Efficiency of Methods for the Delivery of Exogenous DNA to Bovine in vitro Zygotes Using Liposomes
The efficiency of delivery methods for exogenous DNA into bovine in vitro zygotes using liposomes was evaluated. The methods were microinjection of plasmid-liposome complexes into the cytoplasm of zygotes and lipofection of bovine zygotes devoid of a shiny coat. Microinjection was performed in 18-22 h after the start of fertilization, the concentration of the circular plasmid pEGFP-N1 in the injection solution was 18, 26, 250 microg/mL, of the linearized plasmid - 13 microg/mL; the number of embryos was 25, 25, 12 and 25, respectively. Increase in the plasmid concentration did not significantly affect the dynamics of embryo development in vitro and the percentage of embryos expressing the target EGFP gene. All embryos with a fluorescent signal showed a mosaic pattern of expression of the target gene. The fluorescent signal was observed only in 1 blastocyst obtained by microinjection of the linearized plasmid in the complex with liposomes. The efficiency of the method turned out to be low (4.0-8.3%), with the exception of the group where the plasmid concentration was 18 microg/mL (20.0%). For the first time, the effectiveness of the transfection method of zygotes, devoid of the shiny coat, by lipofection for cattle was shown. The start time of transfection with a circular plasmid was in 18, 22, 24, 27 hours from the start of fertilization, the number of embryos was 12, 15, 35, 33; for a linearized plasmid the start time was in 18 and 27 hours from the start of fertilization, and the number was 12 and 13 embryos, respectively. The highest percentage of embryos expressing the TurboGFP gene (51.5%) was observed when transfected in 27 h after the start of fertilization using the circular plasmid. When lipofection was performed using the linearized plasmid, the change in the time of transfection from 18 to 27 h did not significantly affect the value of this indicator (33.3 and 30.8%, respectively). The lipofection method seems promising for non-invasive transfection of bovine zygotes devoid of the shiny coat.