Author(s): SOUZA, A. P. de; BASTOS, A. P. A.; FONSECA, F. M. da; PANDOLFI, J. R. C.; COSTAMILAN, C. A. da V. L. R.; MARQUES, M. G.

Summary: Abstract: Polyethylenimine (PEI) has been explored as an efficient non-viral system for delivering genes to cells; however, there were no protocols for its use in porcine fetal fibroblasts (PFF). Therefore, we compared different concentrations of FITC-PEI (0.625, 1.25, 2.5, 5, 10, 20, 40, or 80 µg/mL) and incubation times (30 min, 1 h, or 2 h). It was observed that the incubation time did not affect the internalization of the PEI-FITC and that 30 min was sufficient to capture the complex. The concentrations higher than 10 µg/mL could reach many marked PFF (>90%). Then, two PEI concentrations were tested, 10 or 40 µg/mL, combined with an N/P of 2 with the pmhyGENIE-5 for 30 min. The percentage of PFF-GFP positive was similar between the PEI concentrations in the evaluation time points (24 h, 48 h, and 72 h). However, 40 µg/mL caused higher membrane damage rates. Thus, it can be concluded that concentrations between 10 – 80 µg/ml of PEI promote high incorporation rates, even in periods as short as 30 minutes. Furthermore, it can be stated that the transfection condition used in Polyplexes 1 (10 µg/mL of PEI and 37.5 µg/mL of pmhyGENIE5 for 30 min) efficiently produces genetically edited porcine fetal fibroblasts with low cell damage.

Publication year: 2024

Types of publication: Journal article

Unit: Embrapa Swine & Poultry

Keywords: Animal genetics, Cationic polymer, Feto, Fibroblastos, Gene delivery, Genética Animal, Non-viral vectors, Nuclear transfer, Primary fibroblast, Suinocultura, Swine, Transfecção genética