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

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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/Ovary Bladder– Mouse Embryo – Entire Brain – Knee Joint Tissue/Brain Slice
In Utero: Mouse/Rat Embryo	MOUSE/RAT EMBRYO: – Cerebral – Cortex – Hippocampus – Spinal Cord Cultured Mouse Embryos and Large Tissue Small 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