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Fig. 1
Construction of plasmid encoding humanized TIE2-R849W and CVP defect in zebrafish caused by TIE2-R849W expression. (A) Construction of plasmid encoding mutant TIE2, based on normal humanized TIE2 sequence, via PCR-mediated site-directed mutagenesis. Location of mutation-related primers and plasmid elements are indicated. (B) Traditional gene sequencing identification after construction. As shown by red arrow, nucleotide T in control group had successfully transformed to C in mutant TIE2 group. (C) At approximately 2 days after fertilization (dpf) in control embryos, CVP formed honeycomb-like structures at the tail (white arrowheads). In contrast, mutant human TIE2 mRNA (200 pg) injection resulted in specific defects in caudal vein plexus (CVP) formation (D, white arrowheads). (E, F) Quantification of area and loop formation at CVP (Scale bars are 100 μm for A-C. Error bars, s.e.m.; *** refers to P < 0.0001 by ANOVA. CVP, caudal vein plexus; CA, caudal artery; CV, caudal vein).