Transfection technology for inserting nucleic acids into cells is getting a boost from novel electroporation techniques, new reagents, and easier-to-use transfection platforms. One leading-edge example is work being done by Virginia Tech associate professor Chang Lu, Ph.D. Dr. Lu's approach eliminates the need for expensive pulse generators, achieves uniform poration over the entire cell surface, and reduces toxicity. The result is reduced cost, higher transfection rates, and improved cell viability.
Dr. Lu's work, presented at CHI's recent “Bioprocessing Summit” is representative of a range of transfection innovations that have reached the market or are moving steadily toward commercialization. Gene delivery and gene-silencing applications in bioprocessing, drug research, and gene therapy are all benefiting.
Dr. Lu's research differs from traditional electroporation in two key ways. Electroporation requires cells to experience an electric field strong enough to cause pore formation and long enough to uptake nucleic acids, but not so strong or long as to kill the cells. In the past, doing this required use of pulse generators, which are expensive and tricky to use.
“We were thinking, what if you could use constant voltage to do that? Perhaps a battery that provides constant DC voltage or a cheap $100–$200 power supply to deliver a voltage that is constant at about 100 or 200 volts,” Dr. Lu explained.
Using semiconductor-chip fabrication techniques and microfluidics, his group accomplished the pulsing by varying the width of the channels (wide followed by narrow) through which cells flow. The field intensity experienced by the cells varies depending upon the size of the channel, hence mimicking a pulse, and the duration in the channel is controlled by the cell flow rate.
“In our device we can constantly flow the cells so that we can continuously produce transfected cells. We have demonstrated up to 20 mL per minute of cell samples processed,” Dr. Lu said.
A second important benefit is uniform poration over a greater surface area. Usually, the membrane area directly facing the electric poles undergoes the greatest poration. “We introduced a motion based on fluid mechanics that makes the cells rotate in the channel. Since the field is constant, the cells are actually experiencing more uniform treatment in terms of the pulse or the field intensity for every cell.”
A broad trend, added Dr. Lu, is continued interest in developing easier and more reliable methods for generating cell lines. “Obviously that requires getting genes incorporated into the chromosome. That's a challenge. We hope our techniques and others will improve the efficiency of doing that. It would be nice to have finer control during delivery.”
Speed and Flexibility
“Over the past two to three years my lab has been using Baculovirus and BacMam technology for about 80 percent of the work we do,” said Kim Stutzman-Engwall, Ph.D., associate research fellow at Pfizer Global R&D Labs. “It's not our exclusive approach; we let the project dictate what method we use, but we are high on BacMam.”
The main advantages of BacMam—using double-stranded DNA insect viruses (Baculovirus) to efficiently deliver and express genes in mammalian cells—are that it is fast compared to cell-line creation, consistent in terms of delivery, and able to transduce a wide variety of cells, says Dr. Stutzman-Engwall.
“It takes two to three months to make a stable cell line that's producing the amount of protein you want for assay development. With BacMam we can do that much more quickly and combine both transient and stable cell-line generation with one protocol.
“We make the virus through transfection, which is essentially a transient experiment, and determine if the expressed protein is something we actually want to work on.” Pfizer has engineered its BacMam viruses to have antibiotic selection markers. “If we're happy with the transient aspect of the expression, we can treat it as if it is a stable cell line by using selection pressure to select a stable clone.”
Dr. Stutzman-Engwall's lab currently supports neuroscience projects and previously supported cardiovascular, metabolic and endocrine disease, diabetes, and antibacterials. “We've handled a broad range of projects. We probably use electroporation least and only if it's a cell that is very difficult to transfect or transduce. But I have to say with BacMam we've not found any cells that we can't transduce.”
According to Dr. Stutzman-Engwall, “BacMam is more consistent than doing DNA transfection for transient expression. We titer our virus so we know what the titer is and we can do put the exact amount in each time to get the level of expression we want. For example, if you add less virus, you get a lower level of expression—we found this is very useful in assays where you want to see a more physiological response instead of overexpression.”