Document Type : Original Article
Biology Department, School of Sciences, University of Isfahan, Isfahan, Iran
Department of Cell and Molecular Biology, Royan Institute for Animal Biotechnology, ACECR, Isfahan, Iran
Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
Objective: The aim of this study was to clone PPARγ1 cDNA in an appropriate mammalian expression vector, with a chimeric cDNA form, encompassing PPARγ with enhanced green fluorescent protein (EGFP) cDNA. This recombinant plasmid will be used for further analyses to investigate the molecular mechanism of PPARγ1 for neural differentiation process. Moreover, the nuclear localization of the PPARγ1 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, PPARγ1 cDNA was synthesized and amplified to produce the entire length of ORF. RT-PCR products containing PPARγ1 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 PPARγ1 cDNA were selected for plasmid preparations and additional analysis was performed to ensure that PPARγ1 cDNA was inserted properly. Finally, to confirm the intracellular localization of EGFP-PPARγ1, bovine fibroblast
cells were transfected with the recombinant plasmid.
Results: Our results from enzymatic digestion and sequencing confirmed, as expected, that PPARγ1 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: PPARγ1 cDNA was cloned and sorted into nuclear compartments of bovine fibroblast cells upon transfection.