小鼠非转录间隔区序列对外源基因表达促进作用初步的研究.docVIP

小鼠非转录间隔区序列对外源基因 硕士研究生 导 师孙怀昌 中文摘要 核糖体基因非转录间隔区ribosomal gene nontranscribed spacer，NTS)能表达。本小鼠NTS系列，表达情况来验证NTS促进。首先将NTSpEGFP-N1真核表达载体，pEGFP-N1-NTS。然后在相同的条件下，分别用NTS载体和对照载体转染细胞观察pEGFP-N1-NTS转染组的荧光阳性细胞数和荧光强度明显高于pEGFP-N1转染组，表明小鼠NTS序列可促进外源基因在不同细胞 PB转座系统是新的基因导入系统。NTS 370bp序列插入PB转座子载体pAAAV-PBTR-EGFP中，获得新载体pAAAV-PBTR-EGFP-NTS分别用两种质粒pAAAV-PBase共转染不同细胞 pAAAV-PBTR-EGFP-NTS转染组的荧光阳性细胞数及荧光强度高于pAAAV-PBTR-EGFP转染组，pAAAV-PBTR-EGFP-NTS+pAAAV-PBase转染组的荧光阳性细胞数pAAAV-PBTR-EGFP +pAAAV-PBase转染组，表明 将小鼠NTS 370bp序列插入表达NEO基因的转座子载体pAAAV-PBTR-NEO中，获得新的表达载体pAAAV-PBTR-NEO-NTS。然后两种质粒pAAAV-PBase共转染不同细胞浓度G418 pAAAV-PBTR-NEO-NTS转染组多于pAAAV-PBTR-NEO组，pAAAV-PBTR-NEO-NTS+pAAAV-PBase转染组细胞集落数多于pAAAV-PBTR-NEOpAAAV-PBase转染组，说明 关键：非转录间隔区 Enhancive Effect of Murine Non-Transcribed Sapce Sequence on Foreign Gene Expression M.D. Candidate: Zhang Wu Supervisor: Prof. Sun Huaichang Abstract 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, the 370-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-N1, resulting in the NTS-containing expression vector pEGFP-N1-NTS. Under the same conditions, the two vectors were transfected into PK-15 cells, NIH-3T3 cells and 293T 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-15 cells, NIH-3T3 cells and AAV-293cells, respectively, in the absence or presence of PB transposasee vector pAAV-PBase. Fluorescent micros