Intranuclear Localization of EGFP-mouse PPARy1 in Bovine Fibroblast Cells

Objective: The aim of this study was to clone PPAR?1 cDNA in an appropriate mammalian expression vector, with a chimeric cDNA form, encompassing PPAR? with enhanced green fluorescent protein (EGFP) cDNA. This recombinant plasmid will be used for further analy- ses to investigate the molecular mechanism of PPAR?1 for neural differentiation process. Moreover, the nuclear localization of the PPAR?1 protein linked to EGFP marker was chased by using transient transfection of a constructed plasmid into bovine fibroblast cells. Materials and Methods: Total RNA was extracted from the fatty tissue of an adult mouse. Using specific pair primers, PPAR?1 cDNA was synthesized and amplified to produce the entire length of ORF. RT-PCR products containing PPAR?1 cDNA were treated by enzymatic digestion and inserted into the pEGFP-C1 downstream from EGFP cDNA. The constructed vector was used for transformation into bacterial competent cells. Positive colonies which showed inserted PPAR?1 cDNA were selected for plasmid preparations and additional analysis was performed to ensure that PPAR?1 cDNA was inserted prop- erly. Finally, to confirm the intracellular localization of EGFP-PPAR?1, bovine fibroblast cells were transfected with the recombinant plasmid. Results: Our results from enzymatic digestion and sequencing confirmed, as expected, that PPAR?1 cDNA was amplified and cloned correctly. This cDNA gene encompassed 1428 bp. The related product was entered into the nucleus of bovine fibroblasts after transfection of its cDNA. Conclusion: PPAR?1 cDNA was cloned and sorted into nuclear compartments of bovine fibroblast cells upon transfection.