miyazaki)

–          In Utero Electroporation System: Assay System for Migration of Cerebral Cortical Neurons (Chapter 14, Hidenori Tabata and Kazunori Nakajima)

–          Epidermis-Targeted Gene Transfer Using In Vivo Electroporation (Chapter 41, Hiroki Maruyama, Jun-Ichi Miyazaki, and Fumitake Gejyo)

–          Skeletal muscle targeting in vivo electroporation-mediated HGF gene therapy of bleomycin induced pulmonary fibrosis in mice

–          Modulation of scratching behavior by silencing an endogenous cyclooxygenase-1 gene in the skin through the administration of siRNA

–          Rat and Mouse: Publication List, EP Settings and Electrode Recommendations

 

Please note these links to our website.

IN VIVO

In Vivo: Mouse/Rat Tissue

MOUSE/RAT:
–    Brain
–    Eye
–    Muscle
–    Skin
–    Kidney
–    Liver
–    Tests/OvaryBladder–    Mouse Embryo – Entire Brain
–    Knee JointTissue/Brain Slice

In Utero: Mouse/Rat Embryo

MOUSE/RAT EMBRYO:
–    Cerebral
–    Cortex
–    Hippocampus
–    Spinal CordCultured Mouse Embryos and Large TissueSmall Tissue–    Whole Embryo Culture- Telencephalon (E7-E13)New Born Retina (P0-P3)New Born Cornea

In Ovo: Chick Embryo

CHICK EMBRYO:
–    Neural Tube
–    Limb Bud
–    Digestive Organ
–    Etc

 

For your further information, have a look through this Rat and Mouse Muscle (a useful Rat and Mouse Muscle information resource) and Applications using In Vivo Electrodes links to our site.  They point out further options (which you might wish to consider).

 

Re: In Vivo Mouse Adult Brain

–          In Vivo Adult Brain with CUY650P5 electrode or

–          In Vivo Adult Brain with CUY200S electrode set

–          Brain Slice EP with CUY700P5E & CUY700P5L electrodes

Other useful links include:

–          In Utero Resource

–          Applications with In Utero and Ex Utero Electrodes and

–          NEPA21 In Utero Video Demonstration (an In Utero video demonstration with the NEPA21 device):

 

Please note the following links to articles on our website and attached further resource material:

Focal Electroporation on Slice Cultures and In Utero EP

–       Publication and reference material:

  1.       For Slice Culture

Negative electrode: CUY700 series or CUY701 series

Positive electrode: CUY614 & 615, CUY611P3-1 or CUY195P0.3

Protocols:

    1. The Caudal Migratory Stream: A Novel Migratory Stream of Interneurons Derived from the Caudal Ganglionic Eminence in the Developing Mouse Forebrain
    2. Electroporation-mediated gene transfer system applied to cultured CNS neurons
    3. In utero electroporation.pdf

 

2.       For In Utero

Electrodes: CUY650P3 or CUY650P5

Protocols:

    1. 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.  For Mouse Brain In Utero E10 to E15 we recommend the CUY650P5.  For <E10 we recommend the CUY650P3.

Kindly note that we are also able to custom-manufacture electrodes to your own specifications.

 

Kindly also note the following summary information:

–    In Utero EP Resource on our website

–    Mouse and Rat (Whole Embryo Culture)Neural Tube (Mouse E9.5, Rat 11.5)

–    Mouse and Rat (Whole Embryo Culture)Neural Plate Metencephalon, Somite (Mouse E7-13)

–    Mouse and Rat (Whole Embryo Culture)Telencephalon (Mouse E7-13)

–    Mouse Embryo (In Utero)Spinal Cord (E10.5)

–    Mouse Embryo (In Utero)Cerebral Cortex, All Areas (>E13.5)

 

New Cultures

–        Publication and reference material:

–        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

–        NEPA21 and CUY21 Publication List and Electrode Recommedations for Rat  Mouse Cultured Embryo

–        In utero electroporation.pdf

–        Electroporation for early chick embryos using News culture (gastrula)

 

 

 

 


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