Gene electrode transfer is a physical method used to deliver genes into the cells by application of short and intense electric pulses. In successful gene electrode transfer, several steps are involved formation of a complex between DNA and cell membrane translocation of DNA across the ized membrane transfer of DNA from cytoplasm into the nucleus and gene expression for gene electro transfer. Different electric pulse protocols are used in order to achieve maximum gene transfection.
One of them is changing the electric field orientation during the pulse delivery. In this video, we demonstrate the difference in gene electrode transfer efficiency when all pulses are delivered in the same direction and when pulses are delivered. By changing electric field orientation experiments were performed on Chinese hamster ovary cells, CHOK one For the experiment.
Follow these steps, grow cells in a culture flask in the incubator for 24 hours. On the day of the experiment, prepare cell suspension by trypsin with 0.25%trips in EDTA solution and count the cells centrifuge cells for five minutes at 1000 rotations per minute at four degrees Celsius and resuspend cell pellets in ISO OSM Miller sodium phosphate buffer to a cell density of 5 million cells per milliliter. And move the sample to the appropriate dish cells are exposed to electric field in pipe tip with integrated electrodes connected to a high voltage prototype generator.
These electrodes allow application of a relatively homogeneous electric field in different directions. The tip and the generator were developed at the laboratory of Biocybernetics faculty of electrical engineering. University of Luana add plasmid, E-G-F-P-N one to a cell suspension concentration of 10 micrograms per milliliter.
Mix cells with plasmid DNA thoroughly and incubate the mixture for two to three minutes at room temperature before applying electric pulses. Aspire 100 microliters of cell suspension into the pipee tip with integrated electrodes. In this experiment, a train of eight rectangular pulses is applied using high voltage prototype generator to different electric field protocols are used in the first protocol.
All pulses are delivered in the same direction, whereas in the second protocol, pulses are delivered by changing electric field orientation. The second protocol can only be used with appropriate pulse generator, which allows application of electric pulses in different directions. Immediately after the pulse application, transfer the cells from pipe tip into six well plates and add fetal bovine serum.
25%of sample volume incubate cells for five minutes at 37 degrees CELs. To allow cell membrane resealing add two milliliter of HAMF 12 to each sample in six well and incubate cells for 24 hours at 37 degrees Celsius in a humidified 5%CO2 atmosphere in the incubator. Efficiency of gene electro transfer is determined as the percentage of cells expressing GFP 24 hours after pulse application.
The cells are observed using a fluorescence microscope in the acquisition software. Set the excitation wavelength to 488 nanometers and choose an appropriate band pass filter for green fluorescent image to detect the cells that are expressing GFP. Acquire at least five images, face contrast and green fluorescence at 20 times.
Objective magnification count cells in face contrast image and cells that are expressing GFP in green fluorescence image determine the percentage of gene electro transfer efficiency by dividing the number of cells that are expressing GFP with a number of all cells in each corresponding image. The percentage of cells expressing GFP when all pulses are delivered in the same direction and when pulses are delivered by changing electric field orientation is presented by changing the electric field orientation. The percentage of cells expressing GFP increases significantly.
