Constructing circSOD2 Expression Plasmid

For circSOD2 expression plasmid (pcircSOD2) construction, full length circSOD2 cDNA was amplified from the HASMC mRNA using Phusion High-Fidelity PCR Master Mix (Thermo Scientific, F531S) and was inserted into a pcDNA3.1(+) CircRNA Mini Vector (a gift from Dr. Jeremy Wilusz^{21 (link)}, Addgene plasmid #, 60648) between Hind III and Xho I restriction site by using In-Fusion HD Cloning reagents (Clonetech, 638910). Residual sequences flanking the full length of circSOD2 on plasmid, which could be mistakenly included into circSOD2 after circularization, were removed by QuikChange II XL Site-Directed Mutagenesis Kit (Agilent, 200521). For luciferase reporter assay, the full length circSOD2 was amplified from the human SMC cDNA pool and was inserted downstream of firefly luciferase cassette in a PGL4.23 vector with minimal promoter at the Xba I site. All vectors were verified by sequencing. The primers amplifying circSOD2, used in mutagenesis and infusion cloning were listed in Major Resources Table.

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Mei X., Cui X.B., Li Y, & Chen S.Y. (2021). CircSOD2, a Novel Regulator for Smooth Muscle Proliferation And Neointima Formation. Arteriosclerosis, thrombosis, and vascular biology, 41(12), 2961-2973.

Publication 2021

Phusion High-Fidelity PCR Master Mix QuikChange II XL Site-Directed Mutagenesis Kit

Assay Cdna Circrna Firefly luciferase Human Luciferase Mrna Mutagenesis Pgl4 Plasmid Primers Site directed mutagenesis Vectors

Corresponding Organization :

Other organizations : University of Missouri, University of Georgia

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Variable analysis

independent variables

Insertion of full length circSOD2 cDNA into pcDNA3.1(+) CircRNA Mini Vector
Removal of residual sequences flanking the full length of circSOD2 on plasmid using QuikChange II XL Site-Directed Mutagenesis Kit
Insertion of full length circSOD2 downstream of firefly luciferase cassette in a PGL4.23 vector with minimal promoter

dependent variables

CircSOD2 expression
Luciferase reporter activity

control variables

Phusion High-Fidelity PCR Master Mix used for amplification
In-Fusion HD Cloning reagents used for inserting circSOD2 into plasmid
Sequencing used to verify all vectors

positive controls

Not specified

negative controls

Not specified

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