This article describes a method for transfecting primary mouse neuronal cells using magnetic polyplexes formed from plasmid DNA and cationic polymer-coated magnetic nanoparticles. The process enhances the delivery of genetic material into neurons, facilitating the expression of fluorescent proteins.
Begin with fluorescent protein-encoding plasmid DNA or pDNA suspension in media. Add biocompatible, cationic polymer-coated magnetic nanoparticles to the pDNA suspension and incubate.
The positively-charged cationic polymers electrostatically interact and conjugate with negatively-charged pDNA, resulting in pDNA condensation and formation of 'magnetic polyplexes' with an overall positive charge.
Now, transfer the polyplex mixture into a multi-well plate containing adherent primary mouse neuronal cell culture. Mount the multi-well plate over a magnetic plate and incubate.
The magnetic field generated by the magnetic plate causes the polyplexes to sediment and accumulate over the neuronal cell surface. This close contact with the cell surface facilitates the non-specific binding of positively-charged polyplexes to negatively-charged cell membrane-associated proteoglycans.
Subsequently, the polyplexes get internalized via endocytosis - a process wherein the local regions of the cell membrane invaginate and pinch off to form membrane-bound vesicles called endosomes.
Inside the cytoplasm, the polyplex's cationic polymers induce proton accumulation in the endosome. The simultaneous influx of chloride ions to maintain charge neutrality increases endosomal ionic strength, leading to water influx.
Eventually, the endosomes swell and rupture due to osmotic pressure, releasing polyplexes into the cytoplasm. The condensed pDNA within the polyplexes facilitate increased cytosolic motility, while the cationic polymers protect pDNA from cytoplasmic nuclease degradation.
Following dissociation of the polyplexes, the liberated pDNA - during the appropriate cell-cycle phase when the nuclear membrane temporarily disassembles - get translocated to the nucleus. Cells successfully transfected with pDNA express fluorescent proteins.
To culture motor neurons, dilute them to 5,000 to 10,000 cells in 500 microliters of culture medium per well in a 24-well plate. Then, remove the laminate solution with a P1000 pipette tip. Immediately transfer the motor neurons diluted in culture medium to the coating plates to avoid drying.
To prepare DNA, resuspend 1 microgram of DNA in 50 microliters of neuronal culture medium, and vortex for 5 seconds. To prepare the bead tube, resuspend 1.5 microliters of beads in 50 microliters of neuronal culture medium. Add 50 microliters of bead solution to 50 microliters of DNA solution, and incubate for 20 minutes at room temperature.
During this incubation, withdraw 100 microliters of culture medium from the well that will be transfected. Subsequently, transfer 100 microliters of the DNA-bead mix to each well, and incubate the 24-well plate 20 to 30 minutes on the magnetic plate at 37 degrees Celsius.
繁體中文
