EGFP和大鼠GDNF基因共表达的慢病毒载体构建及转染大鼠骨髓间充质干细胞

为了构建携带增强绿色荧光蛋白(enhanced green fluorescent protein,EGFP)和胶质细胞源性神经营养因子(glial cell line-derived neurotrophic factor,GDNF)基因的慢病毒载体,观察GDNF基因大鼠骨髓间充质干细胞(rat mesenchymal stem cells,rMSC)的表达,本研究采用RT-PCR(reverse transcription-PCR)方法从P0大鼠小脑组织中扩增出GDNF基因编码区636 bp的片段,通过限制性内切酶酶切、T4DNA连接酶连接,将GDNF插入慢病毒转移载体PNL-IRES2-EGFP,构建PNL-GDNF-IRES2-EGFP。在脂质体介导下将构建成功的慢病毒三质粒系统共转染人胚肾细胞系(293T)包装生产慢病毒,测定病毒滴度。感染rMSCs,荧光显微镜下观察EGFP的表达、转导效率,RT-PCR、Western blot方法分别检测GDNF mRNA和蛋白的表达情况。结果显示:GDNF的基因序列经测序后与GeneBank报道的序列完全一致,重组慢病毒载体质粒PNL-GDNF-IRES2-EGFP经双酶切鉴定正确;三质粒共转染293T细胞后荧光激发可见大量绿色荧光,收集、浓缩病毒后测定其滴度为5.3×107pfu/ml;感染rMSCs结果显示:GDNF-rMSCs组5 d转导效率为93.3%±3.17%,传代培养4周,下降到81.1%±3.59%,差异具有统计学意义(P<0.01)。RT-PCR(real time-PCR)、Western blot显示GDNF成功在rMSCs中表达。本结果表明,我们已经成功构建带有EGFP、GDNF基因的慢病毒载体,并获得GDNF-rMSCs基因工程细胞,但提高该工程细胞外源基因的稳定表达技术仍需进一步探讨。

Construction of lentiviral vectors coexpressing EGFP and rat GDNF gene and transfection rat bone mesenchymal stem cells

In order to construct the lentiviral vector carrying enhanced green fluorescent protein(EGFP) and glial cell line-derived neurotrophic factor(GDNF) and to investigate the expression of GDNF in the rat mesenchymal stem cells(rMSC).The study was about a 636 bp of the cDNA encoding the CDS of the rat GDNF gene which was obtained from the cerebellum of a P0 rat by RT-PCR(reverse transcription-PCR),the restricted endonuclease digestion and T4DNA ligase connections were used to construct the recombinant lentiviral transfer vector: PNL-GDNF-IRES2-EGFP by inserting GDNF into PNL-IRES2-EGFP.Lentivirus three plasmid system which has been constructed was cotransfected into human embryonic kidney cell line-293T by lipofectamine 2000 to produce lentiviral particles,then the virus titer was examined.The rMSCs were infected by obtained lentiviral particles,the expression of EGFP and the transfection efficiency were examined under fluorescent microscope after transfection,the expression of GDNF was examined by RT-PCR and Western blot.The results showed that the cloned GDNF gene sequenced was consistent with the sequence reported in GeneBank.The PNL-GDNF-IRES2-EGFP plasmid was identified to be correct by double restriction enzymes;after three plasmids of lentiviral vector co-transfecting to the 293T cells,a great mount of green fluorescence in 293T cells could be observed with the fluorescent microscope.The supernatant was collected and concentrated,the titer of lentiviral vector particles was found to be 5.3×107 pfu/ml.The result of the infection in rMSCs showed that the transduction efficiency in GDNF-rMSCs group was 93.3%±3.17% after 5 days,and was decreasing significantly to 81.1%±3.59% four weeks later(P<0.01),RT-PCR(real time-PCR) and Western blot showed GDNF successful expression in rMSCs.The present results suggest that lentiviral vector carrying EGFP and GDNF gene had been constructed successfully and established GDNF cDNA-engineered rMSC,but the technique that sustained long-term expression of the transgene in the cDNA-engineered cell is needed to keep approach.