Construction Of Ces2 Knockout Rat Model Based On CRISPR/Cas9 System And Related Drug Metabolism Study

Carboxylesterase 2(CES2)is an important drug metabolizing enzyme in human intestine,and plays an important role in the activation of many prodrugs and lipid metabolism.Although many methods were applied to CES2 research,no Ces2knockout(KO)animal models were reported.Rats are commonly used pattern animals in drug metabolism research,and the main Ces2 subtypes in rats' liver and intestine are Ces2 a and Ces2 c.In this study,CRISPR/Cas9 gene editing system was innovatively applied to construct Ces2 KO rat models,through targeting at the Ces2 a and Ces2 c gene.Ces2a/j,Ces2 c and Ces2a/c/j KO rat models were finally constructed.All KO rats grow and reproduce normally.The loss of Ces2 a expression was verified at m RNA level,and diltiazem was used to verify the loss of Ces2 a function.Irinotecan,aspirin and methylprednisolone succinate(MPHS)were also used to identify metabolic functions of KO rats.Results showed weakened metabolic ability for MPHS in all KO rats,while the metabolism of irinotecan and aspirin was normal.In order to determine the compensation effect in KO rats,the expression of major Ces isoforms in the liver and small intestine was detected at m RNA level.Moreover,analysis of liver function and blood lipids showed normal liver function in Ces2a/j and Ces2 c KO rats,while Ces2a/c/j absence caused slight lipid vacuoles accumulation in the liver and downregulated direct bilirubin and total bile acid.Totally,novel Ces2a/j,Ces2 c and Ces2a/c/j KO rat models was successfully constructed.These models can be used as important tools for Ces2 related drug metabolism research,and will be an important animal model for studying the physiological function of Ces2.