Transient Trim25 Knockdown Enhances Transfection Efficiency in Two Teleost Fish Cell Lines.

Efficient transgene expression following nucleic acid transfection remains difficult in many fish cell lines, particularly those derived from marine species. Although extracellular delivery systems have been extensively optimized, intracellular factors that limit the expression of delivered nucleic acids remain poorly understood in teleosts. In mammals, TRIM25 (tripartite motif-containing protein 25) participates in cytoplasmic exogenous RNA surveillance. Here, we examined whether its teleost homolog contributes to the low reporter expression observed after transfection. Transient siRNA-mediated knockdown of trim25 increased the percentage of eGFP-positive cells following Cap1-m¹Ψ-eGFP mRNA transfection from 19.27% to 74.63% in zebrafish embryonic fibroblast cell line (PAC2) and from 10.41% to 36.15% in Chinese tongue sole testis cell line (CST). Reporter expression following pEGFP-N1 plasmid transfection was also increased, although to a lesser extent. In this study, transfection efficiency was operationally defined as the percentage of eGFP-positive cells among total nuclei and did not directly measure nucleic acid uptake. These findings suggest that transient trim25 knockdown may alleviate intracellular restriction of exogenous reporter expression in PAC2 and CST cells and may provide an affordable approach for improving molecular manipulation in selected fish cell systems.