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目的:通过携带绿色荧光蛋白(EGFP)和转录因子Lmx1A(LIM homeobox transcription factor1-alpha)的双顺反子重组腺病毒的构建和包装,评价双顺反子构建方法的可行性,为进一步探讨转录因子Lmx1A在间充质干细胞分化为神经元细胞的潜能提供实验基础。方法:通过PCR方法获得增强型绿色荧光蛋白(EGFP)和转录因子Lmx1A的CDS区序列,将其构建到双顺反子表达质粒pcDNA3.0BA,通过PCR的方法获得双顺反子表达盒,并插入到腺病毒穿梭质粒pShuttleCMV中,经过与腺病毒骨架质粒pAdEasy-1的同源重组,获得阳性重组质粒pAd-EGFP-Lmx1A,并进一步在HEK293细胞中包装为腺病毒。用携带EGFP和Lmx1A基因的重组腺病毒感染人脐带间充质干细(human umbilical cord mesenchymal stem cells,hUC-MSCs),通过RT-PCR、免疫荧光及Western blot的方法检测EGFP和Lmx1A的表达及产物定位。结果:透射电镜检查包装好的重组腺病毒为典型的腺病毒颗粒形态,大小约为75nm。Ad-EGFP-Lmx1A感染hUC-MSCs后检测到EGFP和Lmx1A的转录产物,表达产物互不影响,表达强弱无明显差异。结论:利用双顺反子真核表达质粒pcDNA3.0BA能够构建同时表达两个基因的重组腺病毒,腺病毒感染细胞后能够将两个基因传递至间充质干细胞中并表达。通过EGFP的观察可对腺病毒的表达效率进行实时观察,这些研究结果为任意组合的双基因表达建立了快速有效的构建方法,重组腺病毒Ad-EGFPLmx1A的成功构建也为将来研究Lmx1A在间充质干细胞分化为神经元过程中的作用提供了实验基础。
OBJECTIVE: To evaluate the feasibility of bicistronic constructs by constructing and packaging a bicistronic recombinant adenovirus carrying green fluorescent protein (EGFP) and the transcription factor Lmx1A (LIM homeobox transcription factor1-alpha) The factor Lmx1A provides an experimental basis for the potential of mesenchymal stem cells to differentiate into neuronal cells. Methods: The CDS region of enhanced green fluorescent protein (EGFP) and transcription factor Lmx1A was obtained by PCR and constructed into bicistronic expression plasmid pcDNA3.0BA. The bicistronic expression cassette was obtained by PCR Inserted into adenoviral shuttle plasmid pShuttleCMV. After homologous recombination with the adenovirus backbone plasmid pAdEasy-1, a positive recombinant plasmid pAd-EGFP-Lmx1A was obtained and further packaged into adenovirus in HEK293 cells. Human umbilical cord mesenchymal stem cells (hUC-MSCs) were infected with recombinant adenovirus carrying EGFP and Lmx1A genes, and the expression of EGFP and Lmx1A was detected by RT-PCR, immunofluorescence and Western blot, respectively. Product positioning. Results: The recombinant adenovirus packaged by transmission electron microscopy was typical of adenovirus particles with a size of about 75 nm. After hUC-MSCs were infected with Ad-EGFP-Lmx1A, the transcription products of EGFP and Lmx1A were detected. The products of expression did not affect each other, and there was no significant difference in the expression of EGFP and Lmx1A. CONCLUSION: The recombinant adenovirus expressing both genes can be constructed by using bicistronic eukaryotic expression plasmid pcDNA3.0BA. After being infected by adenovirus, the two genes can be transmitted to mesenchymal stem cells and expressed. The observation of EGFP can be used to observe the expression efficiency of adenovirus in real time. These results provide a fast and effective method for constructing the double gene expression in any combination. The successful construction of the recombinant adenovirus Ad-EGFPLmx1A is also a promising method for the future study of Lmx1A The role of mesenchymal stem cells in differentiating into neurons provides the experimental basis.