Abstract:

Objective To establish stable germline ICN1 （intracellular domain Notch1）transgenic and double tracer T acute lymphoid leckemia, T-ALL zebrafish models by overexpressing human ICN1 gene in fli-EGFP zebrafish through T lymphocytes specific Rag2 promoter. Methods ICN1 plasmid carrying red fluorescent protein “reporter gene” (Rag2-ICN1-EGFP) was constructed and microinjected into zebrafish single-cell embryos, then the F0 (founder) generation was identified by fluorescence screening for establishing the zebrafish stable transgenic germline. The expression of ICN1 gene and protein was determined by fluorescence microscope, RT-PCR and Western blotting, and the phenotype of T-ALL zebrafish models was identified by flow cytometry and peripheral blood smear. Results Twenty of the 867 (2.3%) mosaic F0 zebrafish embryos injected with the constructed vector were identified to be the germline transgenic zebrafish, including 9 males and 11 females. The F0 adult fish of sexual maturity was copulated as one-to-one form, then F1 generation was produced. By using general view fluorescence microscope, RT-PCR and Western blotting, the expression of ICN1 was validated at the general view, mRNA and protein levels. Flow cytometry and peripheral blood smears suggested a significant reduction in the proportion of red blood cells and an increase of lymphocyte ratio in transgenic zebrafish as compared to control group. Conclusion Notch1 gene induced T-ALL zebrafish models with double tracer system have been successfully constructed, which may provide a research platform for the in-depth discussion on the ICN1 in the promotion of regulatory mechanism of T-ALL and high throughput drug screening in vivo.

Key words: Notch1, T acute lymphoid leukemia, zebrafish, double florescence tracer