Generation and characterization of liver-specific knockout mouse of Cereblon

Abstract

Cereblon (CRBN) was initially revealed as a target gene of mild intellectual disability in human. CRBN is the substrate receptor of Cullin-ring E3 ubiquitin ligase complex (CRL) and mediates degradation through the binding to its endogenous substrates. CRBN also can inhibit the AMPK activity by recruiting AMPK complex to CRL. Previously, to understand the physiological roles of CRBN and elucidate relationship between AMPK and CRBN, as well as to test whether Crbn deficiency affected the function of AMPK in vivo, whole-body Crbn knockout (WKO) mice was generated. Subsequently, the energy metabolism-related functions of CRBN have been studied in the WKO mouse. It was observed that CRBN can affect AMPK level towards down-regulation and the Crbn-deficiency prevents high fat diet-induced obesity. WKO mice fed high-fat diet (HFD) show improvement in their metabolic status. Shortly, AMPK is hyperactivated in the absence of Crbn in vivo and acetyl-CoA carboxylase (ACC) which is the downstream to AMPK is also continuously phosphorylation. As a result, CRBN is defined as a negative regulator of AMPK. In WKO mice, hepatic AMPK activation was increased however, it is not yet revealed that this phenomenon prevails due to systemic effects or tissue-specific effects. As AMPK-mediated phenomenon was only studied in the WKO, the tissue-specific roles of CRBN were needed to be investigated in the tissue-specific KO mice. Hence, I generated liver-specific Crbn knockout (LKO) mice using Cre/loxP system. To investigate whether Crbn was knocked out or not, genotyping was performed and presence of both floxed Crbn band and Cre band was confirmed. In addition, through the tissue sampling of LKO mice, it was confirmed that CRBN protein was specifically absent in liver tissues. To obtain further evidences that I obtained LKO mouse, IHC was also performed. However, it was difficult to see difference in the expression pattern of CRBN between Crbnfloxed/floxed mice and LKO mice through the IHC. I also performed several experiments to compare the behavior of LKO mouse and WKO mouse towards metabolism.