February 1, 2008 (Vol. 28, No. 3)
MicroPorator Aims to Eliminate Challenges of Conventional Transfection Methods
The increasing demand for efficient transfection reflects the expanding applications for such technology. Introducing foreign DNA or RNA into mammalian cells has become fundamental for analyzing gene function, producing recombinant gene products, and devising strategies for gene therapy.
Successful results rely on the transfection method. Transfection of hard-to-transfect cells through common methods such as calcium phosphate, liposome-based reagent, and conventional electroporation, however, are problematic because of low transfection efficiency and low cell viability. Also, even though viral methods are efficient, they are time consuming to set up and are not cost effective.
To overcome these disadvantages, a novel electroporation technology called microporation has been developed, which provides high-efficiency transfection results. Microporation uses a specially designed handheld pipette-type device and specially designed pipette tips that utilize a capillary instead of a cuvette chamber.
The technology provides uniform electrical pulses delivered to samples ranging in volume from 10 µl to 100 µl using the pipette-tip electrodes. This method significantly improves transfection efficiency and cell viability versus the traditional cuvette-type sample chambers.
The potential harmful side effects that are caused by cuvette-type electroporation like pH variation, temperature increase, turbulence, and metal ion generation were eliminated by using the pipette technology. Thus, microporation provides an easier, more efficient, and reproducible way to work with hard-to-transfect cells.
Cell Lines, Primary Cells, and Hard-to-Transfect Cells
A number of transfection methods for mammalian cells are available. Many cell lines and primary cells, however, cannot be transfected efficiently or require time-consuming optimization procedures of the recommended protocols.
In the study, BTX Harvard Apparatus’ (www.btxonline.com) MicroPorator was used to transfect 80 cell lines and 10 primary cell types. Results were determined by a fluorescent microscope 24 hours after transfection. The final transfection efficiency averaged 70% and up to 90% for gene transfer (Figure 1). Cell viabilities ranged between 50–90%. To date, over 150 cell lines including hard-to-transfect lines such as primary neurons have been tested.
Typically, primary cells are less robust and difficult to transfect compared to standard cell lines. Few nonviral transfection methods have been reported that were successful for efficient gene transfer in primary human neuronal cells and 3T3-L1 adipocytes cells. For these cell types, GFP expression in primary neuronal cells (Figure 2) and 3T3-L1 (differentiated adipocyte) cells of up to 50% and viabilities of 70% and 80%, respectively (Figure 3), were achieved.
To evaluate the efficiency of siRNA introduction to cells through the microporation technology, siRNA was microporated into several cell lines including CHO, HeLa, SKOV, MCF-7, and PC3. To validate functionality of these cell lines, microporation was done with either pEGFP (enhanced green fluorescent protein) alone or pEGFP with EGFP-targeted siRNA (Figure 4a).
Endogenous gene knockdown by siRNA microporation was tested. For knockdown of endogenous genes, siRNA oligo for the insulin receptor was microporated into MCF-7 and PC3 cells (Figure 4b). These are cancer cells expressing an endogenous insulin receptor (Figure 4b; line 1).
Western blotting with insulin receptor antibody showed almost complete knockdown of insulin receptor expression both in MCF-7 and PC3 cells (Figure 4b; line 4). This result shows that microporation of siRNA can efficiently inhibit endogenous gene expression.
The physical technique of electroporation allows DNA to penetrate the cell membrane and bypass endosomes/lysosomes, thus avoiding DNA degradation. The DNA may also be directly delivered to the nucleus by microporation.
This means that gene expression is detectable within four to six hours after transfection. Another advantage of microporation technology is the short time required to perform the procedure. Microporation itself is performed in less than one minute, thereby reducing the total hands-on time to about 10–15 minutes.
The MicroPorator was developed as a transfection system guaranteeing high efficiency and cell viability. Microporation seems to be the most efficient nonviral transfection method for hard-to-transfect cells. It is a fast method that can be performed within minutes and the results can be analyzed in one day. Microporation may offer new opportunities for various research applications in which gene transfer is required.
MinSung Kim ([email protected]) is technical manager, and Neoncheol Jung, Ph.D., is director/CBO at NanoEnTek. Web: www.nanoentek.com. Phone: (508) 544-2893. NanoEnTek manufactures the MicroPorator for BTX Harvard Apparatus.