In vivo -jetPEI

CatNo.:IC-6015

Description

in vivo-jetPEI is a linear polyethylenimine, which mediates efficient nucleic acid (DNA, shRNA, siRNA, miRNA, oligonucleotides, …) delivery to a wide range of tissues  using  various  delivery  routes:  intravenous  (IV),  intraperitoneal  (IP), intratumoral,  subcutaneous, topical,  intrathecal,  etc.  Upon  IV  administration, high levels of nucleic acid delivery are achieved into the lungs. Other organs such as salivary glands, heart, spleen and liver are also targeted following IV injection.

In addition, in vivo-jetPEI is an effective carrier for local gene and siRNA delivery such as intratumoral or topical application on the skin.

Storage

Store at 4^oC. DO NOT FREEZE.

Size

0.1 ml;0.5ml

1. in vivo transfection protocol

1.1. Reagents required

We recommend using the 10% isotonic glucose solution (w/v) provided in the kit. This is required in order to form small and stable nucleic acid/in vivo-jetPEI complexes. The use of ionic buffers such as PBS or cell culture media for complex preparation should be avoided.

The nucleic acid should be resuspended in low salt buffer since high salt content in the nucleic acid preparation may lead to precipitation upon complexes formation.

For DNA, the best results are achieved with high quality endotoxin free DNA resuspended in ddH2O and a stock solution of 3-7 μg/μL.

For si/miRNA, it is preferable to use high quality grade si/miRNA (PAGE or HPLC purification) and a stock concentration of 5-10 μg/μL.

1.2.   Recommended amount of nucleic acid and injection volume

The amount of nucleic acid to deliver should be determined according to the animal model, the administration route and the targeted organ. Recommendations for delivery of DNA, siRNA, oligonucleotides and shRNA-expressing plasmids in rodents are given in Table 1.

The concentration of nucleic acid in the final injection solution should not exceed 0.5 μg/μL.

The volume of reagent is defined by the N/P ratio and is calculated according to the formula on page 7. As a general guideline, we recommend using: N/P = 6 – 8. (i.e. 0.12 to 0.16 μL of in vivo-jetPEI per μg of nucleic acid). Prior to injections, ensure that in vivo-jetPEI and glucose solution are  equilibrated  at room temperature. 

Table 1. Recommended conditions for most common injection routes in mice and rats.

Depending on the application, multiple injections may be required,and we recommend keeping the frequency of injection to every 2 – 3 days, with a maximum of 3 injections per week per animal.

1.3.   Protocol

The preparation of the in vivo-jetPEI nucleic acid complexes should be performed in a laminar flow hood using a 10% glucose solution. The final concentration of glucose in the injection volume should be 5%.

We recommend preparing a master mix to ensure homogenous complex formation, the smallest mix being minimum 50 μL.

Define the experimental protocol and parameters:

•  Set the injection volume of complexes to be prepared per animal (Table 1).

Note: the final concentration of glucose in the injection volume is 5%.

•  Define the amount of nucleic acid to be delivered per injection (Table 1).

Note: the final concentration of nucleic acid in the injection volume should not exceed 0.5 μg/μL.

•  Choose the N/P ratio. As a general guideline, we recommend using: N/P = 6 – 8 (i.e. 0.12 to 0.16 μL of in vivo-jetPEI per μg of nucleic acid).

•  Calculate the corresponding volume of in vivo-jetPEI (Table 2).

Table 2. Volumes of in vivo-jetPEI to be used according to the N/P ratio and the amount of nucleic acid required.

Protocol overview

For homogeneous complex preparation, the nucleic acid solution should represent one half of the injection volume and the in vivo-jetPEI reagent solution should represent the other half of the injection volume.

1. Dilute the nucleic acid into ½ the injection volume in 5% glucose (final concentration) using the 10% glucose stock solution (provided) and sterile water. Vortex gently or mix by pipetting up and down.

2. Vortex in vivo-jetPEI reagent for 5 sec and spin down before use.

3. Dilute the in vivo-jetPEI reagent into ½ the injection volume in 5% glucose (final concentration) using the 10% glucose stock solution (provided) and sterile water. Vortex gently and spin down.

4. Add the diluted in vivo-jetPEI to the diluted nucleic acid all at once, vortex gently and spin down.

5. Incubate for 15 minutes at room temperature. From this time point, the complexes are stable 4 h at room temperature and for up to 7 days when stored at 4 °C.

6. Perform  injections  into  animals  using  complexes  equilibrated  at  room  temperature.  If  required, injections can be repeated up to 3 times a week.

7. Monitor gene expression as required at the appropriate time point (6 – 72 h after the last injection) depending on the mode of injection and the targeted organ.

Example: IV injection in mouse

Preparation of 200 μL injection volume of 5% glucose containing 40 μg of plasmid DNA and in vivo-jetPEI at N/P = 8

1. Dilute 40 μg of DNA into 50 μL of 10% glucose; add sterile water to 100 μL, vortex gently and spin down.

2. Dilute 6.4 μL of in vivo-jetPEI into 50 μL of 10% glucose; add sterile water to 100 μL, vortex gently and spin down.

3. Add the diluted in vivo-jetPEI to the diluted DNA at once, vortex briefly and spin down.

4. Incubate for 15 minutes at room temperature.

5. Perform injections into animals using complexes equilibrated at room temperature.

6. Monitor gene expression

2. Troubleshooting

3.  Product Information