Promoterless DNA fragments inhibiting the expression of integrated gene in human pancreatic carcinoma cells

Abstract

To investigate whether introducing promoterless DNA containing the cDNA sequence of green fluorescent protein (GFP) induces gene-specific silencing in human pancreatic cancer cell line harboring genomically integrated GFP gene. Using G418 selection and fluorescent separation we established a highly purified monoclonal pancreatic cancer cell line, recombinant PANC-1, which had a steady level of GFP expression. GFP cDNA was amplified by PCR from plasmid pEGFP-C1 and ligated to the promoterless plasmid PUC19. PUC-GFP was then transfected into monoclone cells along or cotransfected with pEGFP-C1 to panc-1 cells. Each had PUC plasmid transfected group as their control to eliminate possibility of plasmid toxicity and GFP small interferent RNA (siGFP) transfected group as positive control. Western blot, flow cytometry and phase contrast fluorescence microscopy were used to detect the changes of GFP expression. The data showed that (1) PUC-GFP inhibited the GFP expression in monoclonal cell line in a dosage dependent manner. The inhibitory effect of 3 microg PUC-GFP did not show significant difference with siGFP. (2) The significant repression appeared on the fourth day after transfecting monoclonal cells with 3 microg PUC-GFP. By the end of the sixth day, GFP expression in PUC-GFP group and siGFP group remained at a low level. (3) Cotransfecting PUC-GFP with pEGFP-C1 plasmids into PANC-1 cells showed a decreased transfection efficiency when compared with transfecting pEGFP-C1 alone. Higher PUC-GFP vs pEGFP-C1 corresponded with lower transfection efficency. (4) When adding new pEGFP-C1 plasmid to cells after inhibition appeared, the GFP expression recovered. Transfection of the promoterless DNA fragment containing full-length cDNA effectively induces a gene-specific silencing in mammalian cells.