Enhancive Effect Of Murine Non-Transcribed Sapce Sequence On Foreign Gene Expression

Nontranscribed spacer (NTS) is a DNA fragment in the rDNA of mouse, which can enhance gene expression levels and prolong gene expression time. In the present study, the370-bp murine NTS fragment was amplified by PCR and subcloned into a series of expression vectors for comparison studies. The NTS sequence was first cloned into the enhanced green fluorescent protein (GFP)-containing vector pEGFP-Nl, resulting in the NTS-containing expression vector pEGFP-N1-NTS. Under the same conditions, the two vectors were transfected into PK-15cells, NIH-3T3cells and293T cells, respectively. Fluorescent microscopy showed that the NTS sequence could enhance GFP gene expression in all three cell types.PiggyBac (PB) transiposition system is a relatively gene transfer system. To investigate its synergistic effect on PB-mediated gene expression, the NTS was inserted into PB transposon vector pAAAV-PBTR-EGFP, resulting in NTS-containing transposon vector pAAAV-PBTR-EGFP-NTS. The two were transfected into PK-15cells, NIH-3T3cells and AAV-293cells, respectively, in the absence or presence of PB transposasee vector pAAV-PBase. Fluorescent microscopy showed that the NTS sequence could enhance transposon-mediated gene expression in all three cell types, particularly in the presence of transposasee vector pAAV-PBase.The NTS sequence was further cloned into neo gene expression transposon vector pAAAV-PBTR-EGFP, insulting in NTS-containing transposon vector pAAAV-PBTR-EGFP-NTS. Under the same conditions, the two vectors were transfected into PK-15cells, NIH-3T3cells and AAV-293cells, respectively, in the absence or presence of PB transposasee vector pAAV-PBase. After selection for two weeks with G418, significantly more G418-resistant cell clones developed from the NTS-containing vector-transfected cell cultures, particularly from the cell cultures co-transfected with PB transposasee vector pAAV-PBase. These data suggest that the mouse NTS sequence can enhance PB transposon-mediated stable gene expression in all three cell types tested.