[Construction of a regulated Crat overexpression system in mouse hippocampal neuronal HT22 cell line]
Abstract:
Objective: To construct an inducible carnitine acetyltransferase (Crat) gene expression system and provide a controllable experimental platform for research on neuroprotection in glaucoma. Methods: This experimental study was conducted from January 2025 to November 2025 using the mouse hippocampal neuronal HT22 cell line. A high-efficiency tetracycline-inducible lentiviral gene expression system was established by optimizing the multiplicity of infection (MOI) and polybrene concentration, as well as comparing the activities of the cytomegalovirus (CMV) promoter and the elongation factor 1α (EF1A) promoter. According to promoter type, the cells were divided into two groups: the CMV promoter group, in which enhanced green fluorescent protein (EGFP) fluorescence was detected, and the EF1A promoter group, in which mCherry fluorescence was detected. Conditional Crat overexpression was induced by doxycycline (Dox). Unmodified HT22 cells were assigned to an uninfected control group and an uninfected Dox-treated group, whereas successfully constructed HT22-TRE-mCrat/rtTA cells were assigned to an infected non-Dox-treated group and an infected Dox-treated group. The neuroprotective effect of the Crat overexpression system was evaluated in an H2O2-induced oxidative injury model by measuring lactate dehydrogenase (LDH) release, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, and the mRNA expression levels of apoptosis-related factors, including caspase-3, B-cell lymphoma 2-associated X protein (Bax), and B-cell lymphoma 2 (Bcl-2), using quantitative PCR (qPCR). Statistical analyses were performed using the independent-samples t-test or one-way analysis of variance, with Tukey's test for pairwise comparisons. Results: A tetracyline-inducible gene expression system was successfully constructed and stably established in HT22 cells. The EF1A promoter showed higher driving efficiency than the CMV promoter, with a significantly greater number of positive cells in the EF1A promoter group than in the CMV promoter group [(303.0±28.0) vs. (88.7±11.4) cells; t=17.37, P<0.001]. In the H₂O₂-induced oxidative injury model, the LDH release rate was significantly lower in the infected Dox-treated group than in the uninfected Dox-treated group (27.1%±4.7% vs. 49.8%±8.2%; P<0.001). The apoptotic cell rate determined by TUNEL staining was significantly lower in the infected Dox-treated group than in the uninfected Dox-treated group (14.1%±3.4% vs. 41.3%±4.1%; t=16.23, P<0.001). The caspase-3 mRNA level was significantly lower in the infected Dox-treated group than in the uninfected Dox-treated group (140.3±17.1 vs. 192.0±24.3; t=4.26, P=0.002). Bax mRNA expression was significantly lower in the infected Dox-treated group than in the uninfected Dox-treated group (132.0±13.1 vs. 169.8±12.6; t=5.09, P<0.001). Bcl-2 mRNA expression was significantly higher in the infected Dox-treated group than in the uninfected Dox-treated group (83.2±6.9 vs. 58.3±8.1; t=5.74, P<0.001). All of these differences were statistically significant. Conclusion: This study successfully established a controllable Crat overexpression platform using the mouse hippocampal neuronal HT22 cell line, enabling temporal regulation of Crat gene expression and cell-specific studies in this cell line.
