Inserting gene sequences in cultured cell lines is a main component of biomedical research, but inserting large sequences or multiple genes at the same time has been difficult. Now, researchers at Baylor College of Medicine, the Michael E. DeBakey VA Medical Center, and Vanderbilt University in Nashville, Tenn., have harnessed a plasmid-based "jumping gene" system called piggyBac transposon to accomplish this in a stable fashion, opening doors for new drug therapies for a variety of diseases, including cancer.
The findings appear in the online addition of Proceedings of the National Academy of Sciences.
"Until now, to insert multiple genes into cells, we had to use a number of different viruses as vectors to take the genes into the cell. Even when we tried to make these cells with plasmids they were not stable, meaning they survived a relatively short amount of time," said Dr. Matthew H. Wilson, assistant professor of medicine at BCM and on staff of the Michael E. DeBakey Veterans Affairs Medical Center. "This new method allows us to transfer multiple and larger segments of genes at the same time, creating a longer lasting cell line simultaneously expressing multiple transgenes."
Wilson and his colleagues used a relatively new kind of system called the piggyBac transposon as a vector to take the genetic material into the cells. The piggyBac system has been used in insect studies, but Wilson and his colleagues demonstrated that it is also valuable in studies of human cells.
Genes or genetic elements called transposons can "jump" easily from one place to another on the genomes of various organisms. The piggyBac system was first derived from a kind of moth. It can insert itself into genomes while carrying more than one genetic sequence, said Wilson.
This system is based on using a plasmid, circular sections of DNA that are often used as vectors to take genes into cells. Scientists engineer a plasmid to contain a foreign gene with a sequence that promotes or turns it on through a process called cloning. The piggyBac transposon system expands the value of that system because it can carry several gene sequences into the cell at the same time.
Creation of cell lines
For this study, Wilson and his colleagues harnessed the piggyBac system to make cell lines expressing sodium channels that are usually found in neurons in the brain.
"The sodium channel gene we delivered was very large, about 10 kilobases, which is larger than what we have tried to transfer in the past," said Wilson, who is senior author on the study. "Another important aspect of the finding is that the cell lines created are basically permanent."
Since the cell line is considered stable, Wilson said it can now be used for tests on a high-throughput apparatus that allows the fundamental cell processes to be viewed. This was not possible with an unstable cell line.
"We will now be able to make cells express all components of a signaling pathway so we can then evaluate, with the use of this apparatus, drug effects on that pathway," said Wilson, who is also a researcher with the Center for Cell and Gene Therapy at BCM. "We hope that this will one day be used during gene therapy for a disease. If a combination of genes is necessary for the most effective treatment, then this method will allow us to utilize more than one gene at the same time."
Wilson said more research will need to be done before using this technique to treat people with genetic diseases.
Others who contributed to the study include Kristopher M. Kahlig and Melissa A. Daniels both of the department of medicine, Vanderbilt University; Sai Saridey and Aparna Kaja, both of the department of medicine at BCM; and Alfred L. George Jr., department of medicine and department of pharmacology at Vanderbilt University. The work of Wilson began while he was with Vanderbilt University, and completed at BCM.
Funding for the study came from the National Institutes of Health and in part by a career development award to Wilson from the Department of Veterans Affairs.