ATP结合盒子亚型B1(ABCB1)编码多药转运蛋白也被称为P-糖蛋白(Pgp)，在人体内对抗异物外排起到重要作用，并与癌症对化疗的抵抗力密切相关。因此，开发高通量动物模型以筛选Pgp功能、底物和抑制剂的生物利用度至关重要。本研究中，我们生成并验证了斑马鱼缺乏abcb4基因的敲除系列，该基因是人类Pgp转运蛋白同源基因。使用CRISPR/Cas9基因组编辑技术，在斑马鱼abcb4的第4外显子中引入了移码突变。斑马鱼abcb4纯合突变体胚胎的肠道和脑部区域内荧光染料罗丹明123（人类Pgp基质）的积累增加。此外，与野生型斑马鱼相比，abcb4敲除胚胎对毒性化合物（如柔红霉素和长春花碱）更加敏感。免疫染色结果表明，斑马鱼Abcb4主要定位于成年斑马鱼的血管内脑细胞上。利用基因集富集分析对abcb4敲除斑马鱼脑部进行了转录组学分析，发现'细胞周期过程'、'有丝分裂细胞周期'和'以微管为基础的过程'在abcb4敲除脑部中呈显著下调，而且下调程度随年龄增长而加深。本研究建立并验证了abcb4敲除斑马鱼作为研究Pgp功能的体内动物模型。不料，研究还发现斑马鱼abcb4在脑部年龄相关变化中可能具有一个新角色。这些斑马鱼系列将为发现Pgp功能调节剂以及对人类突变体进行表征提供一个平台。©2023生物医学中心有限公司，斯普林格自然出版集团的一部分。

The ATP-binding cassette subfamily B member 1 (ABCB1), encoding a multidrug transporter referred to as P-glycoprotein (Pgp), plays a critical role in the efflux of xenobiotics in humans and is implicated in cancer resistance to chemotherapy. Therefore, developing high-throughput animal models to screen for Pgp function and bioavailability of substrates and inhibitors is paramount. Here, we generated and validated a zebrafish knockout line of abcb4, a human Pgp transporter homolog. CRISPR/Cas9 genome editing technology was deployed to generate a frameshift mutation in exon 4 of zebrafish abcb4. The zebrafish abcb4 homozygous mutant exhibited elevated accumulation of fluorescent rhodamine 123, a substrate of human Pgp, in the intestine and brain area of embryos. Moreover, abcb4 knockout embryos were sensitized toward toxic compounds such as doxorubicin and vinblastine compared to the WT zebrafish. Immunostaining for zebrafish Abcb4 colocalized in the endothelial brain cells of adult zebrafish. Transcriptome profiling using Gene Set Enrichment Analysis uncovered that the 'cell cycle process,' 'mitotic cell cycles,' and 'microtubule-based process' were significantly downregulated in the abcb4 knockout brain with age. This study establishes and validates the abcb4 knockout zebrafish as an animal model to study Pgp function in vivo. Unexpectedly it reveals a potentially novel role for zebrafish abcb4 in age-related changes in the brain. The zebrafish lines generated here will provide a platform to aid in the discovery of modulators of Pgp function as well as the characterization of human mutants thereof.© 2023. BioMed Central Ltd., part of Springer Nature.