Comparison of the efficiency of different promoters driving foreign gene expression in microglia

In order to develop promoters suitable for adeno-associated virus (AAV) vector modification and with high neural cell tissue specificity, and achieve the targeting of AAV gene therapy drugs on the nervous system. Three strong promoters, cytomegalovirus (CMV), chicken β-actin (CBA), and a composite promoter containing CMV and CBA (CBH), were each inserted into the AAV viral vector to investigate their ability to drive exogenous gene expression in microglia. Firstly, the sequences of the CMV, CBA and CBH promoters were synthesized in vitro. Secondly, they were inserted into the pSCAAV-EGFP vector, resulting in three eukaryotic expression vectors(pLMV-EGFP, pLBA-EGFP, and pLBH-EGFP) driving the expression of enhanced green fluorescent protein(EGFP). Following transformation into HB101 competent cells and sequencing verification, the recombinant plasmids were transfected into mouse microglial (BV2) cells. Finally, after transfection 48 h, the expression of EGFP in the cells was observed under a fluorescence microscope, and the efficiency of EGFP expression was detected by flow cytometry and polymerase chain reaction (PCR). The results showed that all three promoters could drive exogenous gene expression in BV2 cells, but there were differences in activation efficiency. The percentage of EGFP-positive cells after transfection with pLMV-EGFP was 4.92%, significantly higher than the other two promoters. The results of quantitative PCR were consistent with those of flow cytometry, showing the highest EGFP expression in cells corresponding to the CMV promoter, followed by CBA, and the lowest for CBH. Therefore, the CMV promoter has better specificity for microglia on the AAV vector and can be used as the preferred promoter for the development of new drugs for gene therapy in the nervous system. Additionally, these findings may also provide a reference for subsequent exploration of more AAV gene therapy drugs with lower toxicity and cost.