Efficient Gene Delivery to Neuronal Cells using the Microporator

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Transfection is a convenient and widely used approach to study the control of gene expression. However, neuronal cell lines and primary Neuronal cells are notoriously difficult to transfect using conventional methods. The MicroPorator technology is a novel gene transfer technique designed for hard-to-transfect cells.
The demand for high- efficiency transfections is increasing, reflecting the expanding applications for transfection technology. Introducing foreign DNA into mammalian cells has become fundamental to analyzing gene function, producing recombinant gene products, and devising strategies for gene therapy. Successful transfection results rely on the transfection method. Digital Bio’s MicroPorator is novel electroporation technology, it offers these essential qualitites for achieving successful transfection results.
In this study, we investigated the efficiency of transfection with Micropoator, a unique electroporation technology using pipettete TIP as a electroporation space, in 12 neuronal cell lines (C6, DU145, F-11, GH3, GT1-1, GT1-7, Hib5, PC-12, SCN2.2, SH-SY5Y, T98G, U-87MG) and 2 primary neuronal cells (Mouse glial, Rat schwann).

METHOD FOR MICROPORATION

Using the Microporation technology 1x10^5 cells were transfected with 0.5 µg pEGFP (using 24 well plate) according to the manual’s protocol. Briefly, the cells were resuspended in 11 µl Resuspension buffer R, the DNA were added, and the mixture was pipetteted using MicroPoator pipette. The microporation resuspension buffer R is used to stabilize the cells during electroporation. For electroporation, microporator pipette was inserted into pipette station. The electroporation was performed using the optimized electric parameters for each cell type. After microporation with optimized electric-parameter, the cells were transferred immediately into pre-warmed complete medium.
Microporation itself is performed within less than one minute, thereby reducing the total hand-on time to about 10-15 min. The cells were harvested 24 h after transfection, and GFP expression was evaluated using fluorescence microscopy. All transfection experiments were repeated three times. Measurement of viable cells was performed by cell counting using Automatic cell counter (C-Reader System) 

TRANSFECTION OF NEURONAL CELL LINES

A number of transfection methods for mammalian cells are available; however, many neuronal cell lines can not be transfected efficiently or time consuming optimization procedures of the recommended protocols are required. But, by using microporation technology a highly efficient gene transfer (60~90% of surviving cells) was obtained in 12 neuronal cell lines tested so far: C6, DU145, F-11, GH3, GT1-1, GT1-7, Hib5, PC-12, SCN2.2, SH-SY5Y, T98G, U-87MG (figure 1). Transfection efficiency was determined 24 hours after microporation by fluorescence microscope. Cells were pulsed with optimzed electric-parameter. For all cell lines, transfections by microporation

CONCLUSIONS

We have developed an innovative transfection system, "MicroPorator", based on a novel electroporation technology. This electroporation based technique combines pipette Tip with mild conditions which guarantee high efficiency and cell survival rate. Physical approach allow DNA to penetrate directly the cell membrane and bypass endosomes / lysosomes, thus avoiding DNA degradation. The DNA may also be directly delivered to the nucleus by microporation. The advantage of the microporation technology is the short time required to perform the procedure. Microporation itself is performed within less than one minute, thereby reducing the total hand-on time to about 10–15 min. In addition, expression is detectable as early as 4 hr after transfection, thus allowing transfection and analysis within 1 day. In conclusion, microporation is the most efficient non-viral transfection method for hard-to-transfect neuronal cells. It is a very fast method that can be performed within minutes and the results can be analyzed within 1 day. Therefore, microporation offers new opportunities for various research applications in which gene transfer is required.

REFERENCES

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