4 June 2010. Motor neurons have proved fiendishly difficult to transfect, forcing scientists to deal with finicky viral transduction or simply experiment with related cell lines instead. Now, researchers from Emory University School of Medicine in Atlanta, Georgia, have described a protocol using magnetic beads. The protocol transfects nearly half of cultured primary motor neurons. The study appeared April 21 in the journal Molecular Neurodegeneration. Led by first author Claudia Fallini and senior author Wilfried Rossoll, the scientists used their technique to explore the activity of Smn, a protein linked to the neurodevelopmental disease spinal muscular atrophy (SMA). Researchers exploring other aspects of motor neuron biology, such as amyotrophic lateral sclerosis (ALS), may also want to give the beads a try.
The only efficient way to put new DNA into a primary motor neuron known to date is by viral transduction, which has several limitations. Researchers need special equipment and biosafety clearance to work with the viruses, and it takes extra effort to clone one’s DNA of interest into a virus and determine the appropriate viral titer to use. In addition, the commonly used lentivirus is limited in how long a piece of DNA it can carry, and delivers but one gene at a time.
Rossoll has been looking for a motor neuron transfection technique for quite a while, trying electroporation and all kinds of reagents. “Some of these are pretty good at killing motor neurons, but not really good at transfecting them,” he quipped. When he heard that magnetofection—a technique using magnetic beads—worked in hippocampal neurons, he decided to try it in his cells. After optimizing the protocol, he and Fallini achieved non-toxic transfection of more than 45 percent of the cells in a culture of embryonic primary motor neurons. Rossoll and Fallini kindly contributed this protocol to the Alzforum Protocols database, from where interested scientists can view the protocol.
To transfect the neurons, Fallini incubates the DNA of interest with polymer-coated, biodegradable iron oxide beads. Once the DNA sticks to the beads, she adds them to the cell culture and places the dish on a magnetic plate. The magnetic attraction pulls the beads down toward cells growing on the bottom of the dish, which then endocytose the beads and DNA.
The researchers were able to transfect the DNA for green fluorescent protein and other constructs into their cells, and to transfect more than one construct at a time. They also transfected shRNA to silence genes in the treated cells. Importantly, the cells survived for at least five days following the procedure. Fallini suspects they could live longer, but her cultures became overrun with contaminating glia by that time. They scientists cultured cells from 13.5-day mouse embryos; they have not yet attempted the protocol in older neurons, which would be more directly relevant to ALS.
“Being able to achieve a higher than 14-15 percent transfection efficiency with low mortality in motor neurons is very exciting news,” wrote Kathryn Volkening of the Robarts Research Institute in London, Ontario, in an e-mail to ARF. Volkening works with Michael Strong on the cell biology of ALS. “The methodology described by these authors would be easier to implement in a general lab where there are safety concerns and special protocols that must be followed when working with lentiviral infection,” she wrote.
Fallini and colleagues used their protocol to examine localization of Smn using live-cell imaging. Smn, which is mutated or missing in inherited SMA, is known to help assemble ribonuclear proteins. Since these mutations specifically affect motor neurons, the researchers suspected the affected protein might have an important role in these cells’ extra-long axons. Fallini transfected motor neurons with fluorescently labeled Smn and observed it traveling up and down axons, suggesting it does indeed have a function there.—Amber Dance.
Fallini C, Bassell GJ, Rossoll W. High-efficiency transfection of cultured primary motor neurons to study protein localization, trafficking and function. Mol Neurodegener. 2010 Apr 21;5:17. Abstract