Ps-34: Analysis of Over Expression Using Lenti-X-Advanced Inducible System Carrying Mouse Peroxisomal Protein (PEP) CDS to Investigate The Over Expression Effects of PEP during Neural Differentiation of Mouse Embryonic Stem Cells


Forouzanfar M *, Beheshti S , Ghaedi K , Tanhaei S , Shoaraye Nejati A , Dormiani K , Karbalaei kH , Neamatollahi M , Izadi T , Nasr Esfahani MH ,

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Objective: Peroxisomal protein (PEP) is considered to be a peroxisomal matrix protein was previously identified as Fndc5 (firbonectin type III domain containing 5 proein; NCBI accession number: AK137327). As peroxisomes have been reported to have essential functions in brain, this expression pattern suggests a possible role for PEP in process of neurogenesis. Very recently it was reported that PEP could be cleaved and secreted to the outside of cells and acts as a hormone. The secreted portion was named Irisin. Irisin sequences are highly conserved in all mammalian species and are important for thermogenesis. The therapeutic potential of Irisin was raised as it was shown that would be effective in increasing of the diabetic individual tolerance to Insulin. Furthemore, exogenously administered Irisin induced the browning of subcutaneous fat. Our previous report indicated that PEP involved in the early process of neurogenesis. The results of previous study revealed that increased expression of PEP occured during retinoic acid induction when embryoid bodies were formed. In this study, we have designed a vector for efficient overexpression of PEP in process of neural differentiation whether it affects this process. Materials and Methods: An appropriate lentiviral vector, pLVX-Tight-Puro, containing the coding sequence of PEP was constructed as reported previously. Then mouse embryonic stem cells were transduced with lentiviruses derived from the pLVX-Tet-Off Advanced regulator vector. Transgenic mouse embryonic stem cells were selected with G418 for 14 days. To check the efficiency of lentiviral vectors, transgenic mouse embryonic stem cells which expressed tTA were transduced further with pLVX-Tight-Pur-EGFP vector. The stable line was selected by both of G418 and puromycin application. Overexpression of EGFP was analysed for with flowcytometry and real time PCR. After ensuring of the viral vector function, stably tTA expressing mouse embryonic stem cells were transduced with pLVX-Tight-Pur-PEP vector. Stable cell line selection carried out with G418 and purimycin. Results: The lentiviral vector expressing PEP was constructed correctly as checked by sequencing. Moreover results indicated stably expression of PEP in mouse embryonic cell line which was induced after doxycyclin treatment. Conclusion: Thus we have generated a stable mESCs overproducing PEP which is ready for further analyses.