Transferência gênica mediada por espermatozoides através de eletroporação capilar em zebrafish Danio rerio

Abstract

Sperm-mediated gene transfer (SMGT) is a promising biotechnology for transgenesis in aquatic species, based on the natural ability of spermatozoa to be spontaneous carriers of exogenous DNA. However, it still has some obstacles, which makes it necessary to implement strategies to optimize the SMGT technique. Physical methods, such as electroporation, have been used to improve the efficiency of DNA uptake in spermatozoa, but even conventional electroporation has some negative effects on cell viability. Thus, alternative electroporation systems such as capillary electroporation can be exploited for transfection of sperm cells, which has shown very high efficacy, with high transfection rate, being fast, reproducible and possessing a low rate of cellular loss. The objective of this study was to validate an efficient capillary electroporation method for DNA incorporation in Zebrafish spermatozoa Danio rerio. Capillary electroporation was performed in two distinct groups (with and without exogenous DNA) using five different voltages: 500V, 750V, 1000V, 1250V and 1500V. Kinetic parameters were determined using computerized semen analysis, while membrane integrity, membrane fluidity, mitochondrial functionality, DNA fragmentation index, reactive oxygen species (ROS) concentration, lipid peroxidation at the plasma membrane (LPO) and absorption of DNA were evaluated by flow cytometry. The results showed that all electroporated groups showed significantly higher percentages of transfected spermatozoa when compared to the control group (P <0.05). Electroporated groups at high voltages (1000V, 1250V and 1500V) demonstrated negative effects on cell membrane integrity when compared to other groups and control. Higher voltages (1250V and 1500V) reduced the motility duration by 50% when compared to the control group without DNA. Electroporation with higher voltages, such as 1000V, 1250V and 1500V, led to a 45% increase in the concentration of reactive oxygen species in sperm cells (P <0.05), while the lowest voltages (500V and 750V) did not differ from the control group (P> 0.05). Membrane fluidity and DNA damage increased with increasing voltage. The spermatic mitochondrial functionality was the only one, among the non-kinetic parameters, despite the different stresses, which did not show a negative response after electroporation (P> 0.05). Under the recommendation of using voltages of up to 750 V with 25% transfection rate optimization, this method represents a safe and efficient alternative for the electroporation of Zebrafish spermatozoa Danio rerio.