Study of ultrasound targeted microbubble destruction combined classic nuclear localization signal peptide for facilitating gene transfection

Abstract

Objective To investigate the transfection efficiency of combining ultrasound targeted microbubble destruction (UTMD) and classic nuclear localization signal (cNLS) peptide for facilitating targeted gene into nucleus. Methods The plasmid of enhanced green fluorescent protein (pEGFP)was transfected into the 293T cells by different ways. This study was divided into four groups: control group, UTMD+ pEGFP; cNLS group, UTMD+ pEGFP+ cNLS; mutative group, UTMD+ pEGFP+ mNLS; and retardant WGA group, UTMD+ pEGFP+ cNLS+ WGA.The NLS was labeled by FITC and pEGFP was maked by Cy3. Six hours after transfection, the rate of plasmid into cells was detected as the percentage of Cy3 positive cells by flow cytometry, the rate of plasmid into nuclear was calculated as the percentage of Cy3 fluorescence intensity in nucleus to the whole cell by laser confocal microscope. At 48 after transfection, the transfection efficiency was detected by flow cytometry, the survival rate of cells was measured by CCK8. RT-PCR and Western blot were used to detect the relative expression amount of mRNA and protein. The differences were compared in four groups to evaluate the enhancement effects of cNLS during UTMD mediated gene transfection. Results ①At 6 hours after transfection, almost all green fluorescence showed in the nucleus in cNLS group, it appeared in both the cytoplasm and nucleus in mNLS group, but mostly appeared in the cytoplasm and almost none in nucleus in WGA group. ②At 6 hours after transfection, the rate of pEGFP into the cells were (61±11)%, (80±10)%, (55±9)% and (58±10)%; the rate of pEGFP into the nucleus were (20±4)%, (50±11)%, (18±3)% and (10±3)% in four groups respectively. The rates were highest in cNLS group and lowest in WGA group, the difference was statistically significant (P 80% in all groups; the transfection efficiency, the relative quantity of mRNA and protein were highest in cNLS group and lowest in WGA group, the difference was statistically significant(P<0.05). Conclusions The UTMD combining cNLS can promote pEGFP into the nucleus for improving the transfection efficiency. Key words: Sonication; Microbubbles; Transfection; Gene; Nuclear localization signal