Site-Directed Mutagenesis of Prdx6 Promoter

To generate site-directed specific mutation of binding sites of Bmal1/E-Box (5′−341/−336-3′; T changed to A and A changed to T) and Nrf2/ARE (5′−357/−349-3′; TG changed to GT) present in Prdx6 gene promoter (Prdx6-CAT plasmid), we utilized PCR-based site-directed mutagenesis (Catalog No. 210518; QuikChange^TM lightning Site-Directed Mutagenesis kit, Agilent Technologies; Santa Clara, CA, USA) following the company’s protocol and our published method [5 (link),21 (link),81 (link)]. The double-stranded Prdx6 promoter construct (−918/+30) was used as template DNA with a pair of complementary primers to mutate the Prdx6 promoter construct at E-Box and ARE sites. The primers used for mutation were as follows:

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Chhunchha B., Kubo E, & Singh D.P. (2022). Obligatory Role of AMPK Activation and Antioxidant Defense Pathway in the Regulatory Effects of Metformin on Cellular Protection and Prevention of Lens Opacity. Cells, 11(19), 3021.

Publication 2022

QuikChangeTM lightning Site-Directed Mutagenesis kit

Binding sites Gene promoter Mutation Nrf2 Plasmid Primers Site directed mutagenesis

Corresponding Organization : University of Nebraska Medical Center

Other organizations : Kanazawa Medical University

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Variable analysis

independent variables

Site-directed specific mutation of binding sites of Bmal1/E-Box (5′−341/−336-3′; T changed to A and A changed to T) and Nrf2/ARE (5′−357/−349-3′; TG changed to GT) present in Prdx6 gene promoter (Prdx6-CAT plasmid)

dependent variables

Not explicitly mentioned

control variables

Double-stranded Prdx6 promoter construct (−918/+30) used as template DNA
Pair of complementary primers used to mutate the Prdx6 promoter construct at E-Box and ARE sites

positive controls

Not mentioned

negative controls

Not mentioned

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