Evaluation of the Stress Effect on Expression of BEST-5 Gene in Gultured Rat Hepatocytes and Cloning of This Gene

Abstract

Introduction:Liver has important roles in body metabolic regulation and for this reason hepatocytes are used worldwide.Investigations showed that isolation of hepatocytes causes activation of stress related genes.The aim of this study was to study the stress related expression of BEST-5 following hepatocytes isolation and culture. Method:The BEST-5 gene is cloned and analyzed for the first time from isolation and hepatocytes.very little is known about this gene and almost nothing is known about its function.RAN was isolated from hepatocytes after 3h culture and used for generation of PCR products corresponding to the BEST-5.cDNA generated was cloned into pCR2.1 plasmid vector.following transformation into TOPO10oneshot cells were grown in LB agar plates containing X-Gal and ampicillin,overnight at 37C.to confirm that the plasmids contained inserts of the correct size,the vectors obtained from mini-perparations were digested with the desired restriction enzymes. Results:sequencing was performed for the gene.RT-PCR and Northern blotting analysis showed that Best-5 mRAN is expressed 3h after isolation and culture of primary hepatocytes (3h)BEST-5 mRNA was observed unitil 5h of culture and then there waz no detectable band of BEST-5 at further time points.Comparison of expression of the level of mRNA of BEST-5,when data statistically were analyzed showed a significant difference between the expression of BEST-5 mRNA expression at 3h with 0h,24h,35h and 48h of culture(p<0.001). Conclusion:According to the results the stress induced by hepatocytes isolation and culture leads to the expression of Best-5 time-dependently.