Figure 1.

Design and Screening of Engineered miATXN3 Constructs

(A) Schematic representation of the human ATXN3 gene and miATXN3 binding sites. The ATXN3 gene (NG_008198.2) consists of 11 exons shown by the numbered black boxes. The white boxes represent the 5′ and 3′ UTRs. The CAG expansion in exon 10 is depicted by a red triangle. The position of the miATXN3 candidates are shown on top of the exons and indicated with numbers 1–11. miATXN3_3, miATXN3_8, and miATXN3_9 are exon spanning. (B) Schematic representation of the miATXN3 constructs. Each construct was expressed by the CAG promotor, followed by the primary miATXN3 sequence in the miR-451 scaffold, and a human growth hormone polyadenylation (hGH poly(A)) signal. (C) Schematic representation of the Luc reporter. The whole sequence of the ATXN3 mRNA (NM_004993.5) was cloned downstream of the RL gene. In addition, FL was co-expressed from the vector as an internal control. (D) Dose-dependent knockdown of ATXN3 Luc reporter by miATXN3 constructs. HEK293T cells were co-transfected with 50 ng of the Luc reporter and 0.1, 1, 10, and 100 ng of the miATXN3 constructs. RL and FL were measured 2 days post-transfection, and RL was normalized to FL expression. Scrambled microRNA (miScr) served as a negative control and was set at 100%.