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Establishment of a Transient NIH‐3T3 Fibroblast Overexpression System to Study Ikaros‐Mediated Downregulation of VPACR‐1

Understanding the molecular mechanisms that regulate tumor suppressor protein activities will provide crucial insight into tumorigenesis. Ikaros, a tumor suppressor protein, has previously been shown to regulate transcription of a receptor termed, vasoactive intestinal peptide receptor 1 (VPACR‐1). VPACR‐1 is expressed in numerous cells that modulate cell differentiation and proliferation. A stable Ikaros NIH‐3T3 system has previously demonstrated a down regulation of endogenous VPACR‐1 expression. Unfortunately, this stable system expressed Ikaros inconsistently. Therefore, there is a critical need for the establishment of a more reliable system to investigate Ikaros mediated downregulation of VPACR‐1. To this end, a transient Ikaros overexpression system was adopted. Cells were transfected with Lipofectamine and collected 48 hours post‐transfection and cDNA generated from total RNA. PCR measurements were performed to measure Ikaros and VPACR‐1 expression. This transient Ikaros technique resulted in the down regulation of VPACR‐1 by approximately 41% (range 15‐58 %, n = 4). A future goal will be to analyze the effects of other Ikaros isoforms and their DNA binding mutants on VPACR‐1. In addition, this transient model system will allow us to conduct chromatin immunoprecipitation assays to verify bound Ikaros protein to consensus sequences in the VPACR‐1 promoter.