MED-JET H4 MULTIJET (MJH4M) Transfection Protocol

Nathan Liu, Hani Abd-Ul-Salam, Noemie Joannette-Lafrance, Jingjing Li, Karim Menassa, Monzur Murshed

Published: 2023-07-04 DOI: 10.17504/protocols.io.kxygx3ekog8j/v1

Abstract

Transfection, a non-viral method of nucleic acid delivery, often shows poor efficiency in vivo . The needle-based in vivo delivery of transfection reagents can be invasive. Here, we report a non-invasive in vivo gene delivery protocol via the needle-free MED-JET H4 MULTIJET (MJH4M) devices using glucose-based and commercial transfection reagents. The objective of this study is to compare the relative transfection efficiencies of the needle-free system to that of the needle-based delivery method. The highest transfection efficiency was noted using 5% glucose as a delivery agent; a 15-fold increase was observed using MJH4M compared to delivery with needles.

Steps

1.

Prepare transfection reagent by diluting renilla-luciferase plasmid DNA (pR-Luc) in 5% glucose (w/v) to a final concentration of 0.1 μg/μl.

2.

Insert the transfection reagent cartridge into the MED-JET H4 MULTIJET (MJH4M) device.

3.

MJH4M device parameters were set to 80 psi and 75 µl.

4.

Adult wild-type C57BL/6 mice were anesthetized via a mixture of inhaled isoflurane and oxygen gas.

5.

The dorsal and ventral regions of the mice hind legs were shaved to expose the skin covering the region below the knee.

6.

The injection landmark was identified on the mice hindleg ventral skin 7mm proximal to the heel.

7.

The tip of the device is positioned directly on the injection landmark and the trigger was pressed. The device was held on the hindleg for 3 seconds to ensure retention of the injected liquid.

8.

Tissue collection occurred 48 hours after the injection. The mice were euthanized using inhaled isoflurane and carbon dioxide followed by cervical dislocation.

9.

Mice hindleg muscles were extracted from the bone and flash frozen in liquid nitrogen.

10.

Luciferase assay and signal normalization were performed to determine the transfection efficiency.

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