Long non-coding RNA Gm15441 attenuates hepatic inflammasome activation in response to metabolic stress

Chad N. Brocker; Donghwan Kim; Tisha Melia; Kritika Karri; Thomas J. Velenosi; Shogo Takahashi; Jessica A. Bonzo; David J. Waxman; Frank J. Gonzalez

A gene targeting strategy was developed to generate a Gm15441 knockout mice without impacting Txnip expression on the opposing strand. To accomplish this goal, CRISPR/Cas9 was used to insert a lox-STOP-lox (LSL) cassette downstream of Gm15441 exon 1 (Figure 4A and B). The LSL cassette prevents transcription of Gm15441 in a Gm15441-floxed mouse but has no effect on transcription of Txnip, which is > 2 kb downstream of the last exon of Txnip. Crossing with a transgenic Cre mouse deletes the LSL cassette and restores Gm15441 expression ( Figure 4C ). All mouse lines were on a pure C57BL/6J background. The genotyping scheme generates an approximately 1200 bp band in Gm15441 wild-type mice (Gm15441 +/+ ) and a 1700 bp band in Gm15441 LSL mice ( Figure 4D ). Basal expression of Gm15441 was significantly lower in livers of Gm15441 LSL when compared to Gm15441 +/+ mice ( Figure 4E ). Expression in heterozygous Gm15441 HET mice was comparable to Gm15441 LSL . To verify the subcellular localization and loss of expression of Gm15441, fluorescence in situ hybridization (FISH) staining was performed on livers from Gm15441 +/+ and Gm15441 LSL mice fed control diet or treated with WY-14643. DAPI staining was used as a counter stain to detect nuclei in liver sections. A pronounced increase in Gm15441 fluorescence was detected in the cytoplasm and nuclei of hepatocytes in livers from WY-14643-treated Gm15441 +/+ mice. The Gm15441 RNA signal was absent in livers from vehicle-treated Gm15441 +/+ and Gm15441 LSL mice, and in livers from WY-14643-treated Gm15441 LSL mice ( Figure 4F ). These data validate Gm15441 LSL mice as an effective knockout mouse model and reveal that hepatic Gm15441 expression is both nuclear and cytosolic evoking several possible mechanisms by which it regulates Txnip.