It is ideal to overexpress a gene of interest coupling to a reporter gene as a single transcript driven by a common promoter to facilitate the identification of transfected cells genuinely overexpressing the desired gene. However, the traditional strategies failed to achieve this aim in case of non-coding RNAs such as miRNAs. Since the processing of pri-miRNA by Drosha enzyme in nuclei generates a destructed mRNA fragment, which could not be transported into cytoplasm, translation of reporter gene will be blocked. To overcome this issue, recent strategies were developed by inserting a miRNA- or shRNA-expressing cassette into an intron sequence located in a marker mRNA, such as a green fluorescent protein (GFP). Following of the splicing of the intron containing miRNA- or shRNA-expressing cassette, subsequent processing of pri-miRNA or shRNA will be facilitated and the resulting mature mRNA encoding a reporter gene will efficiently be exported to cytoplasm and translated into the reporter protein such as GFP. In the current study, we designed an eGFP-intron splicing system in AAV-based vectors to overexpress miR-183 cluster coinciding with eGFP in retinal pigment epithelial (RPE) cells.

To design an eGFP-intron splicing system, the sequences of an artificial intron and eGFP fragments were obtained from Yoel Sadovsky et al. (2016) and flanked by EcoRI and BglII sites for cloning into pAAV-MCS vector. Moreover, a DNA fragment containing miR-183/96/182 sequences was PCR amplified and then inserted between KpnI and XhoI sites located into the intron sequence of eGFP-intron splicing system. Recombinant AAV vectors were transfected into HEK293t cells.

Synthesized eGFP-intron sequence was successfully cloned into pAAV-MCS vector (regarding as control vector) followed by cloning of the miR-183 cluster fragment into intron sequence, resulting in the desired recombinant vector named pAAV.MCS.miR-183.int.eGFP. HEK293t cells were transfected with rAAV vectors. After 24 hours, most cells exhibited bright, intense signal of GFP protein expression.

The eGFP-intron splicing system can be cloned into a wide range of viral and nonviral plasmids and will be applicable as a superior tool to efficiently overexpress miRNAs and shRNAs along with a marker gene for in vitro- and in vivo-gene therapy studies.