中国生物工程杂志

Object: The bicistronic recombinant adenovirus containing enhanced green fluorescent protein (EGFP) and transcription factors Lmx1A (LIM homeobox transcription factor 1-alpha) was constructed and packaged. The feasibility of dicistronic construction methods was evaluated. An experimental basis provides for differentiation potential of mechymal stem cells into neurons. Methods: The CDS of EGFP of Lmx1A, which was obtained by PCR, was inserted into the bicistronic expression plasmid pcDNA3.0BA. The dicistronic expression cassette which obtained by PCR, was inserted into the adenovirus shuttle plasmid pShuttle-CMV and followed homologous recombination with backbone plasmid pAdEasy-1.The adenovirus Ad-EGFP-Lmx1A was packaged in HEK293 cells. Human umbilical cord mesenchymal stem cells(hUC-MSCs) were infected with Ad-EGFP-Lmx1A.The expression and location of Lmx1A and EGFP was identified by RT-PCR, immunofluorescence and Western blot technology. Results: The packaged adenovirus showed typical morphology of an adenoviral particle, about 75nm, through TEM examination. The expression product s of EGFP and Lmx1A could be detected independently of each other. There was no significant difference in the expression strength. In conclusion, two genes can be expressed simultaneously in bicistronic eukaryotic expression plasmid pcDNA3.0BA.Two genes were transmitted into mesenchymal stem cells through adenovirus infection. Adenoviral expression efficiency can be observed in real time by observing the EGFP. These findings establish a rapid and effective method for constructing any two-gene expression. The construction of Ad-EGFP-Lmx1A provides an important basis for the role exploration in mesenchymal stem cells differentiating into neurons.