Methods

Procedures

The method of preparing a mixture of "AteloGene®" & siRNA is common to local and systemic administration.
Please refer to the attached instruction manual in the kit for details of following operating procedures.

1)Preparation of "AteloGene®"

「Pour the whole amount of "AteloGene®" from the prefilled syringe into a microtube, and keep it on cooling device.

2)Preparation of siRNA solution

Prepare 600 µL of 5-10 µM or 20-40 µM siRNA solutions in the case of local or systemic administration, respectively. Keep the prepared solution cold.

3)Preparation of "AteloGene®" & siRNA mixed solution.

While cooling it on cooling device, gently pour the siRNA solution [in 2) above] on "AteloGene®" [in 1) above], and prepare the "AteloGene®" & siRNA mixture by tumbling, rotating and mixing them.

4) Removing bubbles and preparation for administration

Centrifuge the "AteloGene®" & siRNA mixture [in 3) above] to remove bubbles. Suck the mixture into a disposable syringe.

Mechanism of siRNA transfection using "AteloGene®"

Method of local administration

It is effective to inject the "AteloGene®" & siRNA mixture so as to wrap up the whole target site. The standard single dose for a mouse is 200 µL of "AteloGene®" & siRNA mixture.

Example of local administration

<Administration to subcutaneous tumor>

1. Anesthetize the animal, if necessary.
2. Insert the injection needle from about 5 mm this side of the tumor with the cut face of the needle turned upward.
3. Lay the needle parallel to the skin and insert it for 2 to 3 mm in the direction of the tumor. Then, inject 200 µL of the "AteloGene®" & siRNA mixture gently.

Method of systemic administration

The standard single dose for a mouse is 200 µL of "AteloGene®" & siRNA mixture.
The upper limit of the one-time dose is 200 µL. Be sure not to exceed this dose.

1. Anesthetize the animal, if necessary.
2. Fix the mouse.
3. Disinfect the tail of the mouse by wiping it with a cotton swab immersed in ethanol.
4. Insert the needle into the vein at a position 1/4-1/3 from the tail end that is fully inflated.
5. Confirm that the injection needle has entered the vessel and then, slowly inject 200 µL of the "AteloGene®" & siRNA mixture.
6. Confirm the recovery of the mouse.

Confirmation of siRNA transfection effect

Effect of siRNA transfection by "AteloGene®" differs depending on the siRNA sequence, expression level of the target gene, difference in target tissue, etc. Please investigate the duration required after administration for confirmation of effect and the analysis method.

Precautions for operation

1. Perform the procedures in a clean environment.
2. Secure a measure to eliminate RNase to avoid the degradation of siRNA by RNase.
3. During storage, 10×siRNA buffer may generate crystals. In this case, heat it up to about 37℃ and completely dissolve it before use.
4. When the "AteloGene®" content is squeezed out, ensure that bubbles are not hold.