de Souza Andressa Pereira, Bastos Ana Paula, da Fonseca Francisco Noé, Pandolfi José Rodrigo, Costamilan Carlos André da Veiga Lima Rosa, Marques Mariana Groke
Centro de Ciências Agroveterinárias, Universidade do Estado de Santa Catarina, Lages, SC, Brasil.
Empresa Brasileira de Pesquisa Agropecuária, Embrapa Suínos e Aves, Concórdia, SC, Brasil.
Anim Reprod. 2024 Nov 22;21(4):e20240026. doi: 10.1590/1984-3143-AR2024-0026. eCollection 2024.
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 pmhyGENIE-5 for 30 min) efficiently produces genetically edited porcine fetal fibroblasts with low cell damage.
聚乙烯亚胺（PEI）已被探索作为一种将基因传递到细胞的高效非病毒系统；然而，尚无其在猪胎儿成纤维细胞（PFF）中使用的方案。因此，我们比较了不同浓度的异硫氰酸荧光素标记的聚乙烯亚胺（FITC-PEI）（0.625、1.25、2.5、5、10、20、40或80μg/mL）和孵育时间（30分钟、1小时或2小时）。观察到孵育时间不影响PEI-FITC的内化，30分钟足以捕获复合物。高于10μg/mL的浓度可使许多PFF被标记（>90%）。然后，测试了两种PEI浓度，即10或40μg/mL，与pmhyGENIE-5以N/P为2的比例结合30分钟。在评估时间点（24小时、48小时和72小时），不同PEI浓度下PFF-GFP阳性的百分比相似。然而，40μg/mL导致更高的膜损伤率。因此，可以得出结论，即使在短至30分钟的时间内，10 - 80μg/ml的PEI浓度也能促进高掺入率。此外，可以说在多聚体1中使用的转染条件（10μg/mL的PEI和37.5μg/mL的pmhyGENIE-5作用30分钟）能有效地产生基因编辑的猪胎儿成纤维细胞，且细胞损伤低。
