Testing of short interfering RNAs (siRNAs) to knock-down gene expression is complicated in primary differentiated cells due to their limited availability and short in vitro life span. The objective of this study was to bypass these limitations by testing selected siRNAs in a heterologous cell line. A plasmid containing a fragment of porcine IGF-I receptor (pIGF-IR) gene cloned downstream of EGFP sequence was constructed and cotransfected with pIGF-IR/siRNAs in HEK 293T human cell line. The abatement of EGFP fluorescence, measured at mRNA and protein level, was compared with that induced by a EGFP/siRNA. Among the three pIGF-IR/siRNAs tested, one was active against the hybrid reporter gene as EGFP/siRNA, while the other two were inactive. When transfected in primary porcine coronary smooth muscle cells (PC-SMCs) and tested by real-time PCR, immunocytofluorimetry and cell motility assay, pIGF-IR/siRNAs confirmed the results obtained in HEK 293T cells, thus establishing that the search of active siRNAs addressed to a gene of a cell and/or a species of interest can be done in a heterologous cell line.
Identification of active siRNAs against IGF-IR of porcine coronary smooth muscle cells in a heterologous cell line.
NANNIPIERI, MONICA;
2005-01-01
Abstract
Testing of short interfering RNAs (siRNAs) to knock-down gene expression is complicated in primary differentiated cells due to their limited availability and short in vitro life span. The objective of this study was to bypass these limitations by testing selected siRNAs in a heterologous cell line. A plasmid containing a fragment of porcine IGF-I receptor (pIGF-IR) gene cloned downstream of EGFP sequence was constructed and cotransfected with pIGF-IR/siRNAs in HEK 293T human cell line. The abatement of EGFP fluorescence, measured at mRNA and protein level, was compared with that induced by a EGFP/siRNA. Among the three pIGF-IR/siRNAs tested, one was active against the hybrid reporter gene as EGFP/siRNA, while the other two were inactive. When transfected in primary porcine coronary smooth muscle cells (PC-SMCs) and tested by real-time PCR, immunocytofluorimetry and cell motility assay, pIGF-IR/siRNAs confirmed the results obtained in HEK 293T cells, thus establishing that the search of active siRNAs addressed to a gene of a cell and/or a species of interest can be done in a heterologous cell line.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
