piggyBac转座子调节pPiggyBac-Rb质粒的构建

Expression of exogenous Rb gene mediated by piggyBac transposon in retinoblastoma cells

背景:外源性Rb基因的导入是抑制视网膜母细胞瘤细胞增殖的有效途径,piggyBac转座子介导的外源基因表达载体高效安全,在基因治疗方面具有广阔的应用前景。目的:探讨piggyBac转座子介导的外源性Rb基因的转染效率及其对视网膜母细胞瘤细胞的抑制作用。方法:采用RT-PCR技术扩增Rb基因编码区Rb cDNA,定向插入到载体pPiggyBac,获得PiggyBac调节的Rb表达质粒pPiggyBac-Rb;通过单独转染或合并pPiggyBac-helper转染人视网膜母细胞瘤细胞SO-RB50。结果与结论:考马斯亮蓝染色证明piggyBac转座子系统的转染效率最高,荧光定量PCR以及免疫荧光实验证明其介导的目的基因Rb与宿主SO-RB50细胞基因组整合效率最高且可在细胞中长期稳定表达,MTT实验证明经其所介导的外源性Rb基因表达对细胞活性影响最为显著。结果提示piggyBac转座子有可能成为视网膜母细胞瘤基因治疗的安全有效载体。

BACKGROUND： The introduction of exogenous Rb gene into retinoblastoma is an effective way to inhibit growth of the tumor. piggyBac transposon as a vector is safe and highly efficient, which is a potential vector on gene therapy. OBJECTIVE： To explore the transfection efficiency and its effect on inhibiting retinoblastoma by transfecting human retinoblastoma cell line SO-RB50 with an exogenous Rb gene expression system mediated by piggyBac transposon. METHODS： pPiggyBac-Rb plasmid that contains a piggyBac transposon sequence and express normal RB gene was constructed and used to transfect SO-RB50 cells alone or with pPiggyBac-helper. Coomassie brilliant blue staining, quantitative real-time PCR and immunofluorescence were adopted to evaluate the transfect efficiency of exogenous Rb gene mediated by piggyBac transposon. The viability of retinoblastoma cells was determined by 3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide. RESULTS AND CONCLUSION： Transfection efficiency was the highest with help of piggyBac transposon. piggyBac transposon-mediated exogenous Rb gene integrated into genome and obtained a long-term and stable expression.It also had a remarkable inhibition effect on the viability of SO-RB50. All these indicate that piggyBac transposon is a potential vector for retinoblastoma gene therapy.