Construction of expression vectors carrying mouse peroxisomal protein gene (PeP) with GST and Flag labels

The aim of this study was to construct expression vectors carrying mouse peroxisomal protein gene (PEP-cDNA) in prokaryotic and mammalian expression vectors in chimeric cDNA types, encompassingGST and FLAG with PEP-cDNA. PEP-cDNA was sub-cloned in pGEX6p2 prokaryotic expression vector in order to label this gene with GST to purify PEP protein for further biochemical analysis and identifying related proteins thereafter. FLAG-PEP recombinant DNA was produced and sub-cloned inpUcD3 eukaryotic expression vector to express tagged-PEP protein for transient transfection analysis and identifying intracellular localization of PEP protein in future experiments. PEP-cDNA was amplifiedin different PCR reactions using pEGFP-PEP vector and 2 sets of primers introducing specific restriction sites at the ends of PEP. PCR products with BamHI/SalI restriction sites were treated by restriction enzymes and inserted into the pGEX6p2, downstream of GST tag. PEP-cDNA containingBamHI/ApaI restriction sites and FLAG gene (which amplified using pUcD3-FLAG-PEX3 vector) were used as templates in secondary PCR for amplifying FLAG-PEP recombinant DNA. FLAG-PEP fragment was treated by enzymatic digestion and inserted into the pUcD3 eukaryotic expression vector.pGEX6p2-PEP and pUcD3-FLAG-PEP constructed vectors were transformed into the one shot TOP10 and JM105 bacterial competent cells, respectively. Positive colonies were selected for plasmid preparation. Results confirmed correct amplification of the expected products. PEP-cDNA in both PCRreactions encompasses 630 bp. FLAG fragment containing designed sites was 77 bp and FLAG-PEP fragment was 700 bp. Sequencing of constructed vectors confirmed that PEP-cDNA was tagged appropriately and inserted free of mutation and in frame with GST and FLAG

TRANSFECTION OF MOUSE PPARGAMMA1 CDNA IN TO THE BOVINE FIBROBLAST CELLS AND ANALYSIS OF ITS INTRACELLULAR EXPRESSION USING EGFP GENE REPORTER

The main aim of this research is to study nuclear localization of mouse PPARg1 cDNA into pEGFP-C1. To investigate the nuclear localization of PPARg1 protein linked to EGFP marker gene into bovine fibroblast and analysis of intracellular green fluorescency Mouse PPARg1 cDNA in a mammalian expression vector (pEGFP-C1) in a chimeric cDNA type, encompassing PPARg1 with EGFP cDNA. EGFP marker was used for monitoring. To confirm the intracellular localization of EGFP- PPARg1, bovine fibroblast cells were transfected with the 2.4 mg constructed plasmid and 6 ml Lipofectamine 2000. The related product was entered into the nucleus of bovine fibroblasts after transfection of its cDNA. As expected, chimeric cDNA of EGFP-PPARg1 correctly expressed and related protein was dominantly entered to the nuclei of the cells. Thus the recombinant plasmid could be applicable for further studies to unravel the further aspects of PPARg1 function. Moreover fusion of EGFP with PPARg1 does not hamper the nuclear targeting of PPARg1