In vitro analysis of promoter activity in Müller cells

Rational modification of promoter architecture is necessary for manipulation of transgene activity and requires accurate deciphering of regulatory control elements. Identification of minimally sized promoters is critical to the design of viral vectors for gene therapy. To this end, we evaluated computational methods for predicting short DNA sequences capable of driving gene expression in Müller cells.

We measured enhanced green fluorescent protein (eGFP) expression levels driven by "full-length" promoters, and compared these data with computationally identified shorter promoter elements from the same genes. We cloned and screened over 90 sequences from nine Müller cell-associated genes: CAR2, CD44, GFAP, GLUL, PDGFRA, RLBP1, S100B, SLC1A3, and vimentin (VIM). We PCR-amplified the "full-length" promoter (~1500 bp), the proximal promoter (~500 bp), and the most proximal evolutionarily conserved region (ECR; 95-871 bp) for each gene, both with and without their respective 5' untranslated regions (UTRs), from C57BL/6J mouse genomic DNA. We selected and cloned additional ECRs from more distal genomic regions (both 5' and 3') of the VIM and CD44 genes, using both mouse and rat (Sprague-Dawley) genomic DNA as templates. PCR products were cloned into the pFTMGW or pFTM3GW lentiviral transfer vectors. Plasmid constructs were transfected into rat (wMC) or human (MIO-M1) Müller cells, and eGFP expression levels were evaluated by fluorescence microscopy and flow cytometry. Selected constructs were also examined in NIH/3T3 and Neuro-2a cells.

Several ECRs from the nine Müller cell-associated genes were able to drive reporter gene expression as well as their longer counterparts. Preliminary comparisons of ECRs from the VIM and CD44 genes suggested that inclusion of UTRs in promoter constructs resulted in increased transgene expression levels. Systematic comparison of promoter activity from nine Müller cell-expressed genes supported this finding, and characteristic regulation profiles were evident among the different genes tested. Importantly, individual cloned promoter sequences were capable of driving distinct levels of transgene expression, resulting in up to eightfold more cells expressing eGFP with up to 3.8-fold higher mean fluorescence intensity (MFI). Furthermore, combining constructs into single regulatory "units" modulated transgene expression, suggesting that secondary gene sequences provided in cis may be used to fine-tune gene expression levels.

In this study, we demonstrate that computational and empirical methods, when used in combination, can efficiently identify short promoters that are active in cultured Müller cells. In addition, the pFTM3GW vector can be used to study the effects of combined promoter elements. We anticipate that these methods will expedite the design and testing of synthetic/chimeric promoter constructs that should be useful for both in vitro and in vivo applications.