Optimization of Electroporation with Green Fluorescent Protein Gene for Testicular Cell Transplant in Nile Tilapia

Abstract

Transplantation technology can be applied to generate surrogate fish broodstock. Germinal cells as donor used in transplantation are labeled to distinguish with endogenous cells. Donor cells are generally derived from transgenic fish carrying a marker or cells labeled by PKH-26. This study was performed to obtain an alternative method of labeling cells by using electroporation. The method in this study includes the preparation of the recipient, the donor cell preparation and cell transplantation. Recipient was obtained by mating tilapia males and females with a ratio of 1:1. Testicular cells were taken from 4-month-old Nile tilapia. Electroporation was performed with testicular cell density of 104 cells.μL-1, pJfKer-GFP concentration of 50 ng.μL-1, and a pulse length of 20 ms at 0, 100, 200, and 300 volts. In amount of 5x103 cells at 0.5 μL electroporated testicular cells were then injected into the intraperitoneal cavity of 3-day-old Nile tilapia larva. The parameters measured were cell survival, the percentage of GFP fluorescent cells, and cell colonization in recipient fish. The results showed that survival of the electroporated cells with 100 and 200 volts was similar (p>0.05), and higher than that of 300 volt treatment (p<0.05). It shows that the voltage is very influential on cell survival. The increase in voltage will result in the increasing mortality in the cell. Number of fluorescent cells was not significantly different among treatments. While the percentage of fluorescent cells did not differ on the treatment of 100, 200, and 300 volts (p> 0.05) except for the control. In the control treatment luminescence GFP was not visible, it is proved that the GFP gene was not able to pass through the cell membrane in the absence of an electric voltage. Percentage colonization of transplanted cells in the fish after 50 days of rearing gave the highest value at 200 volts (66.67%). As conclusion, electroporation within 100-200 volt was a suitable tool to label testicular cells for transplantation.