Validation of an in Vitro Non-alcoholic Steatohepatitis Cas9 Model (P08-130-19)

Abstract

ObjectivesThe goal of this project was to validate a CRISPR-Cas9 model to examine genes modulating non-alcoholic steatohepatitis (NASH). MethodsAML12 mouse liver cells were maintained in DMEM Advanced medium containing 10% fetal bovine serum, 100 U/mL Pen, and 100 mg/mL Strep (Growth Medium) at 37°C and 5% CO2. To obtain Cas9 expressing clones, cells were infected with ∼100 ng/mL of lentivirus (lentiCas9-Blast, a plasmid containing FLAG tagged Cas9) in the presence of 5 mg/mL polybrene. Blasticidin resistant cells were selected and single colonies were obtained. Cas9 expression was verified by Western blotting using anti-FLAG antibodies. To model NASH in vitro, cells were treated with a BSA-complex of 0.4 mM palmitic acid (PA) or 25 ng/mL TNFα in DMEM with 1 g/L glucose for 24 hours to induce collagen I expression, which is marker for the development of NASH. Collagen I expression was assessed by Western blot using anti-Collagen I antibodies. GAPDH expression was assessed by Western blotting and used to normalize collagen I expression. Additionally, fatty acid induced insulin resistance was examined. Cells were treated with either 0.4 mM, 0.9 mM or 1.2 mM PA in DMEM with 1 g/L glucose for 24 hours, then were stimulated with 100 nM insulin for 15 minutes. Western blot analysis for pAkt (ser473) expression was used assess insulin signaling. ResultsBoth AML12 and Cas9 expressing AML12 cells showed significant elevations in collagen I expression normalized to GAPDH when treated with PA (mean ± SEM 4.6 ± 1.5-fold and 7.9 ± 2.6-fold, respectively), but not when treated with TNFα (1.3 ± 1.1-fold and 1.1 ± 0.2-fold, respectively). A 76% and 36% reduction in fold change expression of pAkt was observed in Cas9 cells treated with 1.2 mM and 0.9 mM PA, respectively, and stimulated with insulin, compared to untreated controls stimulated with insulin, indicating an insulin resistant state. No significant differences were observed between the two cell types. ConclusionsThese experiments demonstrate that the Cas9 does not disrupt PA-induced Collagen I expression or insulin resistance in AML12 cells. Thus, Cas9 AML12 cells can be used with gene specific guide RNAs to mutate putative genes playing a role in NASH in a cost and time efficient manner. Funding SourcesFunding was provided by the Alabama Agricultural Experiment Station and an Auburn University Undergraduate Research Fellowship awarded to PK.