6.2.2. Zebrafish

Recently, Ellis et al. generated an abcb11b knockout zebrafish by using the CRISPR/Cas-9 gene editing technology [95]. Abcb11b is the orthologue of the human BSEP gene in zebrafish. The histological and ultrastructural analysis showed a morphological hepatocyte injury pattern similar to that seen in patients with PFIC2. Similar to the situation in humans, BSEP deficiency induced autophagy in zebrafish hepatocytes. Treatment with rapamycin restored bile acid excretion, attenuated hepatocyte damage and extended the life span

of abcb11b mutant zebrafish. These effects were paralleled by a recovery of the correct canalicular localization of multidrug resistance protein 1.

Due to the transparency of these fish, the system allows monitoring of the bile flow in the intact animal with fluorescently labeled bile salt analogs. Furthermore, P-glycoprotein, which is reported to play a compensatory role (by transporting bile acids, and thereby protecting the hepatocytes from cholestasis induced injury) in BSEP knockout mice [96], is mislocalized to the hepatocyte cytoplasm in mutant zebrafish.

Animal models play an important role in studying cholestatic liver disease [93]. In contrast, identification of drug candidates for the treatment of folding and functionally deficient BSEP-mutants usually relies on in vitro model systems. For such studies, the patient-specific mutations are generated and expressed in cell lines. The impact of small molecules on the structure and function of BSEP is evaluated. Once the drugs have proven a potential in cell models, they are translated to a clinical setting [97,98].

Hydrodynamic tail vein injection in combination with the CRISPR/Cas9 technology has been used to specifically delete BSEP in mice and to study the consequences of the loss of an enzyme of the urea cycle (argininosuccinate lyase) [91]. In a similar way, such a BSEP knockout model system may be used for the study of mice expressing mutant forms of human BSEP in the liver and to monitor the effects of drug candidates on the folding, trafficking and function of the transporter.
