| Summary: | This study tested a method of overexpressing the apical sodium-dependent bile acid transporter (ASBT) in IEC-6 cells to replicate necrotizing enterocolitis (NEC), a gastrointestinal disease prevalent in premature infants. The expression vector pcDNA4/TO was altered to pDR1019, containing the Rattus norvegicus ASBT coding sequence. Sequencing confirmed the ASBT sequence in pDR1019 using forward, reverse, and midsequence primers (respective E-values: 0.0, 0.0, and 2e ⁻¹⁷⁴). IEC-6 cells were transfected with varying ratios of pcDNA6/TR (tetracycline-controlled repression vector) and pDR1019: 6:1, 15:1, and 30:1 pcDNA6/TR:pDR1019. Using relative quantitative real-time PCR (qrt-PCR), the 30:1 transfection had the greatest fold-change difference of ASBT mRNA expression relative to non-transfected IEC-6 cells (overexpression-induced and noninduced trials: 3120.0-fold and 1445.6-fold, respectively). To test the effects of bile acids and cytokines on ASBT expression in IEC-6 cells with overexpressed ASBT, a 30:1 pcDNA6/TR:pDR1019 transfection was performed, followed by treatments of chenodeoxycholic acid (CDCA), tumor necrosis factor-alpha (TNF-α), and interleukin 18 (IL18). According to a qrt-PCR, the ASBT mRNA expression fold-change of non-transfected trials were: CDCA (2.09x10⁹-fold), TNF-α (0.39-fold), IL18 (2.12x10⁹-fold); insufficient cells survived the transfection followed by treatments to yield usable RNA. Using this cell-based model to replicate NEC will aid future molecularly-based investigations of the disease.
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