Construction Of CGAS-STING Regulated Gene Knockout Cell Lines And Interferon-β Gene Promoter-Induced EGFP Transgenic Mice

Background Innate immunity is the first line of the body’s response against foreign invasion including microbial invasion. Zhijian Chen’s group discovered a new DNA sensors, cGAS and the second messenger cGAMP in 2013, which further explains how the body identify the foreign DNA in innate immune signaling in response to foreign invasion. However, the mechanism regarding the cGAS-STING upstream and downstre-am signaling regulation still remains large ly unknown and needs to be further explored.Purpose 1) RNF5 and IFNAR are two components involved in the cGAS-STING path-way function. We plan to construct the RNF5 and IFNAR knockout vectors to studied their corresponding functions in L929 cells by using CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated proteins) gene editing system.2) IFNβ is induced by the cGAS-STING activation in response to foreign invasion. We plan to construct IFNβ promoter-induced EGFP vectors and to establish the corresponding cells and the transgenic mouse line to study the mechanism involved in the IFNβ production in response to foreign invasion.Method Screening the expression of gene components related to cGAS-STING signa-ling by RT-PCR; Constructing gene knockout vectors by using the CRISPR/Cas9 system, and gene-knockout cell line by cell culture to estabolish individual clonal cell lines; Esta-blishing a series of vectors that can detect IFNβ induction and EGFP expression controlled by the IFNβ-promoter; Construction and verification of a transgenic mouse line by microscopic injection and genotyping by PCR and corresponding sequencing.Results We successfully construted and verified the CRISPR/Cas9 knockout vectors PX459-KO-M-RNF5, PX459-KO-M-IFNAR, PX459-rosa26-sg, and generated RIF5、 IFNAR knockout cell lines. We successfully constructed the IFNβ-promoter cotrolled EGFP expression vector, PGL3-Ehancer-IFNβ-promoter-EGFP, And establish a transge-nic mice model which can be used to detect the expression of IFNβin response to immune stimulation.