The Establishment Of Abcb4:EGFP Transgenic Zebrafish For Multidrug Resistance

Objective: Failure of cancer chemotherapy can occur through multidrug-resistance(MDR). A well-established cause of multidrug-resistance involves the increased expression of members of the ATP binding cassette(ABC) transporter superfamily. By the exposure experiment of doxorubicin(DOX), to study the multidrug-resistance mechanism of abcb4 gene in zebrafish. Meanwhile to establish the Tg(abcb4:EGFP) transgenic zebrafish. Finally, providing experimental basis for the establishment of zebrafish drug screening model. Methods: According to the preliminary experiment, zebrafish embryos were treated with 2ml/L Dimethyl sulfoxide(DMSO), 10 μmol Doxorubicin(DOX) and 2ml/L DMSO+10 μmol DOX, respectively. The zebrafish embryos were treated with Eggwater as control group. Exposures were started with 4 to 16 cell stage embryos and terminated after 120 hours-post fertilization(hpf). Drug-induced embryos on 48, 72, 96, 120 hpf were collected to observe the expression of abcb4 gene in zebrafish embryos by whole-mount in situ hybridization(WISH) and quantitative real-time PCR(q PCR). To construte the recombinant plasmid Tol2-abcb4:EGFP with the abcb4 gene promoter, which activity was measured by the detection of luciferase activity. The Tg(abcb4:EGFP) transgenic zebrafish was established by the transposon Tol2 system, and using the apparent analysis and genomic PCR sequencing for its identification. Results: Compared with the control group, we found abcb4 gene was highly expressed in 96,120 hpf drug-induced embryos. In contrast, abcb5 gene did not exert this effect. WISH showed that abcb4 gene was expressed in the gut of 120 hpf wildtype embryos. However, the probe of abcb4 gene was confirmed with 96,120 hpf drug-induced embryos where a strong WISH signal occurred in the brain and heart. The luciferase activity of abcb4 gene promoter was higher than control group. The green fluorescent protein was expressed stably in the brain and gut of theTg(abcb4:EGFP) transgenic zebrafish, and the genomic sequencing is correct. Conclusion: Doxorubicin can increase the expression of abcb4 gene in zebrafish embryos, especially in the heart. For the first time, the high expression of abcb4 gene was found in DMSO-induced embryos. Compared with abcb5 gene in zebrafish, the expression pattern of abcb4 gene is more similar to human ABCB1 gene. With the predicting and cloning abcb4 gene promoter, its functional activity was confirmed by the detection of luciferase activity. The successful establishment of Tg(abcb4:EGFP) transgenic zebrafish provided experimental basis for research on multidrug-resistance mechanism of abcb4 gene and establishment of zebrafish drug screening model.