Li-Ping Shu *, Jin Song *, Yuan-Yuan Tuo,Lu-Jun Shang,Yan-Hua Zhou,Xi-Jun Wu, Zhi-Xu He# ( * co-author # correspongding author((hzx@gmc.edu.cn)
Objective: To establish zebrafish model of G6PD dificiency.With dominant negative, expression of the mutant G6PD can impact the normal G6PD protein expression ,which result G6PD activity reducing.This modle can be used to study G6PD deficiency molecular mechanisms and provide an experimental basis for mass drug screening .Methods: To constrct a gata1:g6pdM118-144-EGFP-PBSK-IsceI recombinant plasmids.The plasmid were microinjected into tüebingen wild-type zebrafish embryos of single-cell stage.zebrafish embryos, which is injected into gata1:g6pdM118-144-EGFP -PBSK-IsceI plasmid, express green fluorescent protein in the blood circulation that be treated as F0 generation G6PD transgenic zebrafish. Feeding F0 generation transgenic zebrafish until breeding period, each F0 generation transgenic zebrafish mate with wild-type zebrafish, the green fluorescent protein expression was observed in blood circulation in F1 generations transgenic zebrafish .Then using genomic PCR,whole mount in situ hybridization (WISH),Western blotting identify the Tg:gata1:g6pdM118-144-EGFP F1 generation transgenic zebrafish on levels of DNA, mRNA, protein .Results: zgata-1-g6pdM118-144-EGFP-pBSK-I-SceI recombinant plasmid is constructed and injected into wild-type zebrafish embryos.We screen zebrafish embryoes,which express green fluorescent protein as Founder 0 generation G6PD transgenic zebrafishs. we got five F0 generation G6PD transgenic zebrafishs. Expressing of the green fluorescent protein in F0 generation transgenic zebrafishs begins in ventral mesoderm at 11 hpf, and circulates with blood after 24 hpf ,and which can be passed on to the offspring. The g6pdM118-144 is amplified from the genome of F1 generation transgenic zebrafish.With WISH,the mRNA of EGFP is detected at erythropoiesis site of F1 generation transgenic zebrafish. With Western blotting, expression of G6PDM118-144-EGFP fusion protein is detected in F1 generation transgenic zebrafish.To sum up,we can identify that the gata-1-g6pdM118-144-EGFP has integrated into the zebrafish genome and transcribed into mRNA, translated purposes fusion protein.Conclusions Identified the Tg:zgata-1:g6pdM118-144-EGFP transgenic zebrafish on levels of DNA, mRNA, protein.Establishment the zebrafish disease models of G6PD deficiency can achieve in-depth study related mechanism in G6PD deficiency and high-throughput screening of antimalaric drugs to treat G6PD deficiency disease.
Acknowledgments This work was supported by the National Natural Science Foundation of China (NO.31360285).