This method demonstrates electroporation-based transfection of primary human pigment epithelial cells using the Sleeping Beauty Transposon System, an evidence for transgene expression and protein secretion. The combination of the Sleeping Beauty Transposon System and electroporation enables efficient transfection of primary human pigment epithelial cells, as well as stable and persistent transgene expression and protein secretion. The overall aim is to establish a cell-based gene addition therapy for the treatment of retinal degenerative diseases, which requires stable and persistent secretion of the therapeutically active protein.
Demonstrating the procedure will be Anne Freialdenhoven and Antje Schiefer, medical technical assistants from the laboratory. To prepare Plasmid DNA for primary human RPE cell electroporation, use a microvolume spectrophotometer to quantify the Plasmid DNA contents, and adjust the concentration to 250 nanograms per microliter in 10 millimolar Tris-HCL. Mix one volume of 250 nanograms per microliter of SB100X transposase Plasmid DNA with 16 volumes of 250 nanograms per microliter of pigment epithelium derived factor transposon Plasmid DNA.
Add two microliters of the resulting plasma mixture into a sterile 1.5 milliliters safe lock micro centrifuge tube on ice and fill a buffer tube with three milliliters of buffer E.Then insert the tube into the pipette station until a click is heard and set the transfection device to 1, 100 volts, a 20 millisecond pulse width and two pulses. To prepare the cells for electroporation. First, check the morphology of the primary RPE cell cultures via phase contrast microscopy to assess their growth and confluency.
Treat the cells with 500 microliters of 0.05%Trypsin EDTA per well for seven to 15 minutes in the cell culture incubator. When the cells have detached collect the cells by centrifugation, resuspend the cells in one milliliter of PBS and centrifuge one to 10 times 10 to the four cell aliquots per transfection reaction. Resuspend the pellets in 11 microliters of buffer R per tube and add two microliters of the prepared plasmid mixture to each tube.
Insert the head of a transfection pipette into a 10 microliter transfection tip until the clamp fully picks up the mounting stem of the piston and load the cell and plasmid solution into the transfection tip. Insert the transfection pipette into the buffer tube placed within the pipette station until a click is heard and press start to begin the electroporation process. After the transfection carefully remove the transfection pipette from the pipette station and immediately pipette the cell and plasmid solution into the prepared wells of the cell culture plate.
To purify his-tagged pigment epithelium derived factor fusion proteins from RPE cell culture supernatants. First, use a bevel cut tip to collect one 30 microliter aliquot of nickel NTA slurry per sample and pellet the nickel NTA resin by centrifugation. Carefully resuspend the nickel NTA resin with 200 microliters of One X incubation buffer and pellet the solution with an additional centrifugation two times.
After the second centrifugation, carefully resuspend the nickel NTA resin pellets with 40 microliters of Four X incubation buffer per sample. Next mix 55 microliters of one aliquot of pretreated nickel NTA slurry with 260 microliters of Four X incubation buffer and 900 microliters of each transefcted RPE cell culture supernatant. Incubate the mixture on a rocking shaker at room temperature for 60 minutes and pellet the nickel NTA resin mixtures again.
Carefully resuspend the pellets in 175 microliters of One X incubation buffer and centrifuge the mixtures two more times. After the second centrifugation carefully resuspend the nickel NTA resin pellets in 30 microliters of Elution buffer for a 20 minute incubation at room temperature with shaking and centrifuge the samples again. Then carefully collect the supernatants and mix them with Two X SDS sample buffer for western blood analysis of the purified proteins.
Cultivated primary RPE cells isolated from human donor eyes demonstrate a typical cobblestone morphology regardless of the donor's age, postmortem time of isolation or time of cultivation. The application of short-term electrical pulses to primary human RPE cells. Using the capillary transfection system does not adversely affect the epithelial morphology.
Western blood analysis of transfected primary human RPE cell culture supernatants demonstrate pigment epithelium derived factor secretion at consistent levels without transgene silencing for more than 500 days. As observed in this representative Western blood analysis of cell culture medium from serially performed transfections, a universally higher pigment epithelium derived factor secretion rate is observed at 21 days after transfection. In a pursued culture, long-term elevated PEDF secretion lasts for at least 165 days.
In addition ELISA-based quantification reveals a 20 fold increase of total pigment epithelium derived factor secretion in transfected primary human RPE cells compared to respective nontransfected control cells. This increment was also affirmed at the gene expression level at which the total PEDF expression was raised more than 30 fold. When attempting this protocol, it is important to use pigment epithelial cells whose morphology corresponds to the In Vivo status.
Also, remember to draw up of the cell solution into the transfection tip without air bubbles. The stably transfected cells can be used in different Ex Vivo or In vivo models to verify the functionality of this cell-based gene addition therapy.
