Mark Fernelius and Dr. Larry Howell, Department of Mechanical Engineering
Technologies for injecting DNA into egg cells have greatly facilitated genetic research. Microinjection is a current standard for this purpose. It uses a hollow glass needle to inject a solution containing DNA into cells. Although widely used, microinjection has several significant challenges. First, the DNA can be damaged when forced through the small opening of the needle. Second, the cell can die from the physical trauma caused by the microinjection needle piercing the cell membrane. Third, the cell can split open because of the increased internal pressure caused by injecting fluid into the cell. Brigham Young University has developed nanoinjection as an alternative approach for DNA injection into cells. The basic description of nanoinjection is illustrated in Figure 1.
The rst nanoinjection devices were microelectromechanical systems (MEMS) using fabrication methods similar to those used to make computer chips. The MEMS nanoinjector has been successfully demonstrated in experiments. However, the nanoinjector is on a solid, opaque, silicon substrate that is not compatible with inverted microscopes currently used in transgenics labs. With an inverted microscope the light source is above the sample and the viewing lens is located beneath the sample. This requires the sample to be placed on a transparent medium for viewing. A stand-alone lance (SAL) is a lance that does not need to be on a chip and would be compatible with the current lab equipment. The development of a stand-alone lance could make nanoinjection technology more available to researchers around the world. A stand-alone lance also has the potential to be used as an experimental resource to test di erent nanoinjection parameters. Even if the SAL did not work as well as the MEMS chip, parameters could be quickly optimized using a stand-alone lance before those same parameters are used in nanoinjections utilizing the MEMS chip.
Several alternatives were considered for the SAL including lances made from gold, tungsten, and platinum. The tungsten probes were selected because their geometry (tip diameter and taper angle) allowed them to be used as SALs and they were readily available at a reasonable cost (approximately $10 per probe). The tungsten probes have a tip diameter of 2 m and a taper angle of 8.1 degrees. A small tip diameter and taper angle are desirable to prevent damaging the egg cell.
Because of its chemical inertness and availability, gold would be an ideal choice for a stand alone lance. However, o -the-shelf gold lances with the desired geometry were not found during a search of commercial sources. Attempts were made to fabricate a gold SAL by pulling gold wire and chemically etching gold acupuncture needles. Both of these methods yielded lances that were too large for nanoinjection. Like gold, platinum is chemically inert and would not oxidize. Therefore, it would also be desirable to use a platinum lance for nanoinjection. However, platinum probes were cost prohibitive (approximately $814.00 for a box of ve probes, or approximately $160 per probe).
Injections with the tungsten lance were carried out in phosphate bu ered solution (PBS). The tungsten lance and an injection can be seen in Figure 2. The preliminary nanoinjection experiments with the tungsten lance were conducted with voltage and DNA. These experiments resulted in survivability rates of approximately 50%. This survivability rate of the injected egg cells was lower than desired. Therefore, it was determined to conduct mock injections without voltage or DNA to isolate the cause of cell death. Because the stand-alone tungsten lance is so much larger than the nanoinjection lance it was thought that the cell deaths might be a result of the larger lance size. Conducting mock injections (no voltage or DNA) isolated the e ect of piercing the cell with the stand-alone tungsten lance. The egg cell survivability rate of mock injections was determined to be higher than 70%.
The mock injection results show a total survivability rate of 78.5% with mock injections performed on 163 egg cells. Thus, piercing the cells with the tungsten lance only results in a 21.5% fatality. The high survivability rate shows that the tungsten SAL has potential as a nanoinjection lance. Adding voltage to the injection procedure is the next step in investigating the potential of the SAL as a nanoinjection lance.
This research was submitted for publication in Proceedings of the ASME 2011 International Mechanical Engineering Congress & Exposition, Denver, CO, Nov 11-17, 2011.