Ji Ming, Yi Shounan, Yu Deling, Wang Wei
Department of Physiology, School of Basic Medicine, Central South University, Changsha, China.
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2011 Dec;36(12):1141-6. doi: 10.3969/j.issn.1672-7347.2011.12.003.
To determine the genetic modification on neonatal porcine islet cell clusters (NICC) by small interfering RNA (siRNA)-mediated tissue factor (TF) knockdown in vitro.
Porcine NICC were transfected with 5 pairs of designed siRNA respectively or in different combinations with lipofectamine 2000. Transfected NICC were analyzed for TF gene by real-time PCR to select the siRNA which worked best. Meanwhile, the viability of NICC after the TF siRNA transfection was examined by FACS. The efficiency of TF gene and protein suppression was measured by real-time PCR and and FACS respectively.
Real-time PCR and FACS showed that a 60% reduction in the TF gene expression and a 50% reduction in the protien level of TF on NICC were achieved by transfecting 3 pairs of selected siRNA. The siRNA transfection had no significant effect on the viability of NICC which was analyzed by FACS.
The expression of TF on porcine NICC is efficiently suppressed by 3 pairs of designed siRNA in vitro.
通过体外小干扰RNA（siRNA）介导的组织因子（TF）基因敲低，确定对新生猪胰岛细胞簇（NICC）的基因修饰。
分别用5对设计好的siRNA或与脂质体2000以不同组合转染猪NICC。通过实时聚合酶链反应（PCR）分析转染后的NICC的TF基因，以选择效果最佳的siRNA。同时，通过流式细胞术（FACS）检测TF siRNA转染后NICC的活力。分别通过实时PCR和FACS测定TF基因和蛋白抑制效率。
实时PCR和FACS显示，转染3对选定的siRNA后，NICC上的TF基因表达降低了60%，TF蛋白水平降低了50%。通过FACS分析，siRNA转染对NICC的活力没有显著影响。
体外3对设计好的siRNA可有效抑制猪NICC上TF的表达。
