cmv promoter  (Vector Biolabs)

Journal: Molecular Therapy. Nucleic Acids

Article Title: Using RNA-targeting CRISPR-Cas13 and engineered U1 systems to target ABCA4 splice variants in Stargardt disease

doi: 10.1016/j.omtn.2025.102789

Figure Lengend Snippet: Evaluation of dCas13e to modulate splicing defects of ABCA4 c.5461-10T>C in ARPE19 (A) A schematic illustration of the pSPL3– ABCA4 c.5461-10T>C minigene and the all-in-one construct of a U6 promoter-driven sgRNA sequence followed by the CMV promoter-driven RBFOX1N-dCas13e-C factor. The sgRNA targeted the same region downstream of E39 (green) as a previously reported antisense oligonucleotide (AON) target site. Positions of SD6 and SA2 primers for splicing product amplification are displayed. (B) RT-PCR results of ABCA4 splice variants in ARPE19 cells transfected with the ABCA4 minigene system (MT), RBFOX1N-dCas13e-C, sgRNAs targeting mCherry (control), or ABCA4 . ACTB was used as a housekeeping gene. Mock control represents ARPE19 without transfection of the ABCA4 minigene system. (C) Gel quantification of relative transcript expression of ABCA4 splice variants compared to MT control without treatment. Data are represented as mean ± SEM ( n = 5). (D) Gel quantification of the ratio of disease transcript to FL transcript. Data are represented as mean ± SEM ( n = 3).

Article Snippet: For lentiviral transduction efficiency testing, ARPE19 was transduced using with lentiviruses with dsRed reporter driven by CMV promoter (Addgene #141148) and the fluorescence signal was analyzed after 4 days using MOI of 30.

Techniques: Construct, Sequencing, Amplification, Reverse Transcription Polymerase Chain Reaction, Transfection, Control, Expressing